<p>Cardiopulmonary bypass (CPB) is associated with inflammatory response and activation of coagulation. Heparin coating of the CPB circuit is shown to improve the biocompatibility of the surface. The biological effects of a new heparin surface, the Corline Heparin Surface (CHS), prepared according to a new principle, have been studied. </p><p>The CHS used during coronary artery bypass grafting with CPB in sixty patients prevented adhesion of cells to the extracorporeal device. The activation of inflammation, coagulation, and fibrinolysis was significantly reduced by the use of CHS. Both a reduced and an increased dose of systemic heparin in combination with the heparin-coated surface resulted in more activation of inflammation and coagulation. </p><p>Photoelectron spectroscopy studies of the molecular structure of the CHS demonstrated that a single layer of the heparin surface, equivalent to what was used in the <i>in vivo</i> studies, did not completely cover the substrate surface. Additional layer of immobilized heparin has resulted in a complete coverage. We examined the biological effects, i.e. activation of inflammation and coagulation, by CHS in one and two layers in an <i>in vitro</i>-study. The data from this study clearly demonstrated that a uniform surface coating of the CHS results in only minor activation of coagulation, inflammation and cell activation. </p><p>Monocytes do not normally express tissue factor (TF), initiator of the coagulation <i>in vivo</i>, but can be induced upon adhesion to artificial surfaces. TF is receptor for coagulation factor VIIa (FVIIa) and binding subsequently leads to formation of thrombin. Other biological effects beyond coagulation, as inflammation and angiogenesis, has recently been associated with the formation of TF·FVIIa. The TF∙FVIIa signal transduction induced an increased sensitivity to PDGF-BB-stimulated migration and an increased production of IL-8 and TNF-α in monocytes. These could be important mechanisms for continued recruitment of cells to sites of inflammation. </p>

<p>In patients with sepsis, the activation of the cascade systems, for example the complement system with the generation of C5a, is followed by a state of immunosuppression with impaired bactericidal capacity caused by suppression of the neutrophil granulocytes. To inhibit the C5a-induced systemic inflammatory and the following anti-inflammatory responses, different anti-C5a strategies have been successful in experimental models of sepsis. In animals and in healthy volunteers after injection of lipopolysaccharide (LPS), an up-regulation of the C5a receptor (C5aR) has been reported. Before designing clinical studies, it was of importance to increase the knowledge of C5a and C5aR regulation in humans. </p><p>At the time when the diagnosis of severe sepsis or septic shock can be established clinically, granulocyte C5aR expression, analysed by flow cytometer, was shown to be reduced, whereas monocyte C5aR expression was unchanged. There was a correlation between granulocyte C5aR expression and the severity of disease, as measured by the APACHE II score. </p><p><i>Ex vivo</i> incubation of whole blood with LPS resulted in a reduction in granulocyte C5aR expression. Such a reduction was not found in isolated cells, indicating that the effect was mediated via plasma factors, such as C5a, IL-8 and TNF-α which all were shown to reduce C5aR expression <i>ex vivo</i>.</p><p>Although there was a trend between chemotaxis, as measured by migration in a modified Boyden chamber, and C5aR expression on granulocytes from patients with severe sepsis or septic shock or from healthy individuals, the correlation failed to reach statistical significance.</p><p>It is concluded that granulocyte C5aR expression is affected by several plasma factors and that a reduction is clinically evident at the time of the sepsis diagnosis. Reduced granulocyte C5aR expression is associated with an impaired chemotaxis but does not alone limit the chemotactic response.</p>

Cardiopulmonary bypass (CPB) is associated with inflammatory response and activation of coagulation. Heparin coating of the CPB circuit is shown to improve the biocompatibility of the surface. The biological effects of a new heparin surface, the Corline Heparin Surface (CHS), prepared according to a new principle, have been studied. The CHS used during coronary artery bypass grafting with CPB in sixty patients prevented adhesion of cells to the extracorporeal device. The activation of inflammation, coagulation, and fibrinolysis was significantly reduced by the use of CHS. Both a reduced and an increased dose of systemic heparin in combination with the heparin-coated surface resulted in more activation of inflammation and coagulation. Photoelectron spectroscopy studies of the molecular structure of the CHS demonstrated that a single layer of the heparin surface, equivalent to what was used in the in vivo studies, did not completely cover the substrate surface. Additional layer of immobilized heparin has resulted in a complete coverage. We examined the biological effects, i.e. activation of inflammation and coagulation, by CHS in one and two layers in an in vitro-study. The data from this study clearly demonstrated that a uniform surface coating of the CHS results in only minor activation of coagulation, inflammation and cell activation. Monocytes do not normally express tissue factor (TF), initiator of the coagulation in vivo, but can be induced upon adhesion to artificial surfaces. TF is receptor for coagulation factor VIIa (FVIIa) and binding subsequently leads to formation of thrombin. Other biological effects beyond coagulation, as inflammation and angiogenesis, has recently been associated with the formation of TF·FVIIa. The TF∙FVIIa signal transduction induced an increased sensitivity to PDGF-BB-stimulated migration and an increased production of IL-8 and TNF-α in monocytes. These could be important mechanisms for continued recruitment of cells to sites of inflammation.

