Anti-inflammatory activity and possible mechanism of extract from Mikania laevigata in carrageenan-induced peritonitis

Objectives The aim was to test the potential use of an extract of Mikania laevigata (popularly known in Brazil as guaco), made from leaves harvested in different months of the year, oil neutrophil migration after all inflammatory Stimulus and investigate the underlying molecular mechanisms. Methods We examined the effect of guaco on vascular permeability and leucocyte function in carrageenan-induced peritonitis in mice. Key findings Our results demonstrated that guaco extract administered subcutaneously (3 mg/kg) decreased the vascular permeability and also leucocyte rolling and adhesion to the inflamed tissues by a mechanism dependent on nitric oxide. Specifically, inhibitors of nitric oxide synthase remarkably abrogated the guaco extract-mediated suppression of neutrophil migration to the inflammatory site. In addition, guaco extract-mediated suppression of neutrophil migration appeared to be dependent on the production of the cytokines interleukin-1 beta and tumour necrosis factor-alpha. One of the major constituents of the guaco extract, coumarin, was able to inhibit the neutrophil migration towards the inflammatory focus. Conclusions In conclusion the anti-inflammatory effect induced by guaco extract may be by inhibition of pro-inflammatory cytokine production at the inflammatory site.

Effect of diode laser on enzymatic activity of parotid glands of diabetic rats

The aim of this study was to evaluate the effect of laser irradiation (LI) on enzymatic activities of amylase, catalase and peroxidase in the parotid glands (PG) of diabetic and non-diabetic rats. Ninety-six female rats were divided into eight groups: D0; D5; D10; D20 and C0; C5; C10; C20, respectively. Diabetes was induced by administration of streptozotocin and confirmed later by the glycemia results. Twenty-nine (29) days after the induction, the PGs of groups D5 and C5; D10 and C10; D20 and C20, were irradiated with 5 J/cm(2), 10 J/cm(2) and 20 J/cm(2) of laser diode (660 nm/100 mW) respectively. On the following day, the rats were euthanized and the enzymatic activity in the PGs was measured. Diabetic rats that had not been irradiated (group D0) showed higher catalase activity (P < 0.05) than those in group C0 (0.14 +/- 0.02 U/mg protein and 0.10 +/- 0.03 U/mg protein, respectively). However, laser irradiation of 5 J/cm(2) and 20 J/cm(2) decreased the catalase activity of the diabetic groups (D5 and D20) to non-diabetic values (P > 0.05). Based on the results of this study, LI decreased catalase activity in the PGs of diabetic rats.

Alpha-oestrogen and progestin receptor expression in the hypothalamus and preoptic area dopaminergic neurones during oestrous in cycling rats

A secretory surge of prolactin occurs on the afternoon of oestrous in cycling rats. Although prolactin is regulated by ovarian steroids, plasma oestradiol and progesterone levels do not vary during oestrous. Because prolactin release is tonically inhibited by hypothalamic dopamine and modulated by dopamine transmission in the preoptic area (POA), the present study aimed to evaluate whether oestrogen receptor (ER)-alpha and progestin receptor (PR) expression in the dopaminergic neurones of arcuate (ARC), periventricular, anteroventral periventricular (AVPe) and ventromedial preoptic (VMPO) nuclei changes during the day of oestrous. Cycling rats were perfused every 2 h from 10-20 h on oestrous. Brain sections were double-labelled to ER alpha or PR and tyrosine hydroxylase (TH). The number of TH-immunoreactive (ir) neurones did not vary significantly in any area evaluated. ER alpha expression in TH-ir neurones increased at 14 and 16 h in the rostral-ARC and dorsomedial-ARC, 14 h in the caudal-ARC and 16 h in the VMPO, whereas it was unaltered in the ventrolateral-ARC, periventricular and AVPe. PR expression in TH-ir neurones of the periventricular and rostral, dorsomedial, ventrolateral and caudal-ARC decreased transitorily during the afternoon, showing the lowest levels between 14 and 16 h; but it did not vary in the AVPe and VMPO. Plasma oestradiol and progesterone concentrations were low and unaltered during oestrous, indicating that the changes in receptors expression were probably not due to variation in ligand levels. Thus, our data suggest that variations in ER alpha and PR expression may promote changes in the activity of medial basal hypothalamus and POA dopaminergic neurones, even under unaltered secretion of ovarian steroids, which could facilitate the occurrence and modulate the magnitude of the prolactin surge on oestrous.

