Spatiotemporal dynamics of PDGFRß expression in pericytes and glial scar formation in penetrating brain injuries in adults.

AIMS: Understanding the spatiotemporal dynamics of reactive cell types following brain injury is important for future therapeutic interventions. We have previously used penetrating cortical injuries following intracranial recordings as a brain repair model to study scar-forming nestin-expressing cells. We now explore the relationship between nestin-expressing cells, PDGFRß+ pericytes and Olig2+ glia, including their proliferation and functional maturation. METHODS: In 32 cases, ranging from 3 to 461 days post injury (dpi), immunohistochemistry for PDGFRß, nestin, GFAP, Olig2, MCM2, Aquaporin 4 (Aq4), Glutamine Synthetase (GS) and Connexin 43 (Cx43) was quantified for cell densities, labelling index (LI) and cellular co-expression at the injury site compared to control regions. RESULTS: PDGFRß labelling highlighted both pericytes and multipolar parenchymal cells. PDGFRß LI and PDGFRß+ /MCM2+ cells significantly increased in injury Zones at 10-13 dpi with migration of pericytes away from vessels with increased co-localization of PDGRFß with nestin compared to control regions (P < 0.005). Olig2+ /MCM2+ cell populations peaked at 13 dpi with significantly higher cell densities at injury sites than in control regions (P < 0.01) and decreasing with dpi (P < 0.05). Cx43 LI was reduced in acute injuries but increased with dpi (P < 0.05) showing significant cellular co-localization with nestin and GFAP (P < 0.005 and P < 0.0001) but not PDGFRß. CONCLUSIONS: These findings indicate that PDGFRß+ and Olig2+ cells contribute to the proliferative fraction following penetrating brain injuries, with evidence of pericyte migration. Dynamic changes in Cx43 in glial cell types with dpi suggest functional alterations during temporal stages of brain repair.

Evaluating dried salted cod amino acid signature for nutritional quality assessment and discriminant analysis.

Aim: Thus, the aim of this study was to answer three scientific questions: (1) Are the protein content and amino acid profile of dried salted cod influenced by species (Gadus morhua and Gadus macrocephalus)? (2) Are the protein content and amino acid profile of dried salted cod influenced by the geographical area of capture (Iceland and Norway)? and (3) Does the amino acid profile have the potential to be used as a discriminator of species and geographical areas of capture? Methods: A total of 45 dried salted cods (2-3 kg of dry weight; n = 15 samples/origin) were used in this study. The Atlantic cod was fished in the Atlantic northeast (FAO 27 area) within the Exclusive Economic zones (EEZ) of Norway (n = 15) and Iceland (n = 15), while the Pacific cod was caught in the Pacific northeast (FAO 67 area) within the Alaska EEZ (n = 15). Total protein content was determined by the Kjeldahl method, in accordance with the AOAC procedures. The amino acid profile was analyzed by HPLC with fluorescence detection (at excitation and emission wavelengths of 338 and 425 nm, respectively). Results: The Atlantic cod presented higher contents of total protein (33.90 versus 33.10 g/100 g of cod edible portion; p = 0.017) and total amino acid contents (32.52 versus 32.04 g/100 g of cod edible portion; p = 0.015) but displayed lower percentage of indispensable amino acids (32.16 versus 32.83 g/100 g of protein; p < 0.001) than Pacific cod. Among the Atlantic cod harvesting locations, the Norwegian cod displayed higher total amino acid contents (96.91 versus 96.81 g/100 g of protein; p = 0.012) and higher percentage of indispensable amino acids (35.38 versus 28.94 g/100 g of protein; p = 0.042) than the Icelandic counterpart. A correct classification of 100% was obtained for the Pacific and Icelandic cod varieties, but the classification accuracy in the Norwegian cod was of just 86.67%, since 2 samples out of 15 were incorrectly classified as Icelandic. Conclusion: The comparison of cod species showed that the Atlantic cod had a significantly lower EAAI than the Pacific cod (p < 0.001; 88.23 versus 88.61). On the other hand, the comparison of the two origins in the Atlantic cod, showed that Norwegian cod displayed a significantly higher EAAI than the Icelandic cod (99.15 versus 77.32). The assessment of the EAAI allows the classification of the protein's nutritional quality, allowing us to classify both cod species as a good protein source to human diet. However, within the Atlantic cod, the Norwegian cod's protein is classified as high quality, while the Icelandic cod attain the classification of useful quality. Regarding the amino acid profile discriminatory potential to classify cod samples. The results show that the AA profile has 100% accuracy in the separation of cod species, but was not globally efficient in the differentiation of the Norwegian from the Icelandic cod.

