An in vivo role for Trypanosoma cruzi calreticulin in antiangiogenesis

Angiogenesis leads to neovascularization from existing blood vessels. It is associated with tumor growth and metastasis and is regulated by pro- and antiangiogenic molecules, some of them currently under clinical trials for cancer treatment. During the last few years we have cloned, sequenced and expressed a Trypanosoma cruzi calreticulin gene (TcCRT). Its product, TcCRT, a 45 kDa protein, is more than 50% identical to human CRT (HuCRT). TcCRT, present on the surface of trypomastigotes, binds both C1q and mannan binding lectin and inhibits the classical activation pathway of human complement. Since TcCRT is highly homologous to a functional antiangiogenic fragment from HuCRT (aa 120–180), recombinant (r) and native (n) TcCRT were tested in their antiangiogenic effects, in the chick embryonic chorioallantoid membrane (CAM) assay. Both proteins mediated highly significant antiangiogenic effects in the in vivo CAM assay. This effect was further substantiated in experiments showing that the plasmid construct pSecTag/TcCRT also displayed significant antiangiogenic properties, as compared to the empty vector. Most likely, the fact that antiangiogenic substances act preferentially on growing neoplasic tissues, but not on already established tumors, is due to their effects on emerging blood vessels. The results shown here indicate that TcCRT, like its human counterpart, has antiangiogenic properties. These properties may explain, at least partly, the reported antineoplasic effect of experimental T. cruzi infection.     

Trypanosoma cruzi histone H1 is phosphorylated in a typical cyclin dependent kinase site accordingly to the cell cycle

Histone H1 of most eukaryotes is phosphorylated during the cell cycle progression and seems to play a role in the regulation of chromatin structure, affecting replication and chromosome condensation. In trypanosomatids, histone H1 lacks the globular domain and is shorter when compared with the histone of other eukaryotes. We have previously shown that in Trypanosoma cruzi, the agent of Chagas' disease, histone H1 is phosphorylated and this increases its dissociation from chromatin. Here, we demonstrate using mass spectrometry analysis that T. cruzi histone H1 is only phosphorylated at the serine 12 in the sequence SPKK, a typical cyclin-dependent kinase site. We also found a correlation between the phosphorylation state of histone H1 and the cell cycle. Hydroxyurea and lactacystin, which, respectively, arrest parasites at the G1/S and G2/M stages of the cell cycle, increased the level of histone H1 phosphorylation. Cyclin-dependent kinase-related enzymes TzCRK3, and less intensely the TzCRK1 were able to phosphorylate histone H1 in vitro. Histone H1 dephosphorylation was prevented by treating the parasites with okadaic acid but not with calyculin A. These findings suggest that T. cruzi histone H1 phosphorylation is promoted by cyclin dependent kinases, present during S through G2 phase of the cell cycle, and its dephosphorylation is promoted by specific phosphatases.     

Study of nanoscale structures in hydrated biomaterials using small-angle neutron scattering

Distribution of water in three classes of biomedically relevant and degradable polymers was investigated using small-angle neutron scattering. In semicrystalline polymers, such as poly(lactic acid) and poly(glycolic acid), water was found to diffuse preferentially into the non-crystalline regions. In amorphous polymers, such as poly(d,l-lactic acid) and poly(lactic-co-glycolic acid), the scattering after 7 days of incubation was attributed to water in microvoids that form following the hydrolytic degradation of the polymer. In amorphous copolymers containing hydrophobic segments (desaminotyrosyl-tyrosine ethyl ester) and hydrophilic blocks (poly(ethylene glycol) (PEG)), a sequence of distinct regimes of hydration were observed: homogeneous distribution (～10 Å length scales) at <13 wt.% PEG (～1 water per EG), clusters of hydrated domains (～50 Å radius) separated at 24 wt.% PEG (1–2 water per EG), uniformly distributed hydrated domains at 41 wt.% PEG (～4 water per EG) and phase inversion at >50 wt.% PEG (>6 water per EG). Increasing the PEG content increased the number of these domains with only a small decrease in distance between the domains. These discrete domains appeared to coalesce to form submicron droplets at ～60 °C, above the melting temperature of crystalline PEG. The significance of such observations on the evolution of micrometer-size channels that form during hydrolytic erosion is discussed.     