Visualising the activity of the cystine-glutamate antiporter in glial cells using antibodies to aminoadipic acid, a selectively transported substrate

The cystine-glutamate antiporter is a transport system that facilitates the uptake of cystine, concomitant with the release of glutamate. The cystine accumulated by this transporter is generally considered for use in the formation of the cysteine-containing antioxidant glutathione, which is abundant in many glial cells. This study used the simple strategy of generating an antibody to aminoadipic acid, a selective substrate for the cystine-glutamate antiporter. Stereospecific accumulation of aminoadipic acid into specific cell types in rat brain slice preparations was detected immunocytochemically. Strong accumulation was detected in astroglial cells in all brain regions studied including those in white matter tracts. Strong accumulation into radial glial cells, including the retinal Muller cells and the Bergmann glial cells was also observed. Glial accumulation was observed not only in cells within the blood brain barrier, but also outside such; anterior pituitary folliculostellate cell and intermediate lobe pituitary glial cells exhibited strong accumulation of aminoadipic acid. Interestingly, some glial cells such as the posterior pituitary glial cells (pituicytes) exhibited very little if any accumulation of aminoadipic acid. Within the brain labelling was not uniform. Particularly strong labelling was noted in some regions, such as the glial cells surrounding the CA1 pyramidal cells. By contrast, neurons never exhibited uptake of aminoadipic acid. Because cystine uptake is associated with glutamate release, it is suggested that this antiporter might contribute to release of glutamate from glial cells under some pathophysiological conditions. (C) 2001 Wiley-Liss, Inc.

Congenital lactic acidosis: Evaluation of the properties of the A199T natural variant of human pyruvate dehydrogenase E1 alpha by in vitro mutation

One cause of congenital lactic acidosis is a mutation in the E1 alpha -subunit of the pyruvate dehydrogenase multienzyme complex. Little is known about the consequences of these mutations at the enzymatic level. Here we study the A199T mutation by expressing the protein in Escherichia coil. The specific activity is 25% of normal and the K-m for pyruvate is elevated by 10-fold. Inhibitors of lactate dehydrogenase might be a useful therapy for patients with such mutations. (C) 2001 Academic Press.

Genomic organization of the CC chemokine MIP-3 alpha/CCL20/LARC/EXODUS/SCYA20, showing gene structure, splice variants, and chromosome localization

We describe the genomic organization of a recently identified CC chemokine, MIP3 alpha /CCL20 (HGMW-approved symbol SCYA20). The MIP-3 alpha /CCL20 gene was cloned and sequenced, revealing a four exon, three intron structure, and was localized by FISK analysis to 2q35-q36. Two distinct cDNAs were identified, encoding two forms of MIP-3 alpha /CCL20, Ala MLP-3 alpha /CCL20 and Ser MIP-3 alpha /CCL20, that differ by one amino acid at the predicted signal peptide cleavage site. Examination of the sequence around the boundary of intron 1 and exon 2 showed that use of alternative splice acceptor sites could give rise to Ata MIP-3 alpha /CCL20 or Ser MIP-3 alpha /CCL20. Both forms of MIP-3cr/CCL20 were chemically synthesized and tested for biological activity. Both flu antigen plus IL-a-activated CD4(+) and CD8(+) T lymphoblasts and cord blood-derived dendritic cells responded to Ser and Ala MIP-3 alpha /CCL20. T lymphocytes exposed only to IL-2 responded inconsistently, while no response was detected in naive T lymphocytes, monocytes, or neutrophils. The biological activity of Ser MIP-3 alpha /CCL20 and Ala MIP-3 alpha /CCL20 and the tissue-specific preference of different splice acceptor sites are not yet known. (C) 2001 Academic Press.

