In vitro culture and morphology of Neoechinorhynchus buttnerae (Eoacanthocephala: Neoechinorhynchidae) collected from the intestine of tambaqui (Colossoma macropomum) farmed in the Brazilian Amazon.

The tambaqui (Colossoma macropomum) is a species of great economic importance for fish farming in the Brazilian Amazon, and acanthocephaliasis caused by Neoechinorhynchus buttnerae (Golvan 1956) represents an obstacle to its production due to it causing severe morphological damage to the intestinal mucosa, thus impairing the absorption of nutrients and causing weight loss in the fish. Therefore, the establishment of in vitro protocols for evaluation of anthelmintic drugs is the first step to development of effective measures for in vivo control of this endoparasite. The present study evaluated the in vitro survival of N. buttnerae maintained in Eagle's minimum essential medium under different culture conditions. Three assays were carried out to evaluate whether temperature, supplementation with the antibiotics penicillin and streptomycin, and culture medium replacement or no replacement would influence the motility and morphology of the acanthocephalans. The results of the Kaplan-Meier analysis indicated that the use of culture in minimum essential medium together with penicillin and streptomycin prolonged the parasite's survival when kept at temperatures of 24 °C or 28 °C. We describe herein for first time an alternative protocol that is ideal for the in vitro culture of N. buttnerae. As such, this protocol ensures greater reliability in further in vitro studies with N. buttnerae.

Acute toxicity of essential oils of Aloysia triphylla (L'Hér.) Britton, Lippia gracilis Schauer, and Piper aduncum L. in Colossoma macropomum (Cuvier, 1818).

The aim of this study was to determine the acute toxicity of the essential oils (EOs) of Aloysia triphylla, Lippia gracilis and Piper aduncum in juvenile tambaqui (Colossoma macropomum), and evaluate the possible histopathological alterations in their gills. For the acute toxicity tests, juvenile tambaqui (n=24/treatment) were distributed in six treatments with three replicates, which comprised the control and five EO concentrations of A. triphylla (60, 80, 100, 120 and 140 mg L-1), L. gracilis (35, 40, 45, 50 and 55 mg L-1) and P. aduncum (42.5, 45, 47.5, 50 and 52.5 mg L-1), with an exposure period of 4 h. The mortality rate and severity of damage to the tambaqui gills were proportional to the increase in the concentration of the EO, with LC50-4 h values estimated at 109.57 mg L -1 for A. triphylla, 41.63 mg L -1 for L. gracilis and 48.17 mg L -1 for P. aduncum. The main morphological damages observed in the gills of the tambaqui exposed to the three EOs, were Grade I: hypertrophy and hyperplasia of lamellar epithelial cells, lamellar fusion, epithelial detachment, capillary dilation and constriction, proliferation of chloride cells and mucosal cells and edema; in low frequency Grade II damage as epithelial rupture and lamellar aneurysm. Necrosis (Grade III damage) was observed only in gill lamellae exposed to P. aduncum EO (47.5, 50.0 and 52.5 mg L-1). Concentrations of EOs below LC50-4 h can be used sparingly, for short periods of exposure for the treatment of diseases in tambaqui breeding.

Outcome clinical audit: analyses of interventional closure of patent ductus arteriosus in dogs.

OBJECTIVES: The objectives of this study were to determine whether conducting a clinical audit was achievable in a group of centres that perform interventional cardiac procedures and to report the success and complications rates in dogs diagnosed with patent ductus arteriosus. METHODS: This was a multicentre, European-wide, prospective study. Patient data were entered into a bespoke database prior to commencing interventional closure of patent ductus arteriosus in all animals undergoing this procedure during the study period. The database was designed to gather clinical audit information, after completion of the procedure, such as discharge outcome, complication rate, and medium-term outcome. RESULTS: A total of 339 cases were included from five participating centres. The process of performing clinical audit was achieved in all centres. Successful discharge outcome was 95.9% with a complication rate of 4.1%. The procedure-related mortality was 0.6%. 149 cases (43.9%) were either lost to follow-up or had not yet had a follow-up within the time period. Of the remaining 169 cases in which follow-up was available, 157 (92.9%) cases had a successful medium-term outcome CONCLUSIONS: This study demonstrates that the process of performing a clinical audit is achievable in veterinary clinical interventions across different centres. These results provide a benchmark for future comparison in our ongoing clinical audit and validate the process of clinical audit for other centres performing cardiac interventions. The use of clinical audit should be considered in other aspects of veterinary medicine.