In patients with sepsis, the activation of the cascade systems, for example the complement system with the generation of C5a, is followed by a state of immunosuppression with impaired bactericidal capacity caused by suppression of the neutrophil granulocytes. To inhibit the C5a-induced systemic inflammatory and the following anti-inflammatory responses, different anti-C5a strategies have been successful in experimental models of sepsis. In animals and in healthy volunteers after injection of lipopolysaccharide (LPS), an up-regulation of the C5a receptor (C5aR) has been reported. Before designing clinical studies, it was of importance to increase the knowledge of C5a and C5aR regulation in humans. At the time when the diagnosis of severe sepsis or septic shock can be established clinically, granulocyte C5aR expression, analysed by flow cytometer, was shown to be reduced, whereas monocyte C5aR expression was unchanged. There was a correlation between granulocyte C5aR expression and the severity of disease, as measured by the APACHE II score. Ex vivo incubation of whole blood with LPS resulted in a reduction in granulocyte C5aR expression. Such a reduction was not found in isolated cells, indicating that the effect was mediated via plasma factors, such as C5a, IL-8 and TNF-α which all were shown to reduce C5aR expression ex vivo. Although there was a trend between chemotaxis, as measured by migration in a modified Boyden chamber, and C5aR expression on granulocytes from patients with severe sepsis or septic shock or from healthy individuals, the correlation failed to reach statistical significance. It is concluded that granulocyte C5aR expression is affected by several plasma factors and that a reduction is clinically evident at the time of the sepsis diagnosis. Reduced granulocyte C5aR expression is associated with an impaired chemotaxis but does not alone limit the chemotactic response.

This work identified an ortholog of Abp1 (actin binding protein 1) in Dictyostelium (Dabp1). In order to analyze the functions of Dabp1 in Dictyostelium, loss-of–function studies and gain-of-function studies were performed by generating cells that either deleted the Dabp1 gene from the genome or overexpressed the Dabp1 protein. In these mutants, most actin-based processes were intact. However, cell motility was altered during early development. During chemotactic streaming, more than 90% of wild type cells had a single leading pseudopodium and a single uropod, whereas more than 27% of Dabp1 null cells projected multiple pseudopodia. Similarly, ~ 90% of cells that overexpressed Dabp1 projected multiple pseudopodia during chemotactic streaming, and displayed reduced rates of cell movement. Expression of the SH3 domain of Dabp1 showed this domain to be an important determinant in regulating pseudopodium number. These results suggest that Abp1 controls pseudopodium number and motility in early stages of chemotactic aggregation in Dictyostelium. This work also revealed an interplay between Dabp1 and MyoB, one of the Myosin I proteins, in controlling pseudopodia formation in Dictyostelium. These two proteins colocalize partially at the cortex in growing cells. The peripheral localization of MyoB was dependent on Dabp1. Depletion of both Dabp1 and MyoB caused defects in organization of the actin cytoskeleton and actin related activities such as formation of small F-actin filled spikes on the cell cortex of growing cells, a higher percentage of multinucleated cells, and an increased number of pseudopodia branching extensively. When MyoB was overexpressed in Dabp1 null mutants, cells had similar phenotypes as Dabp1/MyoB double null mutants, and displayed an increased number of pseudopodia with many branches. Overexpression of Dabp1 in MyoB null mutants rescued the defects in pseudopodia formation. The SH3 of Dabp1 was shown to be important for the rescue of defects caused by depletion of MyoB. Collectively, these data suggest that MyoB and Dabp1 work cooperatively to regulate the uniformity and integrity of the actin extensions during chemotaxis. MyoB requires Dabp1 to function in this process. Dabp1 may function as a scaffold to recruit MyoB to the proper localization. These studies of Dabp1 in Dictyostelium raise broad question about functions of actinassociated proteins in pseudopodia formation and the importance of uniformity and integrity for actin structures in chemotaxis. / text

