Kinetics of parasite distribution after reinfection with genetically distinct strains of Toxoplasma gondii.

Recent data shows that prior infection by Toxoplasma gondii does not protect the host from subsequent reinfection even after the development of immunological memory. Although animal models for T. gondii reinfection were proposed after cases of natural human reinfection were described, little is known about the events that occur immediately after challenge. To further understand these events, BALB/c mice were chronically infected with D8 non-virulent strain (genotype ToxoDB#8 BrIII) and challenged with two different virulent strains: EGS (genotype ToxoDB #229) or CH3 strain (genotype ToxoDB #19). Primary infection protected animals from lethal challenge and morbidity was reduced. Reinfection was confirmed by PCR-RFLP, showing differences in the way the parasites spread in challenged animals. Parasites reached the lungs during early infection and a parasitism delay in the intestine was observed in D8+CH3 group. Parasites from challenge strains were not detected in the brain of D8+CH3 and in the intestine and brain of D8+EGS group. Previous infection with D8 strain of T. gondii protected against lethal challenges, but it did not prevent parasite spread to some organs.

Pathological changes in acute experimental toxoplasmosis with Toxoplasma gondii strains obtained from human cases of congenital disease.

There is a lack of studies using Toxoplasma gondii strains isolated from human patients. Here, we present a pathological study of three strains obtained from human cases of congenital toxoplasmosis in Brazil using inbred mice after oral infection with 10 tissue cysts. Multiplex-nested PCR-RFLP of eleven loci revealed atypical genotypes commonly found in Brazil: toxodb #8 for TgCTBr5 and TgCTBr16 strains and toxodb #11 for the TgCTBr9 strain. BALB/c and C57BL/6 mice were evaluated for survival and histological changes during the acute phase of the disease. All mice inoculated with the non-virulent TgCTBR5 strain survived after 30 days, although irreversible tissue damage was found. In contrast, no mice were resistant to infection with the highly virulent TgCTBR9 strain. The TgCTBr16 strain resulted in 80% survival in mice. However, this strain presented low infectivity, especially by the oral route of infection. Despite being identified with the same genotype, TgCTBr5 and TgCTBr16 strains showed biological differences. Histopathologic analysis revealed liver and lungs to be the most affected organs, and the pattern of tissue injury was similar to that found in mice inoculated perorally with strains belonging to clonal genotypes. However, there was a variation in the intensity of ileum lesions according to T. gondii strain and mouse lineage. C57BL/6 mice showed higher susceptibility than BALB/c for histological lesions. Taken together, these results revealed that the pathogenesis of T. gondii strains belonging to atypical genotypes can induce similar tissue damage to those from clonal genotypes, although intrinsic aspects of the strains seem critical to the induction of ileitis in the infected host.

Real-time PCR as a prognostic tool for human congenital toxoplasmosis.

Real-time PCR (qPCR) was positive in 72/150 (48%) blood samples of newborns with congenital toxoplasmosis. Among infants with active retinochoroiditis, 68% had positive qPCR results, while positivity was 29% (P=0.009) in the absence of ocular involvement. Positive qPCR was associated with the presence of retinochoroidal lesions, with an odds ratio of 2.8.

Heterogeneity of soil surface ammonium concentration and other characteristics, related to plant specific variability in a Mediterranean-type ecosystem.

Heterogeneity and dynamics of eight soil surface characteristics essential for plants--ammonium and nitrate concentrations, water content, temperature, pH, organic matter, nitrification and ammonification rates--were studied in a Mediterranean-type ecosystem on four occasions over a year. Soil properties varied seasonally and were influenced by plant species. Nitrate and ammonium were present in the soil at similar concentrations throughout the year. The positive correlation between them at the time of greatest plant development indicates that ammonium is a readily available nitrogen source in Mediterranean-type ecosystems. The results presented here suggest that plant cover significantly affects soil surface characteristics.

The spectrum of mutations and molecular pathogenesis of hemophilia A in 181 Portuguese patients.