Depolymerisation and rearrangement of actin filaments during exocytosis in rat peritoneal mast cells: involvement of ryanodine-sensitive calcium stores

Cytoskeletal F-actin associated with synaptic vesicles and granules plays an important role during Ca²⁺-mediated exocytosis. In the present work, we have used amperometry and confocal fluorescence to study the role of internal Ca²⁺ in the rearrangement of F-actin (visualised with phalloidin-Alexa 546) during exocytosis in rat mast cells. The F-actin-depolymerising drug, latrunculin A, and the ryanodine receptor agonists ryanodine and caffeine that, per se did not induce exocytosis, enhanced the exocytotic responses elicited by compound 48/80 (C48/80). They also induced cortical actin depolymerisation in the presence or absence of external Ca²⁺. Degranulation induced by C48/80 was accompanied by the formation of a cytoplasmic F-actin network. Depletion of internal Ca²⁺ with cyclopiazonic acid inhibited latrunculin potentiation of C48/80-stimulated exocytosis and completely blocked the formation of the cytoplasmic F-actin network. This indicates that the mobilisation of Ca²⁺ from ryanodine-sensitive intracellular stores plays an important role in the depolymerisation of the cortical F-actin barrier and possibly in the formation of the internal F-actin network during exocytotic activation of peritoneal mast cells.     

Currant allergy and the Rosaceae-grass pollen allergy syndrome: a case report.

BACKGROUND: Despite the increasing use of currants in culinary recipes, currant allergy has rarely been reported. OBJECTIVES: To study a case of currant allergy and to explore cross-reactivity between grass pollen and Rosaceae family fruit allergens. METHODS: Skin prick tests to pollen and skin prick-to-prick tests with currants and peach were performed. Specific IgE levels were determined using the CAP method. We prepared a protein extract of 0.1 mg/mL in phosphate-buffered saline using red currant in the presence of protease inhibitors. Immunoblot inhibition studies were performed to explore cross-reactivity between grass pollen and currant allergens. RESULTS: Skin prick test results were positive to Dactylis, arizonic, and olive pollens. Results of skin prick-to-prick tests with fresh red and black currants were negative and positive, respectively, to peach. The specific IgE level was 5.7 KU/L to red currant and 2.92 KU/L to peach (CAP). Western blot analysis with red currant extract revealed specific IgE protein bands of 37 and 26 kDa. Preincubation of sera with extracts from red currant and peach inhibited both IgE bands, and preincubation with Dactylis pollen inhibited the 37-kDa band only. CONCLUSIONS: We report a case of allergy to grass pollen with an oral allergy syndrome involving several fruits from 2 different families of the Rosidae subclass confirmed by in vitro tests. Inhibition studies demonstrated cross-reactivity between different fruits (currant and raspberry) from the Rosidae subclass and were incomplete with grass pollen allergens.     

Impact of exercise on neuroplasticity-related proteins in spinal cord injured humans

