Assessment of right ventricular systolic function by tissue motion annular displacement in healthy dogs.

INTRODUCTION/OBJECTIVES: There are few parameters for assessment of right ventricular (RV) systolic function on echocardiographic examination. Morphofunctional studies are limited by the irregular shape of the RV. Recently, tissue motion annular displacement (TMAD), a technique that evaluates valve annulus displacement toward the cardiac apex, has shown a good correlation with left ventricular global longitudinal strain (GLS) in healthy dogs. Therefore, the aim of this study was to evaluate the longitudinal systolic function of the RV of healthy dogs using TMAD. ANIMALS: A hundred healthy client-owned dogs. MATERIALS AND METHODS: Cross-sectional study. Physical examination, electrocardiogram, blood pressure measurement, and echocardiography were recorded. The systolic function of the RV was evaluated by GLS free wall and TMAD. Data were compared with those derived from conventional echocardiography. RESULTS: TMAD values varied according to body weight. There was a correlation of TMAD in millimeters with all indices of RV systolic function, including GLS free wall (R:-0.239; p:0.017). TMAD had a correlation with age and heart rate; whereas there was no relationship with sex and blood pressure. The coefficient of variation for the intraobserver evaluation was lower for the TMAD in millimeters (9.9%) compared with the GLS free wall (17.9%). In addition, the mean time to perform TMAD (8.1 s) was lower than that of the GLS free wall (37.7 s). CONCLUSIONS: TMAD is a fast, reproducible, and promising method for assessing RV systolic function in healthy dogs. However, further studies are needed to understand the applicability of this technique in patients with heart disease.

Evaluation of effects of Poincianella bracteosa (Tul.) L.P. Queiroz leaves in Allium cepa and Mus musculus.

POINCIANELLA BRACTEOSA: (Tul.) L.P. Queiroz. (Fabaceae) traditionally is used in Brazilian medicine to treat catarrhal infections, diarrhea, hepatitis and anemia. We investigated the phytochemical profile, and mutagenicity and anti-mutagenicity of aqueous extracts of leaves of P. bracteosa in A. cepa cells and in mice. We investigated four concentrations of extract for the A. cepa bioassay and three doses of extract for administration to mice. For the A. cepa assay, we analyzed 5,000 meristematic cells to determine the mitotic index, mean number of chromosome alterations and percentage of damage reduction. For each animal assay, 2,000 normochromatic erythrocytes were evaluated per mouse to determine the number of micronuclei and the protective effect of the extract. Phytochemical analysis of the extract revealed reducing sugars, tannins and alkaloids, which likely did not interfere with the cell cycle of A. cepa or cause damage to the DNA of A. cepa or mice. The extract exhibited a protective effect in both organisms.

IL4-10 fusion protein: a novel immunoregulatory drug combining activities of interleukin 4 and interleukin 10.

The objective of this study was to test the capacity of a newly developed fusion protein of interleukin 4 (IL-4) and IL-10 [IL4-10 fusion protein (FP)] to shift multiple pro-inflammatory pathways towards immune regulation, and to inhibit pro-inflammatory activity in arthritis models. The effects of IL4-10 FP in comparison with IL-4, IL-10 and IL-4 plus IL-10 on pro- and anti-inflammatory mediators, T cells and immunoglobulin (Ig) receptors in favour of immunoregulatory activity were studied. In addition, the capacity of IL4-10 FP to inhibit pro-inflammatory activity in ex-vivo and in-vivo arthritis models was investigated. IL4-10 FP robustly inhibited pro-inflammatory cytokine [IL-1ß, tumour necrosis factor (TNF)-α, IL-6 and IL-8] production in whole blood cultures, mediated by both the IL-10 and the IL-4 moiety. IL4-10 fusion protein induced IL-1 receptor antagonist (IL-1RA) production and preserved soluble TNF receptor (sTNFR) levels, strongly increasing IL-1RA/IL-1ß and sTNFR/TNF-α ratios. In addition, IL4-10 FP strongly inhibited T helper (Th) type 1 and 17 cytokine secretion, while maintaining FoxP3 expression and up-regulating Th2 activity. In addition, while largely leaving expression of activating Fc gamma receptor (FcγR)I, III and Fc epsilon receptor (FcεR) unaffected, it significantly shifted the FcγRIIa/FcγRIIb ratio in favour of the inhibitory FcγRIIb. Moreover, IL4-10 FP robustly inhibited secretion of pro-inflammatory cytokines by rheumatoid arthritis synovial tissue and suppressed experimental arthritis in mice, without inducing B cell hyperactivity. IL4-10 fusion protein is a novel drug, signalling cells to induce immunoregulatory activity that overcomes limitations of IL-4 and IL-10 stand-alone therapy, and therefore has therapeutic potential for inflammatory diseases such as rheumatoid arthritis.