Disease-causing alterations within the F8 gene were identified in 177 hemophilia A families of Portuguese origin. The spectrum of non-inversion F8 mutations in 101 families included 67 different alterations, namely: 36 missense, 8 nonsense and 4 splice site mutations, as well as 19 insertions/deletions. Thirty-four of these mutations are novel. Molecular modeling allowed prediction of the conformational changes introduced by selected amino acid substitutions and their correlation with the patients' phenotypes. The relatively frequent, population-specific, missense mutations together with de novo alterations can lead to significant differences in the spectrum of F8 mutations among different populations.

Genotype-phenotype correlation in a cohort of Portuguese patients comprising the entire spectrum of VWD types: impact of NGS.

The diagnosis of von Willebrand disease (VWD), the most common inherited bleeding disorder, is characterised by a variable bleeding tendency and heterogeneous laboratory phenotype. The sequencing of the entire VWF coding region has not yet become a routine practice in diagnostic laboratories owing to its high costs. Nevertheless, next-generation sequencing (NGS) has emerged as an alternative to overcome this limitation. We aimed to determine the correlation of genotype and phenotype in 92 Portuguese individuals from 60 unrelated families with VWD; therefore, we directly sequenced VWF. We compared the classical Sanger sequencing approach and NGS to assess the value-added effect on the analysis of the mutation distribution in different types of VWD. Sixty-two different VWF mutations were identified, 27 of which had not been previously described. NGS detected 26 additional mutations, contributing to a broad overview of the mutant alleles present in each VWD type. Twenty-nine probands (48.3 %) had two or more mutations; in addition, mutations with pleiotropic effects were detected, and NGS allowed an appropriate classification for seven of them. Furthermore, the differential diagnosis between VWD 2B and platelet type VWD (n = 1), Bernard-Soulier syndrome and VWD 2B (n = 1), and mild haemophilia A and VWD 2N (n = 2) was possible. NGS provided an efficient laboratory workflow for analysing VWF. These findings in our cohort of Portuguese patients support the proposal that improving VWD diagnosis strategies will enhance clinical and laboratory approaches, allowing to establish the most appropriate treatment for each patient.

Atomic force microscopy-based molecular recognition of a fibrinogen receptor on human erythrocytes.

The established hypothesis for the increase on erythrocyte aggregation associated with a higher incidence of cardiovascular pathologies is based on an increase on plasma adhesion proteins concentration, particularly fibrinogen. Fibrinogen-induced erythrocyte aggregation has been considered to be caused by its nonspecific binding to erythrocyte membranes. In contrast, platelets are known to have a fibrinogen integrin receptor expressed on the membrane surface (the membrane glycoprotein complex alpha(IIb)beta(3)). We demonstrate, by force spectroscopy measurements using an atomic force microscope (AFM), the existence of a single molecule interaction between fibrinogen and an unknown receptor on the erythrocyte membrane, with a lower but comparable affinity relative to platelet binding (average fibrinogen--erythrocyte and --platelet average (un)binding forces were 79 and 97 pN, respectively). This receptor is not as strongly influenced by calcium and eptifibatide (an alpha(IIb)beta(3) specific inhibitor) as the platelet receptor. However, its inhibition by eptifibatide indicates that it is an alpha(IIb)beta(3)-related integrin. Results obtained for a Glanzmann thrombastenia (a rare hereditary bleeding disease caused by alpha(IIb)beta(3) deficiency) patient show (for the first time) an impaired fibrinogen--erythrocyte binding. Correlation with genetic sequencing data demonstrates that one of the units of the fibrinogen receptor on erythrocytes is a product of the expression of the beta(3) gene, found to be mutated in this patient. This work demonstrates and validates the applicability of AFM-based force spectroscopy as a highly sensitive, rapid and low operation cost nanotool for the diagnostic of genetic mutations resulting in hematological diseases, with an unbiased functional evaluation of their severity.