Placental transfer of pyrimethamine studied in an ex vivo placental perfusion model.

Pyrimethamine is used as and anti-infectious agent because of its antifolate properties. Its action is synergistic with that of dapsone and sulfamides on Toxoplasma gondii. The goal of the present study was to evaluate the placental transfer of pyrimethamine in an ex vivo model of perfused human placental cotyledon at term. Human placentas were perfused according to the slightly modified method of Schneider. The pyrimethamine fetal transfer rate was approximately 30%, while cotyledon clearance was about 1.8 ml/min. The placental transfer of pyrimethamine seems to be independent of the maternal concentrations of pyrimethamine, suggesting passive diffusion mechanisms or a nonsaturable active transport at the tested concentrations.

Evaluation of the new photosensitizer Stakel (WST-11) for photodynamic choroidal vessel occlusion in rabbit and rat eyes.

PURPOSE: To evaluate the photodynamic potential of a new hydrosoluble photosensitizer (WST-11, Stakel; Steba Biotech, Toussus-Le-Noble, France), for use in occlusion of normal choroidal vessels in the rabbit eye and CNV (choroidal neovascularization) in the rat eye. METHODS: Occlusive and nonocclusive parameters of Stakel and verteporfin photodynamic therapy (PDT) were investigated in pigmented rabbits. Eyes were followed by fluorescein angiography (FA) and histology at various intervals after PDT. RESULTS: When occlusive parameters (fluence of 50 J/cm(2), 5 mg/kg drug dose and DLI [distance to light illumination] of 1 minute) were used, Stakel PDT was efficient immediately after treatment without associated structural damage of the RPE and retina overlying the treated choroid in the rabbit eye. Two days later, total occlusion of the choriocapillaries was seen in 100% of the treated eyes, along with accompanying histologic structural changes in the overlying retina. When the occlusive parameters (fluence, 100 J/cm2; drug dose, 12 mg/m2; and DLI, 5 minutes) of verteporfin PDT were used, occlusion of the choriocapillaries was observed in 89% of the treated eyes. Histology performed immediately after treatment demonstrated structural damage of the overlying retina and RPE layer. Weaker, nonocclusive Stakel PDT parameters (25 J/cm2, 5 mg/kg, and DLI of 10 minutes) did not induce choriocapillary occlusion or retinal lesions on FA or histology. Weaker, nonocclusive verteporfin PDT parameters (10 J/cm2, 0.2 mg/kg, and DLI of 5 minutes) did not induce choriocapillary occlusion. However, histology of these eyes showed the presence of damage in the retinal and choroidal tissues. Moreover, preliminary results indicate that selective CNV occlusion can be achieved with Stakel PDT in the rat eye. CONCLUSIONS: Unlike verteporfin PDT, Stakel PDT does not cause direct damage to the RPE cell layer or retina. These observations indicate that Stakel PDT may have a high potential for beneficial therapeutic outcomes in treatment of AMD.

Effectiveness of anti-psychotics and related drugs in the Huntington French-speaking group cohort.

PURPOSE: Huntington's disease is a rare condition. Patients are commonly treated with antipsychotics and tetrabenazine. The evidence of their effect on disease progression is limited and no comparative study between these drugs has been conducted. We therefore compared the effectiveness of antipsychotics on disease progression. METHODS: 956 patients from the Huntington French Speaking Group were followed for up to 8 years between 2002 and 2010. The effectiveness of treatments was assessed using Unified Huntington's Disease Rating Scale (UHDRS) scores and then compared using a mixed model adjusted on a multiple propensity score. RESULTS: 63% of patients were treated with antipsychotics during the survey period. The most commonly prescribed medications were dibenzodiazepines (38%), risperidone (13%), tetrabenazine (12%) and benzamides (12%). There was no difference between treatments on the motor and behavioural declines observed, after taking the patient profiles at the start of the drug prescription into account. In contrast, the functional decline was lower in the dibenzodiazepine group than the other antipsychotic groups (Total Functional Capacity: 0.41 ± 0.17 units per year vs. risperidone and 0.54 ± 0.19 vs. tetrabenazine, both p<0.05). Benzamides were less effective than other antipsychotics on cognitive evolution (Stroop interference, Stroop color and Literal fluency: p<0.05). CONCLUSIONS: Antipsychotics are widely used to treat patients with Huntington's disease. Although differences in motor or behavioural profiles between patients according to the antipsychotics used were small, there were differences in drug effectiveness on the evolution of functional and cognitive scores.