Dendritic cells rapidly undergo apoptosis in vitro following culture with activated CD4(+) V alpha 24 natural killer T cells expressing CD40L

Human V alpha 24 natural killer T (V alpha 24NKT) cells are activated by -glycosylceramide-pulsed dendritic cells (DCs) in a CDld-dependent and T-cell receptor-mediated manner. There are two major subpopulations of V alpha 24NKT cells, CD4(-) CD8(-) V alpha 24NKT and CD4(+) V alpha 24NKT cells. We have recently shown that activated CD4(-) CD8 V alpha 24NKT cells have cytotoxic activity against DCs, but knowledge of the molecules responsible for cytotoxicity of V alpha 24NKT cells is currently limited. We aimed to investigate whether CD4(+) V alpha 24NKT cells also have cytotoxic activity against DCs and to determine the mechanisms underlying any observed cytotoxic activity. We demonstrated that activated CD4(+) V alpha 24NKT cells [CD40 ligand (CD40L) -positive] have cytotoxic activity against DCs (strongly CD40-positive), but not against monocytes (weakly CD40-positive) or phytohaemagglutinin blast T cells (CD40-negative), and that apoptosis of DCs significantly contributes to the observed cytotoxicity. The apoptosis of DCs following culture with activated CD4(+) V alpha 24NKT cells, but not with resting CD4(+) V alpha 24NKT cells (CD40L-negative), was partially inhibited by anti-CD40L mAb, Direct ligation of CD40 on the DCs by the anti-CD40 antibody also induced apoptosis of DCs. Our results suggest that CD40-CD40L interaction plays an important role in the induction of apoptosis of DCs following culture with activated CD4+ Va24NKT cells. The apoptosis of DCs from normal donors. triggered by the CD40-CD40L interaction, may contribute to the homeostatic regulation of the normal human immune system, preventing the interminable activation of activated CD4(+) V alpha 24NKT cells by virtue of apoptosis of DCs.

Cationic drug pharmacokinetics in diseased livers determined by fibrosis index, hepatic protein content, microsomal activity, and nature of drug

The disposition kinetics of six cationic drugs in perfused diseased and normal rat livers were determined by multiple indicator dilution and related to the drug physicochemical properties and liver histopathology. A carbon tetrachloride (CCl4)induced acute hepatocellular injury model had a higher fibrosis index (FI), determined by computer-assisted image analysis, than did an alcohol-induced chronic hepatocellular injury model. The alcohol-treated group had the highest hepatic alpha(1)- acid glycoprotein, microsomal protein (MP), and cytochrome P450 (P450) concentrations. Various pharmacokinetic parameters could be related to the octanol-water partition coefficient (log P-app) of the drug as a surrogate for plasma membrane partition coefficient and affinity for MP or P450, the dependence being lower in the CCl4-treated group and higher in the alcohol-treated group relative to controls. Stepwise regression analysis showed that hepatic extraction ratio, permeability-surface area product, tissue-binding constant, intrinsic clearance, partition ratio of influx (k(in)) and efflux rate constant (k(out)), and k(in)/k(out) were related to physicochemical properties of drug (log P-app or pK(a)) and liver histopathology (FI, MP, or P450). In addition, hepatocyte organelle ion trapping of cationic drugs was evident in all groups. It is concluded that fibrosis-inducing hepatic disease effects on cationic drug disposition in the liver may be predicted from drug properties and liver histopathology.

Characterization of the retinoid orphan-related receptor-alpha coactivator binding interface: A structural basis for ligand-independent transcription