Relation of pretreatment sequence diversity in NS5A region of HCV genotype 1 with immune response between pegylated-INF/ribavirin therapy outcomes.

Hepatitis C virus (HCV) infection frequently persists despite substantial virus-specific immune responses and the combination of pegylated interferon (INF)-α and ribavirin therapy. Major histocompatibility complex class I restricted CD8(+) T cells are responsible for the control of viraemia in HCV infection, and several studies suggest protection against viral infection associated with specific HLAs. The reason for low rates of sustained viral response (SVR) in HCV patients remains unknown. Escape mutations in response to cytotoxic T lymphocyte are widely described; however, its influence in the treatment outcome is ill understood. Here, we investigate the differences in CD8 epitopes frequencies from the Los Alamos database between groups of patients that showed distinct response to pegylated α-INF with ribavirin therapy and test evidence of natural selection on the virus in those who failed treatment, using five maximum likelihood evolutionary models from PAML package. The group of sustained virological responders showed three epitopes with frequencies higher than Non-responders group, all had statistical support, and we observed evidence of selection pressure in the last group. No escape mutation was observed. Interestingly, the epitope VLSDFKTWL was 100% conserved in SVR group. These results suggest that the response to treatment can be explained by the increase in immune pressure, induced by interferon therapy, and the presence of those epitopes may represent an important factor in determining the outcome of therapy.

Immunoprotective Leishmania major synthetic T cell epitopes.

Using the predictive algorithm of Rothbard and Taylor (1988. EMBO J. 7:93) and the primary structure of gp63 (Button, L., and M.R. McMaster. 1988. J. Exp. Med. 167:724; Miller, R.A., S.G. Reed, and M. Parsons. 1990. Mol. Biochem. Parasitol. 39:267) we have been able to delineate the structures of a number of gp63 T-cell epitopes which stimulate the proliferation of CD4+ cells. One of these synthetic antigens, inoculated subcutaneously with adjuvant, was shown to specifically induce proliferation of the Th1 subset and provided immunoprotection against two species of Leishmania parasites.

Insights into the antiviral activity of phospholipases A2 (PLA2s) from snake venoms.

Viruses are associated with several human diseases that infect a large number of individuals, hence directly affecting global health and economy. Owing to the lack of efficient vaccines, antiviral therapy and emerging resistance strains, many viruses are considered as a potential threat to public health. Therefore, researches have been developed to identify new drug candidates for future treatments. Among them, antiviral research based on natural molecules is a promising approach. Phospholipases A2 (PLA2s) isolated from snake venom have shown significant antiviral activity against some viruses such as Dengue virus, Human Immunodeficiency virus, Hepatitis C virus and Yellow fever virus, and have emerged as an attractive alternative strategy for the development of novel antiviral therapy. Thus, this review provides an overview of remarkable findings involving PLA2s from snake venom that possess antiviral activity, and discusses the mechanisms of action mediated by PLA2s against different stages of virus replication cycle. Additionally, molecular docking simulations were performed by interacting between phospholipids from Dengue virus envelope and PLA2s from Bothrops asper snake venom. Studies on snake venom PLA2s highlight the potential use of these proteins for the development of broad-spectrum antiviral drugs.

Routine libraries for pattern recognition in quasispecies.

The results obtained through biological research usually need to be analyzed using computational tools, since manual analysis becomes unfeasible due to the complexity and size of these results. For instance, the study of quasispecies frequently demands the analysis of several, very lengthy sequences of nucleotides and amino acids. Therefore, bioinformatics tools for the study of quasispecies are constantly being developed due to different problems found by biologists. In the present study, we address the development of a software tool for the evaluation of population diversity in quasispecies. Special attention is paid to the localization of genome regions prone to changes, as well as of possible hot spots.

Primary Heligmosomoides polygyrus bakeri infection induces myeloid-derived suppressor cells that suppress CD4+ Th2 responses and promote chronic infection.