Portuguese consensus document for the management of alpha-1-antitrypsin deficiency.

Alpha-1-antitrypsin deficiency (AATD) is a genetic autosomal codominant disorder caused by mutations in SERPINA1 gene. It is one of the most prevalent genetic disorders, although it remains underdiagnosed. Whereas at international level there are several areas of consensus on this disorder, in Portugal, inter-hospital heterogeneity in clinical practice and resources available have been adding difficulties in reaching a diagnosis and in making therapeutic decisions in this group of patients. This raised a need to draft a document expressing a national consensus for AATD. To this end, a group of experts in this field was created within the Portuguese Pulmonology Society - Study group on AATD, in order to elaborate the current manuscript. The authors reviewed the existing literature and provide here general guidance and extensive recommendations for the diagnosis and management of AATD that can be adopted by Portuguese clinicians from different areas of Medicine. This article is part of a supplement entitled "Portuguese consensus document for the management of alpha-1-antitrypsin deficiency" which is sponsored by Sociedade Portuguesa de Pneumologia.

Is body weight-support treadmill training effective in increasing muscle trophism after traumatic spinal cord injury? A systematic review.

STUDY DESIGN: Systematic review. OBJECTIVE: To determine the effectiveness of body weight-support treadmill training (BWSTT) for muscle atrophy management in people with spinal cord injury (SCI). SETTING: Studies from multiple countries were included. METHODS: The following databases were consulted from January to October 2013: PubMed, Institute for Scientific Information (ISI), Science Direct and Lilacs. The methodological quality of the articles included was classified according to Jovell and Navarro-Rubio. RESULTS: A total of five studies were included. These studies reported a significant association between BWSTT and increased trophism of the lower limb muscles of humans with SCI, which was observed as an increase in the cross-sectional area. Moreover, improvements in the ability to generate peak torque, contract the knee extensors and ankle plantarflexors with reduction of body weight support were observed after BWSTT. CONCLUSION: The results were considered inconclusive because of the low methodological quality of the articles, which was because of the absence of sample homogeneity, thereby providing a low level of evidence for clinical practice.

Epidemiological review of Toxoplasma gondii infection in humans and animals in Portugal.

Toxoplasmosis is a worldwide zoonosis. However, data from Portugal are limited and a considerable part of the literature is in Portuguese. Currently, the rate of congenital infection in Portugal is unknown, and almost nothing is known of sequelae of congenital toxoplasmosis. There is no recent general population-based serological survey of Toxoplasma gondii in humans in Portugal. In addition, there is little information on genetic characteristics of T. gondii in animals and humans. In the present paper, we review prevalence, clinical spectrum and epidemiology of T. gondii in humans and animals in Portugal. This knowledge should be useful to biologists, public health workers, physicians and veterinarians.

Prevalence of Babesia microti-like infection in red foxes (Vulpes vulpes) from Portugal.

The prevalence of piroplasm (order Piroplasmida) infection was assessed in blood and bone marrow samples from 91 red foxes (Vulpes vulpes) from northern, central and southern Portugal by means of molecular methods. PCR for the 18S rRNA gene of Babesia spp. followed by sequencing revealed 63 foxes positive for the Babesia microti-like piroplasm (syn. Theileria annae) (69.2%; 95% confidence interval [CI]: 58.7-78.5%) and one fox positive for Babesia canis (1.1%; 95% CI: 0.0-6.0%). Positivity to the B. microti-like piroplasm or B. canis in 43 blood samples (83.7%) was significantly higher (p<0.001) than in 43 paired bone marrow samples (20.9%). There were no statistically significant differences in the prevalence of infection between genders (p=0.219) or age groups (<2 years vs. ≥ 2 years) (p=1.0). This is the first report of the B. microti-like piroplasm in foxes from Portugal as well as the first report on detection by PCR and genotyping of B. canis in a red fox worldwide. A natural cycle of the B. microti-like piroplasm is suggested in red fox populations based on the high prevalence of the protozoan. Red foxes might be a reservoir of the B. microti-like piroplasm and a source of infection to dogs.