Abrogation of HMX1 function causes rare oculoauricular syndrome associated with congenital cataract, anterior segment dysgenesis, and retinal dystrophy.

PURPOSE: To define the phenotypic manifestation, confirm the genetic basis, and delineate the pathogenic mechanisms underlying an oculoauricular syndrome (OAS). METHODS: Two individuals from a consanguineous family underwent comprehensive clinical phenotyping and electrodiagnostic testing (EDT). Genome-wide microarray analysis and Sanger sequencing of the candidate gene were used to identify the likely causal variant. Protein modelling, Western blotting, and dual luciferase assays were used to assess the pathogenic effect of the variant in vitro. RESULTS: Complex developmental ocular abnormalities of congenital cataract, anterior segment dysgenesis, iris coloboma, early-onset retinal dystrophy, and abnormal external ear cartilage presented in the affected family members. Genetic analyses identified a homozygous c.650A>C; p.(Gln217Pro) missense mutation within the highly conserved homeodomain of the H6 family homeobox 1 (HMX1) gene. Protein modelling predicts that the variant may have a detrimental effect on protein folding and/or stability. In vitro analyses were able to demonstrate that the mutation has no effect on protein expression but adversely alters function. CONCLUSIONS: Oculoauricular syndrome is an autosomal recessive condition that has a profound effect on the development of the external ear, anterior segment, and retina, leading to significant visual loss at an early age. This study has delineated the phenotype and confirmed HMX1 as the gene causative of OAS, enabling the description of only the second family with the condition. HMX1 is a key player in ocular development, possibly in both the pathway responsible for lens and retina development, and via the gene network integral to optic fissure closure.

Possible new ways in the pharmacological treatment of bipolar disorder and comorbid alcoholism.

Possible new ways in the pharmacological treatment of bipolar disorder and comorbid alcoholism. Azorin JM, Bowden CL, Garay RP, Perugi G, Vieta E, Young AH. Source Department of Psychiatry, CHU Sainte Marguerite, Marseilles, France. Abstract About half of all bipolar patients have an alcohol abuse problem at some point of their lifetime. However, only one randomized, controlled trial of pharmacotherapy (valproate) in this patient population was published as of 2006. Therefore, we reviewed clinical trials in this indication of the last four years (using mood stabilizers, atypical antipsychotics, and other drugs). Priority was given to randomized trials, comparing drugs with placebo or active comparator. Published studies were found through systematic database search (PubMed, Scirus, EMBASE, Cochrane Library, Science Direct). In these last four years, the only randomized, clinically relevant study in bipolar patients with comorbid alcoholism is that of Brown and colleagues (2008) showing that quetiapine therapy decreased depressive symptoms in the early weeks of use, without modifying alcohol use. Several other open-label trials have been generally positive and support the efficacy and tolerability of agents from different classes in this patient population. Valproate efficacy to reduce excessive alcohol consumption in bipolar patients was confirmed and new controlled studies revealed its therapeutic benefit to prevent relapse in newly abstinent alcoholics and to improve alcohol hallucinosis. Topiramate deserves to be investigated in bipolar patients with comorbid alcoholism since this compound effectively improves physical health and quality of life of alcohol-dependent individuals. In conclusion, randomized, controlled research is still needed to provide guidelines for possible use of valproate and other agents in patients with a dual diagnosis of bipolar disorder and substance abuse or dependence.

Sheep abortion associated with Neospora caninum in Mato Grosso do Sul, Brazil

Canids are the main hosts of Neospora caninum, but cattle, (sheep, goats and horses may serve as intermediary hosts. N. caninum infection of pregnant intermediary hosts may provoke abortion and neonatal infections. This study is the first to report lamb abortion associated with N. caninum in Mato Grosso do Sul. Epidemiological data were obtained from interviews with sheep producers. For microscopic examination, fragments of different organs removed from 4 sheep fetuses, aborted and necropsied, were fixed in 10% formaldehyde, embedded in paraffin and subjected to the hematoxylin-eosin staining protocol and immunohistochemistry (IHC) to test for N. caninum and Toxoplasma gondii. The abortion outbreak studied was reported from a herd of 268 Santa Inês sheep (including 186 pregnant ewes), with 10 abortion cases in the last third of gestation. Four fetuses were examined, 3 from a same ewe. At necropsy, one fetus exhibited crackling in the lung and all its organs were reddish. Histological findings detected mononuclear cell infiltrates among myocardium fibers and around blood vessels, in addition to circular structures with basophilic points resembling protozoans. IHC tests revealed strongly positive staining for N. caninum and weakly positive for T. gondii, characterizing N. caninum infection.

