Evaluation of a Cooperia oncophora double-domain ASP-based vaccine against Cooperia spp. infections in cattle and sheep.

A double-domain activation-associated secreted protein (dd-Co-ASP) isolated from the bovine small intestinal parasite Cooperia oncophora was previously shown to be an effective vaccine candidate to protect calves against a homologous challenge infection. The aim of this study was to investigate whether the dd-Co-ASP protein, purified from a Belgian C. oncophora isolate, would offer protection against a C. oncophora isolate from the southern hemisphere as well as other Cooperia species such as C. punctata in cattle and C. curticei in sheep. Two vaccination studies were performed, i.e. one in cattle and one in sheep, in which the protective effects of dd-Co-ASP, supplemented with Quil A as an adjuvant, were compared with an adjuvant control. Whereas our results showed a 75 % reduction in Cooperia spp. cumulative faecal egg counts, the results obtained in sheep demonstrated that dd-Co-ASP was ineffective in raising a protective immune response against a C. curticei challenge infection. Even though sequence analysis of the dd-Co-ASP gene revealed restricted sequence heterogeneity in the double domain ASP within and between bovine Cooperia species, the results of the vaccine study suggest that there is sufficient conservation at the protein level to yield cross-protection, holding promise for the development of a general Cooperia vaccine for use in cattle.

Fasciolosis in South America: epidemiology and control challenges.

Fasciolosis caused by Fasciola hepatica severely affects the efficiency of livestock production systems worldwide. In addition to the economic impact inflicted on livestock farmers, fasciolosis is an emergent zoonosis. This review emphasizes different aspects of the disease in South America. Available data on epidemiology in bovines and ovines in different countries, as well as a growing body of information on other domestic and wildlife definitive hosts, are summarized. The issue of drug resistance that compromises the long-term sustainability of current pharmacological strategies is examined from a regional perspective. Finally, efforts to develop a single-antigen recombinant vaccine in ruminants are reviewed, focusing on the cases of leucine aminopeptidase or thioredoxin glutathione reductase.

Quiste primario de bazo / Primary splenic cyst

No disponible

[Epidemiology of post-partum haemorrhage]. / Épidémiologie de l'hémorragie du post-partum.

OBJECTIVES: To synthesize the available evidence regarding the incidence, causes, risk factors of post-partum haemorrhage (PPH) and the associated maternal morbidity. METHODS: Consultation of the Medline database and of national reports on maternal mortality. RESULTS: PPH is defined as a post-partum blood loss≥500mL, and severe PPH as a post-partum blood loss≥1000mL, whatever the delivery route. In population-based studies, the incidence of PPH is around 5% of deliveries when blood loss is not precisely assessed and around 10% when it is. The incidence of severe PPH is around 2%. Uterine atony if the main cause of PPH. Maternal mortality due to obstetric haemorrhage has decreased in France (currently 1.6 death/100,000 live births) but remained the first cause of maternal death (16%) and the most avoidable (80%). In high-resource countries, PPH is the main cause of acute severe maternal morbidity, and of pregnancy-related ICU admissions. In addition to the direct consequences of acute hypovolemia, PPH exposes the women to the complications of transfusion, of intensive care and to infertility in case of hysterectomy. The main risk factors for PPH are factors of uterine atony, but they are globally poorly predictive. CONCLUSION: PPH is the principal cause of severe maternal morbidity, most often due to uterine atony. Risk factors related to components of care during labor and delivery are amenable to change, and the assessment of their risks-benefits balance should take into account the associated risk of PPH.

A new approach for the characterization of proliferative cells in cestodes.

Cestodes show a remarkable proliferative capability that sustains the constant growth and differentiation of proglottids essential for their lifestyle. It is believed that a separate population of undifferentiated stem cells (the so-called germinative cells) are the only cells capable of proliferation during growth and development. The study of this particular cell subpopulation is hampered by the current lack of methods to isolate it. In this work, we developed a reproducible flow cytometry and cell sorting method to quantify and isolate the proliferating cells in the tetrathyridia larvae of the model cestode Mesocestoides corti, based on the DNA content of the cells. The isolated cells display the typical germinative cell morphology, and can be used for RNA isolation with a yield in the ng to µg range. We expect that this approach may facilitate the characterization of the germinative cells in M. corti and other model tapeworms.