Seroepidemiology of Toxoplasma gondii infection in women from the North of Portugal in their childbearing years.

Seroprevalence of Toxoplasma gondii infection and associated risk factors were investigated in 401 women of childbearing age from the North of Portugal. Of the 98 (24·4%) seropositive women, 92 (93·9%) only had immunoglobulin (Ig)G, two (2·0%) only had IgM, and four (4·1%) others had both IgG and IgM. Risk factors for T. gondii infection in women were: engaging in soil-related activities without gloves [odds ratio (OR) 8·4], consumption of unwashed raw vegetables or fruit (OR 7·6), and consumption of smoked or cured (non-cooked) processed pork products (OR 2·5). Most women of childbearing age from the North Portugal are susceptible to primary infection with T. gondii and, therefore, the risk of congenital toxoplasmosis remains high.

Prevalence of antibodies to Toxoplasma gondii in dogs from northeastern Portugal.

Prevalence of antibodies to Toxoplasma gondii was investigated in 673 domestic dogs from northeastern Portugal, using the modified agglutination test (MAT) with 1 : 20 as cutoff for seropositivity; antibodies were found in 256 dogs (38.0%). Differences between seroprevalence levels in males (36.7%) and females (41.8%) and between pure-breed (42.1%) and mixed-breed dogs (35.2%) were not statistically significant. Multiple logistic regression analysis identified age above 12 mo (odds ratio [OR]  =  4.0), chance of eating birds or small mammals (OR  =  4.0), housing exclusively outdoors (OR  =  1.5), home-cooked meals (OR  =  3.0), and eating raw meat or viscera (OR  =  7.7) as risk factors for the canine T. gondii infection. Some control measures are suggested based on these findings.

Fusion proteins containing family 1 and family 2 PspA fragments elicit protection against Streptococcus pneumoniae that correlates with antibody-mediated enhancement of complement deposition.

PspA is an important pneumococcal vaccine candidate that is capable of inducing protection in different animal models. Because of its structural diversity, a PspA-based vaccine should contain at least one fragment from each of the two major families (1 and 2) in order to elicit broader protection. In the present work, we have tested the potential of PspA hybrids containing fused portions of family 1 and 2 (PspA1ABC-4B and PspA1ABC-3AB) PspA fragments to induce protection against pneumococci bearing distinct PspA fragments. Sera from mice immunized with these hybrid PspA fragments were able to increase C3 deposition on pneumococci bearing PspA fragments from both families, in contrast with sera made against the PspA family 1 (PspA1ABC) and PspA family 2 (PspA3ABC) fragments, which were effective only within the same family. Although PspA hybrids were able to extend protection against pneumococcal infection with strains bearing diverse PspA fragments, the immunity elicited by family 2 was clade dependent, suggesting that PspA fragments from family 2 clades 3 and 4 should both be included in a comprehensive PspA vaccine. These results indicate that PspA fusion proteins constitute an efficient immunization strategy for future PspA-based antipneumococcal vaccines since they are able to extend protection provided by a protein derived from a single transcript.

An associated process for the purification of immuno globulin G, catalase, superoxide dismutase and albumin from haemolysed human placenta blood.