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Mannheimia haemolytica (M. haemolytica)-induced bovine respiratory disease causes millions of dollars in economic losses to Canadian cattle industry. Contemporary management strategies built around the use of antimicrobials are proving to be increasingly unavailing and lead to drug residues in meat which may contribute to the development of multi drug resistant bacteria. Many M. haemolytica vaccines are effective in stimulating antibody responses but studies of vaccina-tion in young calves and the cattle exposed to M. haemolytica (high-risk cattle) have shown poor vaccine efficacy. Antimicrobial peptides (AMPs) may help in the management of respiratory disease caused by M. haemolytica while minimizing the risk of drug residues in animal-derived food products. AMPs are positively charged molecules that can kill bacteria primarily through the electrostatic interactions with the anionic bacterial lipid bilayer. Since the primary target of AMPs is the bac-terial surface charge, which is evolutionarily conserved, the development of resistance towards AMPs seems less likely. These peptides hold potential to replace or reduce the use of antibiotics. Human β-Defensin 3 (HBD3) and Microcin J25 (MccJ25) are cationic peptides that have shown good activity against many Gram-negative bacteria. Five peptides, namely native HBD3, three synthetic HBD3 analogues (28 amino acid, 20AA, and 10AA), and MccJ25 were selected for microbicidal activity against M. haemolytica. Three C-terminal analogues of HBD3 with all cysteines replaced with valines were manually synthesized using solid phase peptide synthesis (SPPS). In all the three analogue, replacement of cysteine with valine rendered them linear and increased their antibacterial activity. Minimum Bactericidal concentration (MBC) assays were performed with the final inoculum size of 1-5x105 cells/ml, with the exception of the 10AA analogue which was incubated with 104 cells/ml final inoculum size. The antimicrobial assay showed that M. haemolytica was intermediately sensitive to HBD3, 28AA and 20AA analogue with an MBC of 50 µg/ml. MccJ25 had limited effect with an MBC greater than 100 µg/ml. The MBC value of 6.3 µg/ml achieved with the 10AA analogue is likely a result of lower final inoculum size. AMPs have several immunomodulatory functions, and these peptides can act as chemoattractant, induce cytokine release that in turn leads to chemotaxis of monocytes and neutrophils. Since neutrophils play an important role in the pathogenesis of BRD, the chemotactic effect of HBD3, 20AA and 28AA peptides on bovine neutrophils was studied using Boyden chamber. Peripheral blood neutrophils isolated from normal healthy cattle showed chemotaxis towards HBD3 and 20AA peptides (P<0.05) but not towards 28AA analogue. Co-incubation of neutrophils with any of the peptides did not affect their chemotaxis towards N-formyl-L-methionyl-L-leucyl-phenylalanine (fMLP). Based on these data, it can be concluded that HBD3 and its analogues showed antimicrobial ef-fects against M. haemolytica but MccJ25 had limited microbicidal activity against M. haemolytica. While HBD3 and 20AA analogue were chemotactic for bovine peripheral blood neutrophils, none of the peptides inhibited fMLP-induced migration of neutrophils. These peptides hold potential for further in vivo testing to develop them for use to manage M. haemolytica-induced respiratory disease in cattle.