Primary infection with the gastrointestinal nematode Heligmosomoides polygyrus bakeri is chronic in C57BL/6 (B6) mice whereas challenge infection is rapidly eliminated. F4/80-CD11b+Gr+ cells, presumed to be neutrophils, were reported to accumulate around encysting larvae in intestinal tissue during primary infection, but their exact identity and role remain unclear. We observed significant increases in F4/80-CD11bhiGr1hi cells in mesenteric lymph nodes (MLNs) and spleen after primary but not challenge infection; a high proportion of these cells expressed Ly6G and Ly6C. These cells, which phenotypically resemble myeloid-derived suppressor cells (MDSC), increased in lamina propria (LP) early during primary infection. Increased MDSC were associated with low numbers of alternatively activated macrophages (AAMØ) in LP and CD4+GATA3+ T cells and AAMØ in MLN and spleen. Purified CD11c-CD11b+Gr1+ cells from H. polygyrus bakeri-infected mice suppressed OVA-specific CD4+ T-cell proliferation via a nitric oxide-dependent mechanism and parasite-specific IL-4 secretion in vitro. Adoptive transfer of CD11c-CD11b+Gr1+ cells from mice with primary infection resulted in significantly higher adult worm burdens and increased egg production in naïve B6 recipients infected with H. polygyrus bakeri. Altogether, these findings indicate that primary H. polygyrus bakeri infection induces a novel subset of MDSC that suppress CD4+ Th2 responses and promote chronic infection.

HFE gene mutations in Brazilian thalassemic patients.

Hereditary hemochromatosis is a disorder of iron metabolism characterized by increased iron intake and progressive storage and is related to mutations in the HFE gene. Interactions between thalassemia and hemochromatosis may further increase iron overload. The ethnic background of the Brazilian population is heterogeneous and studies analyzing the simultaneous presence of HFE and thalassemia-related mutations have not been carried out. The aim of this study was to evaluate the prevalence of the H63D, S65C and C282Y mutations in the HFE gene among 102 individuals with alpha-thalassemia and 168 beta-thalassemia heterozygotes and to compare them with 173 control individuals without hemoglobinopathies. The allelic frequencies found in these three groups were 0.98, 2.38, and 0.29% for the C282Y mutation, 13.72, 13.70, and 9.54% for the H63D mutation, and 0, 0.60, and 0.87% for the S65C mutation, respectively. The chi-square test for multiple independent individuals indicated a significant difference among groups for the C282Y mutation, which was shown to be significant between the beta-thalassemia heterozygote and the control group by the Fisher exact test (P value = 0.009). The higher frequency of inheritance of the C282Y mutation in the HFE gene among beta-thalassemic patients may contribute to worsen the clinical picture of these individuals. In view of the characteristics of the Brazilian population, the present results emphasize the need to screen for HFE mutations in beta-thalassemia carriers.

Natural compounds isolated from Brazilian plants are potent inhibitors of hepatitis C virus replication in vitro.

Compounds extracted from plants can provide an alternative approach to new therapies. They present characteristics such as high chemical diversity, lower cost of production and milder or inexistent side effects compared with conventional treatment. The Brazilian flora represents a vast, largely untapped, resource of potential antiviral compounds. In this study, we investigate the antiviral effects of a panel of natural compounds isolated from Brazilian plants species on hepatitis C virus (HCV) genome replication. To do this we used firefly luciferase-based HCV sub-genomic replicons of genotypes 2a (JFH-1), 1b and 3a and the compounds were assessed for their effects on both HCV replication and cellular toxicity. Initial screening of compounds was performed using the maximum non-toxic concentration and 4 compounds that exhibited a useful therapeutic index (favourable ratio of cytotoxicity to antiviral potency) were selected for extra analysis. The compounds APS (EC50=2.3µM), a natural alkaloid isolated from Maytrenus ilicifolia, and the lignans 3(∗)43 (EC50=4.0µM), 3(∗)20 (EC50=8.2µM) and 5(∗)362 (EC50=38.9µM) from Peperomia blanda dramatically inhibited HCV replication as judged by reductions in luciferase activity and HCV protein expression in both the subgenomic and infectious systems. We further show that these compounds are active against a daclatasvir resistance mutant subgenomic replicon. Consistent with inhibition of genome replication, production of infectious JFH-1 virus was significantly reduced by all 4 compounds. These data are the first description of Brazilian natural compounds possessing anti-HCV activity and further analyses are being performed in order to investigate the mode of action of those compounds.