Induction of cell-mediated immunity during early stages of infection with intracellular protozoa

Toxoplasma gondii and Trypanosoma cruzi are intracellular parasites which, as part of their life cycle, induce a potent cell-mediated immunity (CMI) maintained by Th1 lymphocytes and IFN-g. In both cases, induction of a strong CMI is thought to protect the host against rapid parasite multiplication and consequent pathology and lethality during the acute phase of infection. However, the parasitic infection is not eliminated by the immune system and the vertebrate host serves as a parasite reservoir. In contrast, Leishmania sp, which is a slow growing parasite, appears to evade induction of CMI during early stages of infection as a strategy for surviving in a hostile environment (i.e., inside the macrophages which are their obligatory niche in the vertebrate host). Recent reports show that the initiation of IL-12 synthesis by macrophages during these parasitic infections is a key event in regulating CMI and disease outcome. The studies reviewed here indicate that activation/inhibition of distinct signaling pathways and certain macrophage functions by intracellular protozoa are important events in inducing/modulating the immune response of their vertebrate hosts, allowing parasite and host survival and therefore maintaining parasite life cycles.

The use of confocal laser scanning microscopy to analyze the process of parasitic protozoon-host cell interaction

In this communication we review the results obtained with the confocal laser scanning microscope to characterize the interaction of epimastigote and trypomastigote forms of Trypanosoma cruzi and tachyzoites of Toxoplasma gondii with host cells. Early events of the interaction process were studied by the simultaneous localization of sites of protein phosphorylation, revealed by immunocytochemistry, and sites of actin assembly, revealed by the use of labeled phaloidin. The results obtained show that proteins localized in the interaction sites are phosphorylated. The process of formation of the parasitophorous vacuole was monitored by labeling the host cell surface with fluorescent probes for lipids (PKH26), proteins (DTAF) and sialic acid (FITC-thiosemicarbazide) before interaction with the parasites. Evidence was obtained indicating transfer of components of the host cell surface to the parasite surface in the beginning of the interaction process. We also analyzed the distribution of cytoskeletal structures (microtubules and microfilaments visualized with specific antibodies), mitochondria (visualized with rhodamine 123), the Golgi complex (visualized with C6-NBD-ceramide) and the endoplasmic reticulum (visualized with anti-reticulin antibodies and DIOC6) during the evolution of intracellular parasitism. The results obtained show that some, but not all, structures change their position during evolution of the intracellular parasitism.

The anti-IRBP IgG1 and IgG2a response does not correlate with susceptibility to experimental autoimmune uveitis

Susceptibility to experimental autoimmune uveitis (EAU) in inbred mice has been associated with a dominant Th1 response. Elevated anti-inter-photoreceptor retinoid-binding protein (anti-IRBP) IgG2a/IgG1 antibody ratios have been implicated as candidate markers to predict disease severity. In the present study, both the anti-IRBP antibody isotype and severity of EAU phenotypes were examined in 4 non-isogenic genetically selected mouse lines to determine if they can be used as general markers of disease. Mice between 8 and 12 weeks old selected for high (H III) or low (L III) antibody response and for maximum (AIR MAX) or minimum (AIR MIN) acute inflammatory reaction (AIR) were immunized with IRBP. Each experiment was performed with at least 5 mice per group. EAU was evaluated by histopathology 21 days after immunization and the minimal criterion was inflammatory cell infiltration of the ciliary body, choroid and retina. Serum IgG1- and IgG2a-specific antibodies were determined by ELISA. EAU was graded by histological examination of the enucleated eyes. The incidence of EAU was lower in AIR MIN mice whereas in the other strains approximately 40% of the animals developed the disease. Low responder animals did not produce anti-IRBP IgG2a antibodies or interferon-gamma. No correlation was observed between susceptibility to EAU and anti-IRBP isotype profiles. Susceptibility to EAU is related to the intrinsic capacity to mount higher inflammatory reactions and increased production of anti-IRBP IgG2a isotype is not necessarily a marker of this immunologic profile.