Maternal and health care determinants of preconceptional use of folic acid supplementation in France: results from the 2010 National Perinatal Survey.

OBJECTIVES: To estimate the national prevalence and analyse the factors associated with preconceptional folic acid supplementation, including maternal sociodemographic characteristics, region of residence, birth control use and chronic diseases requiring medical care before conception. DESIGN: Cross-sectional population-based study. SETTING: All maternity units in France. POPULATION: A nationally representative sample of women giving birth in 2010 (n = 12,646). METHODS: Data came from mothers' interviews 2-3 days after delivery. Statistical analyses included multivariable logistic regressions. MAIN OUTCOME MEASURE: Folic acid supplementation starting at least 1 month before conception. RESULTS: 14.8% (95% confidence interval [95% CI] 14.2-15.4) of women used folic acid before pregnancy; this percentage varied from 10.4% to 18.7% across regions. Supplementation was more frequent in primiparae, French citizens, women with higher educational levels and those needing medical monitoring or treatment before conception. Women who stopped contraception to become pregnant (75% of our population) used folic acid more often (intrauterine device or implant: 19%, pill: 17%, other methods which did not need medical monitoring: 17%) than other women (7%). The adjusted odds ratios were 3.3 (95% CI 2.6-4.3) for intrauterine device and implant, 2.2 (95% CI 1.8-2.6) for pill and 1.9 (95% CI 1.5-2.4) for other methods, compared with women who did not use birth control. CONCLUSION: The absence of preconceptional folic acid supplementation for most women, even those needing consultations with healthcare professionals before pregnancy, shows that campaigns to promote folic acid supplementation should address not only women but also healthcare professionals involved in birth control and obstetric care before pregnancy.

Leucine aminopeptidase is an immunodominant antigen of Fasciola hepatica excretory and secretory products in human infections.

The liver fluke Fasciola hepatica parasitizes humans and ruminant livestock worldwide, and it is now being considered a reemerging zoonotic disease, especially in areas in which it is endemic, such as South America. This study investigates the immune response to excretory and secretory products produced by F. hepatica in a group of patients from the Peruvian Altiplano, where the disease is highly endemic. Using a proteomic approach and immunoblotting techniques, we have identified the enzymes leucine aminopeptidase (LAP) and phosphoenolpyruvate carboxykinase as immunodominant antigens recognized by sera from fasciolosis patients. An indirect enzyme-linked immunosorbent assay using recombinant LAP as the antigen was developed to check sera from individuals of this region. Our results demonstrate that LAP produces a specific and strong reaction, suggesting its potential use in the serologic diagnosis of F. hepatica infections in humans.

Nonrandom spatial distribution of synonymous substitutions in the GP63 gene from Leishmania.

In this work we analyze the variability in substitution rates in the GP63 gene from Leishmania. By using a sliding window to estimate substitution rates along the gene, we found that the rate of synonymous substitutions along the GP63 gene is highly correlated with both the rate of amino acid substitution and codon bias. Furthermore, we show that comparisons involving genes that represent independent phylogenetic lines yield very similar divergence/conservation patterns, thus suggesting that deterministic forces (i.e., nonstochastic forces such as selection) generated these patterns. We present evidence indicating that the variability in substitution rates is unambiguously related to functionally relevant features. In particular, there is a clear relationship between rates and the tertiary structure of the encoded protein since all divergent segments are located on the surface of the molecule and facing one side (almost parallel to the cell membrane) on the exposed surface of the organism. Remarkably, the protein segments encoded by these variable regions encircle the active site in a funnel-like distribution. These results strongly suggest that the pattern of nucleotide divergence and, notably, of synonymous divergence is affected by functional constraints.

Phylogenetic relationships and theoretical model of human cathepsin W (lymphopain), a cysteine proteinase from cytotoxic T lymphocytes.