An investigation into the significance of the N-linked oligosaccharides of Leishmania gp63.

Leishmania major promastigotes, when grown in the presence of tunicamycin (TM), produced a plasma membrane-bound, proteolytically active gp63 with a lower molecular weight than the native glycoprotein. However, this lower molecular weight form of gp63 continued to be recognized by concanavalin A (Con A), suggesting that inhibition of N-linked glycosylation was not complete. Metabolic labeling of gp63, using [35S]methionine, demonstrated that in the range of 5-10 micrograms ml-1 TM, only the lower molecular weight form was synthesized, suggesting that inhibition was complete and that lectin binding was likely due to the GPI anchored sugars. Removal of the oligosaccharides from L. major and L. mexicana amazonensis promastigotes using endoglycosidase F, caused the gp63 molecular weight to decrease to the same value observed in the presence of TM, once again without affecting the proteolytic activity. However, this deglycosylated enzyme continued to bind Con A until subsequently treated with periodate. The latter oxidation reaction resulted in complete loss of Con A binding without inhibiting the protease activity or the substrate specificity of gp63. Further investigations revealed that both glycosylated and deglycosylated gp63 were resistant to proteolytic digestion by either autolysis or cathepsin D. These findings indicate that the N-linked oligosaccharides of gp63 are not essential for folding, transport, maintenance of enzyme activity or resistance to proteolysis.

Genetic analysis of spermidine synthase from Leishmania donovani.

The polyamine biosynthetic pathway of protozoan parasites has been validated as a target in antiparasitic chemotherapy. To investigate this pathway at the biochemical and genetic level in a model parasite, the gene encoding spermidine synthase (SPDSYN), a key polyamine biosynthetic enzyme, has been cloned and sequenced from Leishmania donovani. The L. donovani SPDSYN gene encodes a polypeptide of 300 amino acids that exhibits 56% amino acid identity with the human counterpart. SPDSYN is present as a single copy gene in the leishmanial genome and encodes a 1.6 kb transcript. Employing SPDSYN flanking sequences to construct drug resistance cassettes, a Deltaspdsyn knockout strain of L. donovani was created by double targeted gene replacement. This Deltaspdsyn line could not convert putrescine to spermidine and was auxotrophic for polyamines. The polyamine auxotrophy could be circumvented by exogenous spermidine but not by putrescine (1,4-diaminobutane), cadaverine (1,5-diaminopentane), 1,3-diaminopropane, or spermine. Incubation of the null mutant in polyamine-deficient medium resulted in a rapid depletion in the intracellular spermidine level with a concomitant elevation of the putrescine pool. In addition, the level of trypanothione, a spermidine-containing thiol, was reduced, whereas the glutathione pool increased 3-4-fold. These data establish that SPDSYN is an essential enzyme in L. donovani promastigotes. The molecular and cellular reagents created in this investigation provide a foundation for subsequent structure-function and inhibitor design studies on this key polyamine biosynthetic enzyme.

Cloning and expression of the hypoxanthine-guanine phosphoribosyltransferase from Leishmania donovani.

The gene encoding the hypoxanthine-guanine phosphoribosyltransferase (HGPRT) enzyme from Leishmania donovani has been cloned and sequenced. The hgprt open reading frame encoded a polypeptide of 211 amino acids that exhibited 3 regions of significant homology with other eukaryotic HGPRTs and a C-terminal tripeptide compatible with a glycosomal targeting signal. Northern blot analysis of L. donovani RNA revealed two hgprt transcripts, a 1.9-kb mRNA and a 1.7-kb transcript. The expression of the 1.7-kb hgprt mRNA and the activity of HGPRT enzyme were both augmented approx. 5-fold in parasites incubated in the absence of purines. Southern blots of genomic DNA indicated only a single hgprt locus within the L. donovani genome. Overexpression of L. donovani hgprt in E. coli complemented genetic deficiencies in hypoxanthine and guanine phosphoribosylating activities and yielded abundant quantities of enzymatically active HGPRT. The recombinant HGPRT was purified to homogeneity and recognized hypoxanthine, guanine and allopurinol, but not adenine or xanthine, as substrates. The hgprt clone and pure HGPRT protein provide essential reagents for validating HGPRT as a therapeutic target for the treatment of leishmaniasis and other diseases of parasitic origin.