Caracterización de la proteína del cuello de las roptrias 5 en plasmodium falciparum (PfRON5)

El conocimiento de las proteínas implicadas en el proceso de invasión de los merozoitos a los eritrocitos por Plasmodium es el punto de partida para el desarrollo de nuevas estrategias para controlar la malaria. Muchas de estas proteínas han sido estudiadas en Toxoplasma gondii, donde se han identificado las proteínas que pertenecen al Tight Junction (TJ), el cual permite una interacción fuerte entre las membranas de la célula huésped y el parásito, necesaria para la invasión parasitaria. En este género, cuatro proteínas del cuello de las roptrias (RON2, RON4, RON5 y RON8) y una proteína de micronemas (TgAMA-1) se han encontrado como parte del TJ. En Plasmodium falciparum, se han caracterizado las proteínas PfRON2 y PfRON4. En el presente estudio se realiza la identificación de la proteína PfRON5, una proteína de ~110 kDa que se expresa en las etapas de merozoitos y esquizontes de la cepa FCB-2 utilizando técnicas de biología molecular, bioinformática e inmuoquímica.

Prevenção da toxoplasmose congénita em Portugal

A toxoplasmose congénita é uma doença infecciosa, causada pelo parasita Toxoplasma gondii e, adquirida por transmissão materno-fetal, a qual pode acarretar sequelas neurológicas e oculares muito graves, no recém-nascido. O presente estudo incide sobre as linhas de prevenção da doença, em Portugal. A base da prevenção define-se como primária, através da determinação do estatuto imunológico da mulher, do aconselhamento e adopção de medidas higiénico-dietéticas das mulheres seronegativas, de forma a evitar a infecção materna. A vigilância serológica, na detecção de uma possível infecção materna, e a instituição da terapêutica de profilaxia, constituem a prevenção secundária, de modo a evitar a infecção fetal. A prevenção terciária recai, sobre o estabelecimento de um novo esquema terapêutico, dotado de alguma teratogenicidade, com o intuito de minimizar as sequelas da infecção. Em Portugal, existem muitas mulheres seronegativas, mal informadas acerca da doença, e que não tomam medidas preventivas correctas, para evitar a infecção. Esta problemática é decrescente, de norte para sul do país. A prevenção da doença pode ser bem-sucedida, através da implementação de directrizes específicas, dirigidas aos diferentes grupos de risco e da orientação correcta, pelos profissionais de saúde. A realização de estudos, em várias áreas de intervenção da doença, optimiza a sua prevenção e a sua relação de custo-benefício.

Identification of Hammondia heydorni oocysts by a heminested-PCR (hnPCR-AP10) based on the H-heydorni RAPD fragment AP10

Toxoplasma gondii, Hammondia hammondi, Neospora caninum, Neospora hughesi and Hammondia heydorni are members of the Toxoplasmatinae sub-family. They are closely related coccidians with similarly sized oocysts. Molecular diagnostic techniques, especially those based on polymerase chain reaction (PCR), can be successfully applied for the differentiation of Hammondia-like oocysts. In this paper, we describe a rapid and simple method for the identification of H. heydorni oocysts among other members of the Toxoplasmatinae sub-family, using a heminested-PCR (hnPCR-AP10) based on a H. heydorni RAPD fragment available in molecular database. DNA of oocysts of H. heydorni yielded a specific fragment of 289-290 bp in the heminested-PCR assay. No product was yielded when the primers were used for the amplification of DNA extracted from T. gondii, N. caninum, N. hughesi and H. hammondi, thus allowing the differentiation of H. heydorni among other members of the Toxoplasmatinae sub-family. The hnPCR-AP10 was capable of detecting H. heydorni genetic sequences from suspensions with at least 10 oocysts. In conclusion, the hnPCR-AP10 proved to be a reliable method to be used in the identification of H. heydorni oocysts from feces of dogs. (C) 2010 Elsevier B.V. All rights reserved.