Validation of heart rate spot-check protocol to measure circadian variation and heart rate in healthy dogs and dogs with atrial fibrillation.

INTRODUCTION: Alternatives for out-of-clinic heart rate (HR) measurement are required to optimise the management of atrial fibrillation (AF) in dogs. Additionally, the presence of circadian variation (CV) in HR in pet dogs remains unknown. We aimed to identify the number and duration of spot-checks required for an accurate estimation of 24-hour HR in canine AF. Circadian variation in HR was examined in healthy dogs and dogs with AF, and spot-check-derived HR was compared with a CV-derived gold standard. MATERIALS AND METHODS: Ambulatory electrocardiogram data from healthy dogs and dogs with AF were retrospectively analysed. Heart rate was calculated from the entire recording and pre-defined periods (spot-checks) of one hour to 30 and 60 s in duration. Circadian variation in HR was determined by cosinor analysis. Bias and limits of agreement of means and median HR with mesor HR were determined by correlation and Bland-Altman analysis. RESULTS: Circadian variation in HR was identified not only in 18/22 healthy dogs and 14/21 AF dogs but only on ambulatory electrocardiogram recordings. Four-hourly spot-checks provided the most accurate estimate of mesor HR in healthy dogs (bias of the median over 30 s 7.70, limits of agreement 7.48), whereas, in dogs with AF, four, six and eight-hourly spot-checks provided reliable estimates of mesor HR (bias within -1.29 and -29.5). CONCLUSIONS: Four, six and eight-hourly HR spot-checks can estimate 24-hourly HR in dogs with AF. There was CV in HR in most healthy pet dogs and dogs with AF. Spot-check protocols cannot identify CV in HR.

Thrombocytopenia in pregnancy, a challenge in the intensive care unit (ICU). / Trombocitopenia en el embarazo, un desafío en la unidad de cuidados intensivos (UCI).

Thrombocytopenia is a common alteration during pregnancy and the multiple possible aetiologies make the differential diagnosis challenging. Physiological changes of pregnancy can mask severe conditions until advanced stages. This case refers to a pregnant woman who presented fever and thrombocytopenia during labour. In the postpartum period she developed rapidly progressive hemodynamic instability requiring admission to the ICU. There was progression to acute kidney injury, persistent thrombocytopenia and platelet dysfunction. The differential diagnosis included DIC in the context of sepsis, PPH and other causes of thrombotic microangiopathies in pregnancy.

Antiviral activity of hemolymph of Podalia against rubella virus.

Many active principles produced by animals, plants and microorganisms have been employed in the development of new drugs for the treatment of human diseases. Among animals known to produce pharmacologically active molecules that interfere in human cell physiology. Rubella virus (genus Rubivirus, family Togaviridae) is a single stranded RNA virus of positive genome polarity. Rubella virus infection of susceptible women during the first trimester of pregnancy often results in long-term virus persistence in the fetus causing multiple organ abnormalities. Potent antiviral activity against rubella virus (RV) has been observed in the hemolymph of Podalia sp. (Lepidoptera: Megalopygidae). This study evaluated the effect of hemolymph on RV infected Statens Serum Institute Rabbit Cornea (SIRC) cells. Results of cell viability and cell proliferation assays indicated that hemolymph was not toxic to cultured SIRC cells. Viral binding assay, antiviral assay, PCR, real-time PCR, and transmission electron microscopy were used to demonstrate that hemolymph in post-treatment could inhibit the production of infectious RV particles. Specifically, hemolymph was found to inhibit RV adsorption to the SIRC cells.