The human placenta is a rich raw material for production of many biopharmaceutical products. Here we describe a co-purification process for the production of four different proteins from haemolysed human placenta blood: IgG, catalase (Cat), superoxide dismutase (Sod) and albumin (Alb). The process can be divided in two parts: the common steps and the specific separation techniques for each protein. The common steps are: extraction, haemoglobin precipitation, concentration/diafiltration and the first Q-Sepharose chromatography step. At this chromatography step the process is branched: while IgG and Cat were recovered in the flow-through, Sod and Alb were eluted separately. IgG and Cat were separated in a second Q-Sepharose chromatography step during which IgG was recovered in the flow-through, whereas Cat bound to the resin. IgG was purified by S-Sepharose chromatography, followed by selective precipitation with n-octanoic acid, yielding about 0.4 g of IgG per kg of placenta. Cat was eluted at the second Q-Sepharose chromatography step and was purified by Blue Sepharose chromatography. A total of 1.8 x 10(6) units of Cat were recovered/kg of placenta, with a specific activity of 45000 units/mg of protein. Sod was further purified by S-Sepharose and Phenyl-Sepharose chromatography steps and recovered in the non-adsorbed fractions. The yield of Sod was 2.1 x 10(5) units/kg of placenta, with a specific activity of 1194 units/mg of protein. Alb purification was followed by a combined process including thermocoagulation and treatment with activated charcoal. The final step was Phenyl-Sepharose chromatography. The process yielded 3.1 g of Alb/kg of placenta. The described methodology was designed to be easily scaled-up for industrial production.

Cloning, purification, crystallization and preliminary X-ray diffraction analysis of the antistasin-type inhibitor ghilanten (domain I) from Haementeria ghilianii in complex with porcine beta-trypsin.

Ghilanten, isolated from the leech Haementeria ghilianii, is a potent two-domain anticoagulant protein homologous to the factor Xa inhibitor antistasin. A synthetic gene encoding the amino-terminal domain of ghilanten (ghilanten-D1) was constructed, expressed in the methylotrophic yeast Pichia pastoris and purified by heparin-Sepharose chromatography. Recombinant ghilanten-D1 inhibits bovine trypsin and human factor Xa with equilibrium inhibition constants (K(i)) of 126 and 1.2 nM, respectively. Ghilanten-D1 has been crystallized in complex with porcine beta-trypsin; three different-looking but isomorphous crystal forms were obtained, each belonging to the orthorhombic space group P2(1)2(1)2(1). These crystals diffracted to beyond 3.6 A resolution using a rotating-anode X-ray source. A data set complete to 3.7 A resolution was collected.

Albumin purification from human placenta.

Albumin is the human protein used mainly for therapeutic purposes. Besides the traditionally used plasma, blood from placenta is an alternative source for albumin purification. We describe here an industrial process for purification of albumin from human placenta. The proposed albumin-purification process, for 50 kg of placentas, comprises: (i) extraction of haemolysed blood with saline and solid/liquid separation by basket centrifugation; (ii) selective precipitation of haemoglobin by ethanol/chloroform and precipitate removal by filtration in a press filter; (iii) concentration/diafiltration of the filtrate in a 30 kDa cross-flow ultrafiltration (CFUF) membrane; (iv) thermo-coagulation at 70 degrees C with sodium octanoate/EDTA; (v) treatment with activated charcoal at pH 3; (vi) concentration/diafiltration of the filtrate in a 30 kDa CFUF membrane; (vii) anion-exchange chromatography Q-Sepharose; (viii) hydrophobic-interaction chromatography with phenyl-Sepharose; and (ix) conditioning and pasteurization. The process yields an average of 4.5 g of albumin/kg of placenta with a purity of 97.1% and A(403) of 0.05 (1% protein). The final product passes pyrogen and toxicity tests in vivo and it does not contain polymers or aggregates, even after the accelerated stability test, as judged by gel filtration, as required by the Brazilian Pharmacopoeia.

Kinetics of eosinophil cationic protein release in mite-allergic rhinitis after specific nasal provocation.

Nasal allergen challenges, despite not reproducing exactly natural allergen exposures, are a useful method to try to understand the complex mediator and cellular kinetics as well as the cellular interactions triggered by allergen exposure in allergic rhinitis. In this article we evaluated the kinetics of Eosinophil Cationic Protein (ECP) release into nasal lavage fluid after nasal allergen challenge. Our results have shown heterogeneity in ECP kinetics, probably due to heterogeneous activation of the eosinophils already present in the nasal mucosa of perennial allergic rhinitis patients. More studies in this field are needed to evaluate the effect of specific immunotherapy and/or prolonged topical steroid therapy on the kinetics of ECP liberation after allergen challenge. These studies will ultimately lead to a more clear definition of the clinical usefulness of this procedure.