Kinetoplastid membrane protein-11 (KMP-11) is differentially expressed during the life cycle of African trypanosomes and is found in a wide variety of kinetoplastid parasites.

An abundant 11-kDa membrane protein was purified from African trypanosomes by organic solvent extraction and octyl-Sepharose chromatography. This protein cross-reacts with monoclonal antibodies originally generated against the lipophosphoglycan-associated protein of Leishmania donovani. Immunoblot analysis showed that the 11-kDa molecule was present in a variety of species of kinetoplastids. It was found in several species and subspecies of African trypanosomes and was present in low amounts in bloodstream forms and in larger amounts in procyclic, epimastigote and metacyclic life cycle stages. Expression of the 11-kDa molecule rapidly increased during transformation from bloodstream forms to procyclic forms, paralleling expression of the major surface glycoprotein of Trypanosoma congolense, the glutamic acid/alanine-rich protein, an analogue of T. brucei procyclin. The molecule was present in procyclic trypanosome membranes at approximately 2 x 10(5)-1 x 10(6) molecules per cell, suggesting it may have an important role in parasite membrane organization and function. Amino-acid analysis of the trypanosome 11-kDa protein showed it had a different composition than that of its leishmania counterpart. Its wide distribution in kinetoplastids and its membrane disposition suggest a name for this class of molecules: kinetoplastid membrane protein-11 (KMP-11).

Production of interferon-gamma and interleukin-4 by human T cells recognizing Leishmania lipophosphoglycan-associated protein.

The Leishmania protein LPGAP which is co-isolated with lipophosphoglycan is a specific activator of T cells from individuals who have recovered from American leishmaniasis. We have tested the effect of LPGAP on peripheral blood mononuclear cells (PBMC) from Kenyan donors cured from L. donovani infections. LPGAP induced vigorous proliferation and production of interferon-gamma (IFN-gamma) by the cells. In addition PBMC incubated with LPGAP released interleukin-4 (IL-4) after pulsing with ionomycin and phorbol myristate acetate. Single cells were isolated from LPGAP-stimulated cell lines and expanded as T-cell clones. LPGAP-reactive T-cell clones were activated by crude preparations of both promastigotes and axenic grown amastigote-like parasites. Among 9 CD4+ T-cell clones recognizing LPGAP, cells secreting predominantly IFN-gamma as well as cells secreting predominantly IL-4 were identified. The results show that both IFN-gamma producing (Th1-like) and IL-4 producing (Th2-like) T cells recognizing LPGAP are expanded after infection with L. donovani in humans.

Does lipophosphoglycan enhance the T cell-stimulatory activity of lipophosphoglycan-associated proteins?

Peripheral blood mononuclear cells (PBMC) from 13 American tegumentary leishmaniasis (ATL) patients and two healthy controls were tested in in vitro proliferative response assays with crude lipophosphoglycan (LPG), purified LPG, LPG-associated proteins (LPGAP) and synthetic LPGAP from L. donovani. Cells from another group of six ATL patients were similarly tested with LPGAP obtained from L. major. L. major LPGAP was more stimulatory than L. donovani LPGAP. Crude LPG from L. donovani was significantly more stimulatory than all the other L. donovani antigens tested, including L. donovani LPGAP. The present results indicate that the association with the glycolipid (LPG) may enhance the antigenicity of LPGAP for human T lymphocytes.

Randomized prospective study of local transdermic anesthetic in fine needle aspiration biopsy for breast lumps.