Antiviral activity of hemolymph of Podalia against rubella virus

Many active principles produced by animals, plants and microorganisms have been employed in the development of new drugs for the treatment of human diseases. Among animals known to produce pharmacologically active molecules that interfere in human cell physiology. Rubella virus (genus Rubivirus, family Togaviridae) is a single stranded RNA virus of positive genome polarity. Rubella virus infection of susceptible women during the first trimester of pregnancy often results in long-term virus persistence in the fetus causing multiple organ abnormalities. Potent antiviral activity against rubella virus (RV) has been observed in the hemolymph of Podalia sp. (Lepidoptera: Megalopygidae). This study evaluated the effect of hemolymph on RV infected Statens Serum Institute Rabbit Cornea (SIRC) cells. Results of cell viability and cell proliferation assays indicated that hemolymph was not toxic to cultured SIRC cells. Viral binding assay, antiviral assay, PCR, real-time PCR, and transmission electron microscopy were used to demonstrate that hemolymph in post-treatment could inhibit the production of infectious RV particles. Specifically, hemolymph was found to inhibit RV adsorption to the SIRC cells.

Macrophages stimulate gastric and colorectal cancer invasion through EGFR Y(1086), c-Src, Erk1/2 and Akt phosphorylation and smallGTPase activity.

The interactions between cancer cells and their microenvironment are crucial for malignant progression, as they modulate invasion-related activities. Tumor-associated macrophages are generally considered allies in the process of tumor progression in several types of cancer, although their role on gastric and colorectal carcinomas is still poorly understood. In this report, we studied the influence of primary human macrophages on gastric and colorectal cancer cells, considering invasion, motility/migration, proteolysis and activated intracellular signaling pathways. We demonstrated that macrophages stimulate cancer cell invasion, motility and migration, and that these effects depend on matrix metalloproteinase (MMP) activity and on the activation of epidermal growth factor receptor (EGFR) (at the residue Y(1086)), PLC-γ (phospholipase C-gamma) and Gab1 (GRB2-associated binding protein-1), as evidenced by siRNA (small interference RNA) experiments. Epidermal growth factor (EGF)-immunodepletion impaired macrophage-mediated cancer cell invasion and motility, suggesting that EGF is the pro-invasive and pro-motile factor produced by macrophages. Macrophages also induced gastric and colorectal cancer cell phosphorylation of Akt, c-Src and ERK1/2, and led to an increase of RhoA and Cdc42 activity. Interestingly, whereas macrophage-mediated cancer cell c-Src and ERK1/2 phosphorylation occurred downstream EGFR activation, Akt phosphorylation seems to be a parallel event, taking place in an EGFR-independent manner. The involvement of EGF, EGFR-downstream signaling partners and MMPs in macrophage-mediated invasion provides novel insights into the molecular crosstalk established between cancer cells and macrophages, opening new perspectives for the design of new and more efficient therapeutic strategies to counteract cancer cell invasion.

Physical characteristics of the eggs of red-legged partridge (Alectoris rufa) reared in captivity / Características físicas dos ovos da perdiz vermelha (Alectoris rufa) criada em cativeiro

O objetivo do presente estudo foi avaliar os efeitos da idade das fêmeas (um, dois e três anos) e do mês de postura (março, abril e maio) sobre as características físicas dos ovos da perdiz vermelha (Alectoris rufa) criada em cativeiro. O peso (W), o comprimento máximo (L) e a largura máxima (B) de 2878 ovos foram determinados diretamente, enquanto o índice de forma (B/L), o volume (V) e a superfície (S) foram calculados com base nos parâmetros determinados diretamente. A análise mostrou diferenças significativas (P<0,01) no peso dos ovos entre as diferentes idades e entre meses de postura, com menor peso nas fêmeas mais jovens. Observaram-se diferenças significativas (P<0,01) no comprimento do ovo entre as classes de idade, mas não entre os meses de postura (P>0,05). Observaram-se diferenças significativas (P<0,01) na largura máxima e no índice de forma do ovo entre as diferentes classes de idades, com valores mais elevados nas fêmeas mais velhas e no período de postura mais tardio. O volume dos ovos estimados por meio de V1= 0,51LB2e V2=0,913W foi afetado significativamente (P<0,01) pela idade e pelo mês de postura, bem como as áreas, S1=4.835W0,662, S2=4,951V10,666e S3=4,951V20,666, as quais apresentaram os mesmos efeitos.