O gênero Psychotria destaca-se na família RUBIACEAE pela produção de alcalóides bioativos e por sua taxonomia complexa, sendo muitas vezes relacionado aos gêneros Palicourea, Cephaelis, Calycodendron e Calycosia. A divisão de Psychotria nos subgêneros Psychotria, Tetramerae e Heteropsychotria foi proposta com o objetivo de auxiliar a classificação quimiotaxonômica do gênero. Estudos demonstram que o subgênero Psychotria (espécies pantropicais) produz alcalóides poliméricos, formados por duas ou mais unidades de triptamina; enquanto o subgênero Heteropsychotria parece estar envolvido com a produção de alcalóides indol monoterpênicos, de acordo com pesquisas realizadas com diferentes espécies coletadas no Sul do Brasil. O isolamento e purificação dos alcalóides estrictosamida, ácido estrictosidínico e miriantosina, a partir de Psychotria myriantha, corroboram com esta hipótese, permitindo a inclusão da espécie no subgênero Heteropsychotria. A literatura descreve várias atividades para extratos e alcalóides isolados de espécies de Psychotria, como antimicrobiana e analgésica, por exemplo. Neste trabalho, o extrato n-butanólico de alcalóides de P. myriantha, além de seus alcalóides isolados, apresentaram atividade inibidora da migração de leucócitos, sugerindo um efeito antiinflamatório, e capacidade de inibir a ação da enzima acetilcolinesterase. Extratos e alcalóides isolados da espécie foram avaliados quanto à atividade antioxidante em CCD, frente ao DPPH, apresentando resultado negativo. O extrato EBA e o alcalóide ácido estrictosidínico aumentaram o tempo de latência no teste da retirada da cauda frente ao estímulo térmico, indicando uma atividade analgésica do tipo opióide. A influência do ácido estrictosidínico, alcalóide isolado em maior quantidade em massa, sobre os níveis de DA, DOPAC, 3-MT, HVA, 5-HT e 5-HIAA em estruturas cerebrais de ratos foi verificada. Hipocampos de animais que receberam injeção intra-hipocampal bilateral de ácido estrictosidínico (20 μg/μL) apresentaram redução de 83,4 % nos níveis de serotonina, em comparação ao grupo controle, enquanto os córtices desses animais apresentaram redução nos níveis de DOPAC (35,9%), 3-MT (24,7%) e 5-HIAA (9%). Hipocampos e estriados de ratos tratados com injeção i.p. de ácido estrictosidínico (10 mg/kg) demonstraram diminuição de 63,4 e 28,7% nos níveis de 5-HT, respectivamente. As alterações nos níveis de aminas biogênicas nas estruturas avaliadas, além das atividades analgésica e inibidora da acetilcolinesterase, indicam que P. myriantha e espécies do subgênero Heteropsychotria constituem uma potencial fonte de substâncias bioativas no tratamento de distúrbios do sistema nervoso central. / Psychotria genus is an important in RUBIACEAE due to bioactive alkaloids production and complex taxonomy, being related to Palicourea, Cephaelis, Calycodendron and Calycosia genera. The division of Psychotria in Psychotria, Tetramerae and Heteropsychotria subgenera was proposed with the aim of aiding the genus chemotaxonomic classification. Studies demonstrate that Psychotria subgenus produce polyindoline alkaloids formed by two or more triptamine units; whereas Heteropsychotria subgenus seems to be involved with indole monoterpene alkaloids production, according to researches with different species collected in Southern Brazil. Isolation and purification of strictosamide, strictosidinic acid and miriantosine from Psychotria myriantha corroborated this hypothesis, allowing its inclusion into Heteropsychotria subgenus. The scientific literature describes several activities to Psychotria extracts and alkaloids, such as antimicrobial and analgesic, for example. In this work, P. myriantha n-butanolic alkaloid extract and isolated compounds inhibited the leukocyte migration, suggesting an antiinflammatory activity, and a weak ability to inhibit the action of acetylcholinesterase enzyme. Alkaloids and extracts from P. myriantha were evaluated as regards their antioxidant activity using DPPH, with no positive results. EBA and strictosidinic acid increased the latency in the tail flick model, indicating an opioid analgesic activity. The influence of strictosidinic acid on the levels of DA, DOPAC, 3-MT, HVA, 5-HT and 5-HIAA in brain structures of rats was verified. Hippocampus with intra-hippocampal injection of strictosidinic acid (20 μg/μL) displayed a decrease of 83.4% in serotonin levels, in comparison with control group; whereas the cortex showed a decrease in the levels of DOPAC (35.9%), 3-MT (24.7%) and 5-HIAA (9%). Hippocampus and striatum that received intraperitoneal injection of strictosidinic acid (10 mg/kg) showed 5-HT levels reduction of 63.4 e 28.7%, respectively. The biogenic amine levels alterations in the studied structures, associated with the analgesic and acetylcholinesterase inhibitor activities, suggest that P. myriantha and species from the Heteropsychotria subgenus constitute a font of bioactive compounds in the treatment of central nervous system disturbs.