The recently described cysteine proteinase cathepsin W, also known as lymphopain, which is expressed specifically by CD8+ T lymphocytes, is phylogenetically related to the cruzipain-like group of the C1 family of peptidases. We have constructed sequence alignments and a theoretical three dimensional homology model of cathepsin W. These have allowed the characterization of signature features of cathepsin W in particular and the cruzipain lineage in general. The signature features are (1) an extended loop structure, Gly 170-Trp 200, in the second or beta-sheet domain; (2) an additional disulfide bond, Cys 25/Cys 60; (3) an additional "orphan" cysteine, Cys 5; (4) an additional residue. Ala 11, inserted after the first beta-sheet sheet; and (5) an S2 pocket lined with Phe 68 and Phe 230 which explains the preference for substrates containing Leu at P2. Further, the model suggested that cathepsin W could exist as a dimer with the Cys 5 of each monomer forming a disulfide bond and the Arg 40 Phe 46 loop (RISFWDF) forming part of the dimeric interface. By comparing cathepsin W with other members of the cruzipain group and with other C1 peptidases, six conserved residues were identified which appear in general to be characteristic of the cruzipain group, and which differentiate cruzipain group members from other C1 peptidases including those of the related cathepsin L lineage. The signature residues of the cruzipain lineage are (cruzipain numbering) Asn 33, Trp 38, Ala 124, Leu 127, Leu 164, and Pro 174.

Proteinases and associated genes of parasitic helminths.

Many parasites have deployed proteinases to accomplish some of the tasks imposed by a parasitic life style, including tissue penetration, digestion of host tissue for nutrition and evasion of host immune responses. Information on proteinases from trematodes, cestodes and nematode parasites is reviewed, concentrating on those worms of major medical and economical importance. Their biochemical characterization is discussed, along with their putative biological roles and, where available, their associated genes. For example, proteinases expressed by the various stages of the schistosome life-cycle, in particular the well-characterized cercarial elastase which is involved in the penetration of the host skin and the variety of proteinases, such as cathepsin B (Sm31), cathepsin L1, cathepsin L2, cathepsin D, cathepsin C and legumain (Sm32), which are believed to be involved in the catabolism of host haemoglobin. The various endo- and exoproteinases of Fasciola hepatica, the causative agent of liver fluke disease, are reviewed, and recent reports of how these enzymes have been successfully employed in cocktail vaccines are discussed. The various proteinases of cestodes and of the diverse superfamilies of parasitic nematodes are detailed, with special attention being given to those parasites for which most is known, including species of Taenia, Echinococcus, Spirometra, Necator, Acylostoma and Haemonchus. By far the largest number of papers in the literature and entries to the sequence data bases dealing with proteinases of parasitic helminths report on enzymes belonging to the papain superfamily of cysteine proteinases. Accordingly, the final section of the review is devoted to a phylogenetic analysis of this superfamily using over 150 published sequences. This analysis shows that the papain superfamily can be divided into two major branches. Branch A contains the cathepin Bs, the cathepsin Cs and a novel family termed cathepsin Xs, while Branch B contains the cruzipains, cathepsin Ls, papain-like and aleurain/cathepsin H-like proteinases. The relationships of the helminth proteinases, and similar proteinases from protozoan parasites and other organisms, within these groups are discussed.

Cathepsin C from Schistosoma japonicum--cDNA encoding the preproenzyme and its phylogenetic relationships.

A cDNA encoding preprocathepsin C was isolated from adults of the asian blood fluke Schistosoma japonicum. The deduced amino acid sequence of S. japonicum cathepsin C comprised 458 amino acid residues; 22 NH2-terminal residues corresponding to the signal peptide, 199 residues corresponding to the propeptide and 237 COOH-terminal residues corresponding to the mature enzyme region. The amino acid sequence of this preprocathepsin showed 43% and 50% identity to that of human and rat, respectively. The preproenzyme shared only 59% identity with the sequence for a cathepsin C reported from Schistosoma mansoni, differing from it in active-site residues and in its potential N-glycosylation sites. Northern-blot analysis showed that S. japonicum cathepsin C was expressed in greater quantities in female than in male parasites. Phylogenetic analysis utilizing the mature enzyme sequences of S. japonicum and other cathepsin Cs demonstrated that cathepsin Cs and cathepsin Bs shared a common ancestry. The unusually long prosegment observed in cathepsin C from S. japonicum and from other species was compared to that of cathepsin Bs and cathepsin Ls. The extension contained two blocks of residues which were highly conserved among cathepsin Cs. The COOH terminus of the prosegment exhibited a composite of features present in the prosegments of cathepsin Ls and cathepsin Bs. Most significantly, given the common ancestry of cathepsin B and cathepsin C, the prosegment of cathepsin C included ERFNIN-like motifs and other residues more characteristic of non-cathepsin-B-like members of the papain superfamily such as cathepsin L.