Influenza virus A(H3N2) strain isolated from cerebrospinal fluid from a patient presenting myelopathy post infectious.

BACKGROUND: Neurological involvement during influenza infection has been described during epidemics and is often consistent with serious sequelae or death. OBJECTIVE: To investigate the etiologic agent involved in myelopathy post influenza-like syndrome. STUDY DESIGN: This investigation focuses on virus isolation from the cerebrospinal fluid (CSF) collected from a 19-year-old male student presenting with clinical diagnosis of myelopathy post influenza-like syndrome. To achieve this goal, different cell cultures and molecular methodologies were carried out. RESULTS: Influenza virus A(H3N2) strain was isolated in MDCK cell culture; virus particles were observed under electron microscopy. Phylogenetics analyses showed that the Brazilian influenza A(H3N2) strains were closely related to the A/Perth/16/2009-like. CONCLUSION: This study demonstrates that influenza virus A(H3N2) strain was the cause of illness of the students. According to the Brazilian influenza virus sentinel surveillance data A/Perth/16/2009-LIKE (H3N2) strain has predominated during the 2010 influenza virus season in Brasília-DF.

Anatomy and pathology of the masticator space.

OBJECTIVE: This article reviews and illustrates the anatomy and pathology of the masticator space (MS). BACKGROUND: Pathology of the masticator space includes inflammatory conditions, vascular lesions, and tumours. Intrinsic tumours of this space can be benign and malignant, and they may arise from the mandibular ramus, the third division of the trigeminal nerve, or the mastication muscles. Malignant tumours may appear well defined and confined by the masticator fascia, without imaging signs of aggressive extension into neighbouring soft tissues. Secondary invasion of the masticator space can also occur with tumours of the nasopharynx, oropharynx, oral cavity, and parotid glands. Perineural tumour spread (PNS), especially along the trigeminal nerve, can also occur with masticator space malignancies. CONCLUSION: Masses of the MS are difficult to evaluate clinically, and computed tomographic (CT) and magnetic resonance (MR) images are essential for the diagnosis and characterisation of these lesions. Malignant tumours may appear well defined and confined by the fascia. Thus, when a mass is identified, a biopsy should be done promptly. PNS may occur in tumours involving the MS and its recognition on imaging studies is essential to plan the appropriate treatment. TEACHING POINTS: • Differentiating between intrinsic and extrinsic lesions is essential to the differential diagnosis • Infections of the MS may cross the fascia and mimic neoplasms on imaging studies • Malignant tumours may show no aggressive signs, such as bone erosion or violation of the fascia • Perineural spread (PNS) is often clinically silent and frequently missed at imaging and leads to tumour recurrence.

Adsorbed fibrinogen leads to improved bone regeneration and correlates with differences in the systemic immune response.

Designing new biomaterials that can modulate the inflammatory response instead of attempting just to reduce it constitutes a paradigm change in regenerative medicine. This work aimed to investigate the capacity of an immunomodulatory biomaterial to enhance bone regeneration. For that purpose we incorporated a molecule with well-established pro-inflammatory and pro-healing roles, fibrinogen, in chitosan scaffolds. Two different incorporation strategies were tested, leading to concentrations of 0.54±0.10mg fibrinogen g(-1) scaffold immediately upon adsorption (Fg-Sol), and 0.34±0.04mg fibrinogen g(-1) scaffold after washing (Fg-Ads). These materials were implanted in a critical size bone defect in rats. At two months post-implantation the extent of bone regeneration was examined by histology and the systemic immune response triggered was evaluated by determining the percentages of myeloid cells, T and B lymphocytes in the draining lymph nodes. The results obtained indicate that the fibrinogen incorporation strategy conditioned the osteogenic capacity of biomaterials. Fg-Ads scaffolds led to more bone formation, and the presence of Fg stimulated angiogenesis. Furthermore, animals implanted with Fg-Ads scaffolds showed significant increases in the percentages of B lymphocytes and myeloid cells in the draining lymph nodes, while levels of T lymphocytes were not significantly different. Finally, a significant increase in TGF-ß1 was detected in the plasma of animals implanted with Fg-Ads. Taken together the results presented suggest a potential correlation between the elicited immune response and biomaterial osteogenic performance.