The present study investigated the effects of exercise on the serum concentrations of brain-derived neurotrophic factor (BDNF), insulin-like growth factor 1 (IGF-1), prolactin (PRL) and cortisol (COR) in 11 chronically spinal cord-injured athletes. In these subjects BDNF concentration at rest was sixfold higher compared with the concentrations reported earlier in able-bodied persons, while IGF-1, PRL and COR were within normal range. Ten minutes of moderate intensity handbiking (54% of the maximal heart rate) during a warm-up period (W) induced an increase (P<0.05) of BDNF of approximately 1.5-fold from basal level at rest, while a decrease to basal level was found after an immediately succeeding handbiking time trial (89% of the maximal heart rate) over the marathon distance of 42 km (M). An increase (P<0.01) of serum IGF-1 was found after W and this levels remained elevated (P<0.01) until the end of M. W had no significant effects on the serum PRL and COR, however, M induced an increase (P<0.01) of both hormones. This is the first study showing elevated BDNF concentrations at rest in spinal cord-injured athletes. Furthermore, short moderate intensity handbiking but not immediately following long lasting high intensity handbiking further increases serum BDNF concentrations. IGF-1 response to exercise differs to BDNF response as this neuroplasticity-related protein remains elevated during the long lasting physical demand with high intensity. The augmented PRL concentration suggests that a possible mechanism by which exercise promotes neuroplasticity might be the activation of neural serotonergic pathways as 5-HT is the main PRL releasing factor. Elevated COR concentrations after M are unlikely to be deleterious to neuroplasticity as COR concentrations remain within the physiological range. The present study suggests that exercise might be beneficial to enhance neuroprotection and neuroplasticity, thereby improving recovery after spinal cord injury.     

Reduced Frequency of Memory T Cells and Increased Th17 Responses in Patients with Active Tuberculosis

Phenotypic and functional alterations in Mycobacterium tuberculosis T cell subsets have been reported in patients with active tuberculosis. A better understanding of these alterations will increase the knowledge about immunopathogenesis and also may contribute to the development of new diagnostics and prophylactic strategies. Here, the ex vivo phenotype of CD4⁺ and CD8⁺ T cells and the frequency and phenotype of gamma interferon (IFN-γ)- and interleukin 17 (IL-17)-producing cells elicited in short-term and long-term cultures following CFP-10 and purified protein derivative (PPD) stimulation were determined in noninfected persons (non-TBi), latently infected persons (LTBi), and patients with active tuberculosis (ATB). Phenotypic characterization of T cells was done based on the expression of CD45RO and CD27. Results show that ATB had a reduced frequency of circulating CD4⁺ CD45RO⁺ CD27⁺ T cells and an increased frequency of CD4⁺ CD45RO⁻ CD27⁺ T cells. ATB also had a higher frequency of circulating IL-17-producing CD4⁺ T cells than did LTBi after PPD stimulation, whereas LTBi had more IFN-γ-producing CD4⁺ T cells than did non-TBi. The phenotype of IFN-γ-producing cells at 24 h differs from the phenotype of IL-17-producing cells with no differences between LTBi and ATB. At 144 h, IFN-γ- and IL-17-producing cells were mainly CD45RO⁺ CD27⁺ T cells and they were more frequent in ATB. These results suggest that M. tuberculosis infection induces alterations in T cells which interfere with an adequate specific immune response.     

New mutations in the GLA gene in Brazilian families with Fabry disease

Fabry disease (FD) is an X-linked inborn error of glycosphingolipid catabolism that results from mutations in the alpha-galactosidase A (GLA) gene. Evaluating the enzymatic activity in male individuals usually performs the diagnosis of the disease, but in female carriers the diagnosis based only on enzyme assays is often inconclusive. In this work, we analyzed 568 individuals from 102 families with suspect of FD. Overall, 51 families presented 38 alterations in the GLA gene, among which 19 were not previously reported in literature. The alterations included 17 missense mutations, 7 nonsense mutations, 7 deletions, 6 insertions and 1 in the splice site. Six alterations (R112C, R118C, R220X, R227X, R342Q and R356W) occurred at CpG dinucleotides. Five mutations not previously described in the literature (A156D, K237X, A292V, I317S, c.1177_1178insG) were correlated with low GLA enzyme activity and with prediction of molecular damages. From the 13 deletions and insertions, 7 occurred in exons 6 or 7 (54%) and 11 led to the formation of a stop codon. The present study highlights the detection of new genomic alterations in the GLA gene in the Brazilian population, facilitating the selection of patients for recombinant enzyme-replacement trials and offering the possibility to perform prenatal diagnosis.