O gênero Psychotria destaca-se na família RUBIACEAE pela produção de alcalóides bioativos e por sua taxonomia complexa, sendo muitas vezes relacionado aos gêneros Palicourea, Cephaelis, Calycodendron e Calycosia. A divisão de Psychotria nos subgêneros Psychotria, Tetramerae e Heteropsychotria foi proposta com o objetivo de auxiliar a classificação quimiotaxonômica do gênero. Estudos demonstram que o subgênero Psychotria (espécies pantropicais) produz alcalóides poliméricos, formados por duas ou mais unidades de triptamina; enquanto o subgênero Heteropsychotria parece estar envolvido com a produção de alcalóides indol monoterpênicos, de acordo com pesquisas realizadas com diferentes espécies coletadas no Sul do Brasil. O isolamento e purificação dos alcalóides estrictosamida, ácido estrictosidínico e miriantosina, a partir de Psychotria myriantha, corroboram com esta hipótese, permitindo a inclusão da espécie no subgênero Heteropsychotria. A literatura descreve várias atividades para extratos e alcalóides isolados de espécies de Psychotria, como antimicrobiana e analgésica, por exemplo. Neste trabalho, o extrato n-butanólico de alcalóides de P. myriantha, além de seus alcalóides isolados, apresentaram atividade inibidora da migração de leucócitos, sugerindo um efeito antiinflamatório, e capacidade de inibir a ação da enzima acetilcolinesterase. Extratos e alcalóides isolados da espécie foram avaliados quanto à atividade antioxidante em CCD, frente ao DPPH, apresentando resultado negativo. O extrato EBA e o alcalóide ácido estrictosidínico aumentaram o tempo de latência no teste da retirada da cauda frente ao estímulo térmico, indicando uma atividade analgésica do tipo opióide. A influência do ácido estrictosidínico, alcalóide isolado em maior quantidade em massa, sobre os níveis de DA, DOPAC, 3-MT, HVA, 5-HT e 5-HIAA em estruturas cerebrais de ratos foi verificada. Hipocampos de animais que receberam injeção intra-hipocampal bilateral de ácido estrictosidínico (20 μg/μL) apresentaram redução de 83,4 % nos níveis de serotonina, em comparação ao grupo controle, enquanto os córtices desses animais apresentaram redução nos níveis de DOPAC (35,9%), 3-MT (24,7%) e 5-HIAA (9%). Hipocampos e estriados de ratos tratados com injeção i.p. de ácido estrictosidínico (10 mg/kg) demonstraram diminuição de 63,4 e 28,7% nos níveis de 5-HT, respectivamente. As alterações nos níveis de aminas biogênicas nas estruturas avaliadas, além das atividades analgésica e inibidora da acetilcolinesterase, indicam que P. myriantha e espécies do subgênero Heteropsychotria constituem uma potencial fonte de substâncias bioativas no tratamento de distúrbios do sistema nervoso central. / Psychotria genus is an important in RUBIACEAE due to bioactive alkaloids production and complex taxonomy, being related to Palicourea, Cephaelis, Calycodendron and Calycosia genera. The division of Psychotria in Psychotria, Tetramerae and Heteropsychotria subgenera was proposed with the aim of aiding the genus chemotaxonomic classification. Studies demonstrate that Psychotria subgenus produce polyindoline alkaloids formed by two or more triptamine units; whereas Heteropsychotria subgenus seems to be involved with indole monoterpene alkaloids production, according to researches with different species collected in Southern Brazil. Isolation and purification of strictosamide, strictosidinic acid and miriantosine from Psychotria myriantha corroborated this hypothesis, allowing its inclusion into Heteropsychotria subgenus. The scientific literature describes several activities to Psychotria extracts and alkaloids, such as antimicrobial and analgesic, for example. In this work, P. myriantha n-butanolic alkaloid extract and isolated compounds inhibited the leukocyte migration, suggesting an antiinflammatory activity, and a weak ability to inhibit the action of acetylcholinesterase enzyme. Alkaloids and extracts from P. myriantha were evaluated as regards their antioxidant activity using DPPH, with no positive results. EBA and strictosidinic acid increased the latency in the tail flick model, indicating an opioid analgesic activity. The influence of strictosidinic acid on the levels of DA, DOPAC, 3-MT, HVA, 5-HT and 5-HIAA in brain structures of rats was verified. Hippocampus with intra-hippocampal injection of strictosidinic acid (20 μg/μL) displayed a decrease of 83.4% in serotonin levels, in comparison with control group; whereas the cortex showed a decrease in the levels of DOPAC (35.9%), 3-MT (24.7%) and 5-HIAA (9%). Hippocampus and striatum that received intraperitoneal injection of strictosidinic acid (10 mg/kg) showed 5-HT levels reduction of 63.4 e 28.7%, respectively. The biogenic amine levels alterations in the studied structures, associated with the analgesic and acetylcholinesterase inhibitor activities, suggest that P. myriantha and species from the Heteropsychotria subgenus constitute a font of bioactive compounds in the treatment of central nervous system disturbs.