Fibrinogen promotes resorption of chitosan by human osteoclasts.

The osteoconductive and osteoinductive properties of materials intended for bone regeneration have been extensively tested, but the resorbability of these materials is often overlooked. Osteoclasts are responsible for bone resorption and play a crucial role in bone remodeling, which is essential for complete regeneration of bone tissue following injury. In this study we compare, for the first time, the ability of unmodified and fibrinogen (Fg)-modified chitosan (Ch) substrates to support the formation of multinucleated osteoclasts, and the potential of these cells to resorb the two substrates in vitro. Osteoclasts were differentiated from primary human peripheral blood monocytes directly on the substrates being investigated. Our results showed similar cell adhesion to unmodified and Fg-modified Ch substrates. Although the number of multinucleated osteoclasts on both Ch substrates increased throughout the culture period, by 21 days of culture significantly more highly multinucleated osteoclasts (>10 nuclei per cell) were observed on Fg-modified Ch, when compared to Ch alone. In addition, cells were tartrate-resistant acid phosphatase positive and secreted significantly more enzyme on Ch-based substrates than in control conditions. Unmodified and Fg-modified Ch resorption was investigated by fluorescence microscopy and confirmed by electron microscopy. Quantification of results obtained by fluorescence microscopy shows that Fg modification led to significantly higher substrate resorption by 17 days of culture. Our results show that osteoclasts, beyond resorbing mineralized substrates, successfully resorb a polymeric substrate (Ch), with Fg accelerating this process. Thus, in bone tissue regeneration strategies employing polymeric biomaterials, resorption may depend not only on macrophages, but also on osteoclasts.

Retroperitoneal bronchogenic cyst: MRI findings.

The authors describe a case of a retroperitoneal bronchogenic cyst in a 36-year-old female. She presented with abdominal pain, nausea, and vomiting. An MRI scan revealed an 8 cm cystic lesion in the left upper retroperitoneum, with intermediate signal on T2-weighted images, high signal on T1 weighted images, and lack of internal enhancement after gadolinium. After laparoscopic excision, the histology findings were compatible with a bronchogenic cyst, which is extremely uncommon in the retroperitoneum.

Epidemiological and molecular characterization of rubella virus isolated in São Paulo, Brazil during 1997-2004.

Rubella virus (RV) infection during the early stages of pregnancy can lead to serious birth defects, known as the congenital rubella syndrome (CRS). In 2003, the Pan American Health Organization (PAHO) adopted a resolution calling for the elimination of rubella and the congenital rubella syndrome (CRS) in the Americas by the year 2010. Brazil will have implemented the recommended PAHO strategy for elimination and interruption of endemic rubella virus transmission. The characterization of genotypes during the final stages of rubella elimination is important for determining whether new rubella isolates represent endemic transmission or importations. Samples (blood, urine, cerebrospinal fluid, and throat swabs) collected from patients with symptoms suggestive of rubella infection in 1997-2004 were isolated in cell culture and genotyped. Twenty-eight sequences were analyzed and two genotypes were identified: 1a and 1G. The information reported in this paper will contribute to understanding the molecular epidemiology of RV in São Paulo, Brazil.

Phylogenetic analysis of rubella virus strains during an outbreak in São Paulo, 2007-2008.