Estudo da frequência e perfil epidêmico-sorológico da toxoplasmose ocular em pacientes atendidos no ambulatório de oftalmologia do hospital universitário onofre lopes no município de Natal, Rio Grande do Norte

Toxoplasmosis, provoked by the intracellular parasite Toxoplasma gondii, is one of the most prevalent parasitoses in the world. In humans, transmission occurs by three evolutionary forms of the parasite: oocysts, tissue cysts and tachyzoites. Wild and domestic felines are definitive hosts. The ocular form of toxoplasmosis can be of congenital origin with early or late clinical manifestations, or acquired after birth. T. gondii is considered the main culprit for most cases of infectious uveitis. This study aimed at assessing ocular toxoplasmosis, relating it to factors associated to the patient s lifestyle and describing the epidemic-serological and clinical profile of affected individuals. A cross-sectional study was conducted with a population of 159 patients. Univariate analysis (odds ratio) was used to evaluate the data, with a confidence interval of 95% and p-value < 0.05. A prevalence of 4% of ocular toxoplasmosis was observed in the population of patients treated at an ophthalmological clinic. Of patients directly examined by immunoenzymatic assay (MEIA-AxSYM®- Microparticle Enzyme Immune Assay), considering only uveitis, a frequency of anti-T. gondii of 73%, most of whom exhibited titulation between 40-99 UI IgG/mL. With respect to location of ocular lesions, bilaterality was observed in 57% of patients assessed by the ophthalmoscopy technique. When compared with the results of an active search of medical records, a similarity in ocular toxoplasmosis (74%) and bilateral lesion location (55%) was observed. Type I lesion was the most frequent type observed, with intraocular disposition in the macula. An epidemiological survey revealed that direct contact with cats; consuming raw or poorly cooked meat and direct contact with the soil were significantly associated with greater likelihood of acquiring ocular toxoplasmosis. Sample characterization in relation to age range was significant for patients between 31 and 40 years [χ², chi-square test (p = 0.04)], but population traits such as schooling, sanitary district, and monthly income were not significant. Results confirm that ocular toxoplasmosis is widely distributed in the metropolitan area of Natal, Brazil, with significant prevalence of ocular lesions provoked by T.gondii. It is suggested that sanitary authorities exert greater control in order to minimize the risk of toxoplasmic infection, mainly in pregnant women.

Avaliação das atividades anti-toxoplásmica, antioxidante e antiinflamatória dos monoterpenos timol (lippia sidoides) e estragol (croton zenhtneri)

Toxoplasmosis, a benign disease in normal healthy individuals, can have serious effects in pregnant women and immunocompromised patients. It is a parasitic disease caused by Toxoplasma gondii (Tg), an obligatory intracellular protozoan. The prophylactic and therapeutic arsenal against this parasite is very restricted. Thus, there is an ongoing search for novel drugs and therapeutic strategies. A promising alternative is a rational approach using medicinal plants. This study aimed to standardize methodologies for assessing the toxicological, antiproliferative, antioxidant, antiinflammatory and anti-Toxoplasma effects of Estragole and Thymol compounds isolated from species of plants (Lippia sidoides and Croton zenhtneri) commonly used in the Cariri region of Ceara State, Brazil. First we evaluated in vivo toxicity and conducted a pathological analysis of mice livers. In vivo antiinflammatory activity was assessed using air pouch and paw edema methods. Cytotoxicity assays were performed and antiproliferative, antioxidant and nitric oxide production analyzed. Anti-Toxoplasma activity was evaluated in a congenital experimental model with varying stages of maternal infection using the ME-49 strain and a non- congenital model by using ME-49 and RH strains. The results suggest low to moderate toxicity for both compounds. Thymol was more toxic in vivo and in vitro, having greater pathological repercussion than Estragole. The compounds were inactive for antiproliferative activity. Thymol showed better antioxidant activity, while Estragole stimulated nitric oxide production in macrophages. Both showed significant antiinflammatory activity. In non-congenital Tg infection, both compounds were active only against the ME49 strain. In congenital infection, Estragole (oral route) improved the newborn weight of infected mothers compared with untreated controls. Subcutaneous administration of the two compounds increased the weight of offspring born to infected mothers compared with untreated controls. We concluded that Estragole and Thymol exhibit important biological and anti-Toxoplasma activities. Further studies are needed to elucidate the mechanism of action of these compounds and other possible activities not investigated in the present study

Experimental toxoplasmosis in pregnant bitches

Three Toxoplasma gondii free bitches (No, 3-5) were fed 15 000 sporulated T. gondii oocysts at 56, 40 and 32 day of gestation and the outcome of the pregnancy was monitored. Two of the three dogs infected during pregnancy showed evidence of congenital infection and one aborted. Two control bitches not fed oocysts delivered eight uninfected healthy pups. This study demonstrated that I gondii can be congenitally transmitted in dogs when bitches are infected during pregnancy. (C) 1999 Elsevier B.V. B.V. All rights reserved.