AIM: To evaluate the use of local transdermic anesthetics in fine needle aspiration biopsy (FNAB) in breast lesions. METHODS: Prospective randomized study of 119 patients having breast lesions, all being indicated for FNAB. The patients were divided into three groups: 40 patients entered in the active group (lidocaine + prilocaine); 40 patients underwent the placebo group (aqueous extract of Triticum vulgaris); and a control group of 39 women in whom FNAB was performed without the administration of any substance. Both the anesthetic and placebo were administered an hour before FNAB. Pain was quantified through a visual analogic scale of pain. The type of pain was also classified in terms of occurrence: only during the puncture, only during the movements and both. RESULTS: The visual linear analogic scale of pain showed an average of 3.3 in the active group, 3.5 in the placebo and 4.0 in the control group (NS). Analysis of the type of pain which was referred by the patient showed that 15% of the patients in the active group, 12.5% of those in the placebo group and 5.1% in the control group did not refer to any sensation of pain. Pain, when felt, was similar in all three groups (p < 0.4). CONCLUSIONS: Both the quantification and the type of pain referred to were similar in all three groups. However, there was a tendency of the patient to refer to less pain when the active substance or the placebo were used, when results were compared to the control group.

Dietary risk assessment of organophosphorus and dithiocarbamate pesticides in a total diet study at a Brazilian university restaurant.

In this study ready-to-eat food samples were collected in the production line of the university restaurant of the University of Brasilia, Brazil, which serves non-vegetarian and vegetarian meals daily. Samples were analysed for the presence of ten organophosphorus insecticides (OPs) by GC/FPD, after extraction with ethyl acetate and anhydrous sodium sulfate (LOQ = 0.002 mg kg(-1)), and for dithiocarbamate fungicides, as CS(2), using the spectrophotometric method (LOQ = 0.05 mg kg(-1)). About 43% of the 175 samples analysed contained at least one OP compound at levels up to 1.83 mg kg(-1). Methamidophos was the compound most detected (37.7%), present in most of the soup, soybean and salad samples. No OP residues were found in fruit juice, beans and bran rice samples. The cumulative acute intake of OPs was estimated using methamidophos and acephate as index compounds (IC). The total cumulative intake represented 9.1% and 47.7% of the methamidophos ARfD for the non-vegetarian and vegetarian diets, respectively. When acephate was used as IC, the total intakes represented 20.7% and 116% of the ARfD for the non-vegetarian and vegetarian diets, respectively. Dithiocarbamates were detected in 70% of the 177 samples analysed, at levels up to 0.51 mg kg(-1) CS(2); all salad samples were positive and no residues were found in fruit juice. The chronic intake of dithiocarbamates represented 8.6 and 8.9% of the ADI (mancozeb) for the vegetarian and non vegetarian diets, respectively.

Can we predict dispersal guilds based on the leaf-height-seed scheme in a disjunct cerrado woodland?

Although there have been advances in methods for extracting information about dispersal processes, it is still very difficult to measure them. Predicting dispersal groups using single readily-measured traits would facilitate the emergence of instructive comparisons among ecological strategies of plants and offer a path towards improved synthesis across field experiments. The leaf-height-seed scheme consists of three functional traits: specific leaf area, plant canopy height, and seed mass. We tested, applying logistic regression analysis, whether these traits are potential predictors of dispersal guilds in a disjoint cerrado woodland site in southeastern Brazil. According to our results, none of the plant traits studied could predict dispersal guild; this means that abiotically and biotically dispersed species showed similar values of specific leaf area, height, and seed mass. The species of both guilds exhibited sclerophylly, probably a result of the typical soil nutrient deficiency of cerrado, which also may have placed constraints upon plant canopy height regardless of the dispersal mode. In the cerrado, some abiotically dispersed trees might present higher than expected seed mass as support to the investment in high root-to-shoot ratio at the seedling stage. Seeds of bird-dispersed species are limited in size and mass because of the small size of most frugivorous birds. Since soil nutrient quality might contribute to the similarity between the dispersal guilds regarding the three traits of the scheme, other plant traits (e.g., root depth distribution and nutrient uptake strategy) that detail the former should be considered in future predictive studies.

[The geographic distribution of the Brazilian population and some of its socioeconomic characteristics, 1960-1980]. / Distribuicao espacial da populacao brasileira e algumas caracteristicas socio-economicas entre 1960-1980.

This paper "describes the demographic occupation of the national territory [of Brazil] between 1960 and 1980, taking into account four areas according to their population and area: totally occupied, suboccupied, partially occupied and unoccupied. For each occupied area it describes [the] main economic and demographic features and observes the population redistribution [that] occurred in Brazil during that period." (SUMMARY IN ENG)