Rubella virus (RV) is an important human pathogen that causes rubella, an acute contagious disease. It also causes severe birth defects collectively known as congenital rubella syndrome when infection occurs during the first trimester of pregnancy. Here, we present the phylogenetic analysis of RV that circulated in São Paulo during the 2007-2008 outbreak. Samples collected from patients diagnosed with rubella were isolated in cell culture and sequenced. RV RNA was obtained from samples or RV-infected cell cultures and amplified by reverse transcriptase-polymerase chain reaction. Sequences were assigned to genotypes by phylogenetic analysis using RV reference sequences. Seventeen sequences were analyzed, and three genotypes were identified: 1a, 1G, and 2B. Genotypes 1a and 1G, which were isolated in 2007, were responsible for sporadic rubella cases in São Paulo. Thereafter, in late 2007, the epidemiological conditions changed, resulting in a large RV outbreak with the clear dominance of genotype 2B. The results of this study provide new approaches for monitoring the progress of elimination of rubella from São Paulo, Brazil.

The effect of adsorbed fibronectin and osteopontin on macrophage adhesion and morphology on hydrophilic and hydrophobic model surfaces.

Macrophages play a crucial role in the host response to biomaterials. Here we investigated the effect of adsorbed fibronectin (FN) and osteopontin (OPN), two important proteins for tissue repair, on macrophage adhesion and morphology. Since cell-biomaterial interactions are modulated via proteins adsorbed onto biomaterial surfaces, FN and OPN were adsorbed on model self-assembled monolayers (SAMs) of alkanethiols on gold with different functional terminal groups (CH(3), OH and tetra(ethylene-glycol)). The initial interaction of inflammatory cells with a biomaterial is crucial for the ensuing phases of an inflammatory reaction. For this reason short-term cultures of primary human macrophages were performed. To account for the competitive adsorption of other proteins serum was added to the culture medium and the effect compared with serum-free medium cultures. In the presence of serum hydrophilic surfaces increased macrophage adhesion. In particular, FN induced a higher cell density, while OPN tended to decrease it. In serum-free medium cell adhesion was greater on hydrophobic surfaces, except for OPN-coated SAMs. Importantly, FN no longer enhanced macrophage adhesion, while OPN maintained its inhibitory effect. Cell polarization studies indicated that macrophage morphology variations induced by surface chemistry are overcome by pre-adsorbed OPN. Taken together our results show that in the presence of serum macrophage adhesion is promoted by FN hydrophilic surfaces, but impaired on OPN-coated surfaces. The effects of inhibited macrophage adhesion on macrophage fusion, and its relevance to the initial stages of the inflammatory response to biomaterials are discussed.

On the use of evoked potentials for quantification of pain.

Pain is a subjective and individual sensation causing major discomfort. So, it is necessary to put into practice methods to objectively quantify it. Several studies indicate that evoked potentials (EP) generate responses which may reflect painful processes. This study reports the results of the application of two different protocols by using biopotentials to objectively measure pain. The first (protocol 1) evaluates the relation between pain, induced by electrical stimulation, and subjective perception and also with nociceptive flexion reflex (NFR) represented by muscle activity (electromyography) detected on the femoral biceps after sural nerve stimulation. The second protocol (protocol 2) verifies whether there is some correlation between M-wave parameters and subjective pain sensation. The results obtained from protocol 1 suggest that the area of the EMG envelope and entropy estimated from the EMG activity are correlated with subjective sensation of pain. The analysis of data obtained from protocol 2 shows a correlation between the global minimum of the M-wave and pain increase. These results contribute to studies which seek to objective measures for pain quantification based on the analysis of biopotentials.

Isolation and genotyping of rubella virus from a case of congenital infection in Brazil.

The incidence of CRS and CRI has decreased markedly worldwide with the implementation of efficient vaccination programs. We report a congenital rubella case with fetal death occurred at 29th week of gestation. RV was confirmed in placenta. The results of phylogenetic analysis showed that the RVs/SaoPaulo01.- BRA/08.CRI belongs to the genotype 2B of RV.