Isolation of a cDNA encoding Fasciola hepatica cathepsin L2 and functional expression in Saccharomyces cerevisiae.

Cathepsin L2 is a major cysteine proteinase secreted by adult Fasciola hepatica. The enzyme differs from other reported cathepsin Ls in that it can cleave peptide substrates that contain proline in the P2 position. A cDNA was isolated from an expression library by immunoscreening with antiserum prepared against purified native cathepsin L2. This cDNA was sequenced and shown to encode a complete preprocathepsin L proteinase. Functionally active recombinant cathepsin L proteinase was expressed and secreted by Saccharomyces cerevisiae transformed with the cDNA. The recombinant enzyme was purified from large-scale fermentation broths using ultrafiltration and gel filtration chromatography on Sephacryl S200 HR columns. NH2-terminal amino acid sequencing showed that the cleavage point for activation of the recombinant pro-enzyme is identical to that of the F. hepatica-produced cathepsin L2. The mature active recombinant proteinase behaved similarly to the native enzyme when analysed by SDS-PAGE, immunoblotting and zymography and also cleaved peptides containing proline in the P2 position. Finally, the recombinant cathepsin L2 cleaved fibrinogen to form a fibrin clot, a property we described for F. hepatica cathepsin L2.

Functional expression of Fasciola hepatica cathepsin L1 in Saccharomyces cerevisiae.

A cDNA encoding the complete precursor of a Fasciola hepatica cathepsin L protease was isolated and sequenced. Functionally active enzyme was expressed and secreted by Saccharomyces cerevisiae transformed with a plasmid carrying the complete gene. Experiments with temperature-sensitive yeast mutants showed that the enzyme is trafficked through the yeast secretory pathway. Yeast transformed with a truncated gene, which lacked the pre-peptide-encoding and most of the pro-peptide-encoding sequences, did not express funtionally active enzyme. The yeast-expressed enzyme exhibited physicochemical properties in common with the native enzyme including, pH optimum for activity, stability at 37 degrees C and ability to cleave gelatin and immunoglobulin. Enzyme kinetic data showed that the native and yeast-expressed cathepsin L1 have similar specificities for substrates with hydrophobic residues in the P2 position. This is the first report of the functional expression of a cathepsin L proteinase in S. cerevisiae that did not require the use of yeast secretory signal sequences.

[Booster effect in elderly patients living in geriatric institutions]. / Efecto empuje en pacientes de la tercera edad residentes en instituciones geriátricas.

BACKGROUND: Tuberculinic response is weaker in elderly patients. To know the real tuberculinic reactivity in elderly patients living in geriatric institutions in Catalonia, Spain, and to reduce the probability of false negatives this study was carried out to detect the booster effect. METHODS: Three hundred forty-three individuals with a mean age of 81 +/- 6.5 years, residing in four geriatric centers within the Central Health Care Region of Catalonia, were studied. After performing the first Mantoux intradermoreaction this was repeated at one week in case of negativity. A third test was carried out after one more week on persistence of negativity. Qualitative study was carried out on cell immunity in those patients whose first Mantoux test was negative. RESULTS: The first Mantoux test was positive in 163 patients (47.5%) and the performance of the second and third test in cases of negativity of the previous test increased the number of positivities by 12.5% and 4.1%, respectively, up to a total of 220 patients (64%). In the patients who did not react to the first intradermoreaction and who preserved cell immunity a booster effect was obtained in 57 (52.8%). The prevalence of tuberculous infection in this group of elderly patients calculated in the group with preserved cell immunity was 81.2% while the morbidity of tuberculous disease in the positive cases in the Mantoux test was 1.8%. CONCLUSIONS: Given the high frequency of the booster effect in elderly people, systematic testing is recommended to investigate tuberculinic reactivity in patients who are initially negative on Mantoux intradermoreaction.

Dinucleotide biases in the platyhelminth Schistosoma mansoni.

The analysis of dinucleotide biases in coding and flanking regions, introns, rDNA and repetitive sequences, in the flatworm Schistosoma mansoni is reported. Except for rDNA, all regions display CpG avoidance and TpG plus CpA excess, which might be evidence of the presence of 5mC. The distribution and hierarchies of dinucleotides differ from the data published for invertebrate and vertebrate coding sequences.