The retinoid orphan-related receptor-alpha (RORalpha) is a member of the ROR subfamily of orphan receptors and acts as a constitutive activator of transcription in the absence of exogenous ligands. To understand the basis of this activity, we constructed a homology model of Rill using the closely related TRbeta as a template. Molecular modeling suggested that bulky hydrophobic side chains occupy the RORa ligand cavity leaving a small but distinct cavity that may be involved in receptor stabilization. This model was subject to docking simulation with a receptor-interacting peptide from the steroid receptor coactivator, GR-interacting protein-1, which delineated a coactivator binding surface consisting of the signature motif spanning helices 3-5 and helix 12 [activation function 2 (AF2)]. Probing this surface with scanning alanine mutagenesis showed structural and functional equivalence between homologous residues of RORalpha and TRbeta. This was surprising (given that Rill is a ligand-independent activator, whereas TRbeta has an absolute requirement for ligand) and prompted us to use molecular modeling to identify differences between Rill and TRbeta in the way that the All helix interacts with the rest of the receptor. Modeling highlighted a nonconserved amino acid in helix 11 of RORa (Phe491) and a short-length of 3.10 helix at the N terminus of AF2 which we suggest i) ensures that AF2 is locked permanently in the holoconformation described for other liganded receptors and thus 2) enables ligand-independent recruitment of coactivators. Consistent with this, mutation of RORa Phe491 to either methionine or alanine (methionine is the homologous residue in TRbeta), reduced and ablated transcriptional activation and recruitment of coactivators, respectively. Furthermore, we were able to reconstitute transcriptional activity for both a deletion mutant of Ill lacking All and Phe491 Met, by overexpression of a GAL-AF2 fusion protein, demonstrating ligand-independent recruitment of AF2 and a role for Phe491 in recruiting AF2.

Biochemical characterization of two mutants of human pyruvate dehydrogenase, F205L and T231A of the E1 alpha subunit

Mutations in the E1alpha subunit of the pyruvate dehydrogenase multienzyme complex may result in congenital lactic acidosis, but little is known about the consequences of these mutations at the enzymatic level. Here we characterize two mutants (F205L and T231A) of human pyruvate dehydrogenase in vitro, using the enzyme expressed in Escherichia coli. Wild-type and mutant proteins were purified successfully and their kinetic parameters were measured. F205L shows impaired binding of the thiamin diphosphate cofactor, which may explain why patients carrying this mutation respond to high-dose vitamin B-1 therapy. T231A has very low activity and a greatly elevated K-m for pyruvate, and this combination of effects would be expected to result in severe lactic acidosis. The results lead to a better understanding of the consequences of these mutations on the functional and structural properties of the enzyme, which may lead to improved therapies for patients carrying these mutations.

Haemolytic activity of Actinobacillus actinomycetemcomitans strains on different blood types

Haemolytic activity of sixty nine Actinobacillus actinomycetemcomitans strains on different animal and human blood types was examined by using a trypticase soy agar supplemented with yeast extract (0.5%). Blood types used were: rabbit, sheep and human (A, Rh+; A, Rh-; B, Rh+; B, Rh-; O, Rh+; O, Rh-; AB, Rh+; AB, Rh- groups). Plates were inoculated and, incubated in microaerophilic conditions, at 37ºC, for 48 h. The haemolytic activity of the tested strains was characterized as alpha-haemolysis. Only two isolates were not haemolytic on all blood types (2.9%), two strains were haemolytic only on human blood (one strain on AB, Rh+ group and another one on A, Rh+ and AB, Rh+ groups). No specificity between haemolysin produced by the tested strains and blood type was observed.

Long term follow-up and patterns of response of ALT in patients with chronic hepatitis NANB/C treated with recombinant interferon-alpha

The response to interferon treatment in chronic hepatitis NANB/C has usually been classified as complete, partial or absent, according to the behavior of serum alanine aminotransferase (ALT). However, a more detailed observation of the enzymatic activity has shown that the patterns may be more complex. The aim of this study was to describe the long term follow-up and patterns of ALT response in patients with chronic hepatitis NANB/C treated with recombinant interferon-alpha. A follow-up of 6 months or more after interferon-a was achieved in 44 patients. We have classified the serum ALT responses into six patterns and the observed frequencies were as follows: I. Long term response = 9 (20.5%); II. Normalization followed by persistent relapse after IFN = 7 (15.9%); III. Normalization with transient relapse = 5 (11.9%); IV. Temporary normalization and relapse during IFN = 4 (9.1%); V. Partial response (more than 50% of ALT decrease) = 7 (15.9%); VI. No response = 12 (27.3%). In conclusion, ALT patterns vary widely during and after IFN treatment and can be classified in at least 6 types.

Chronic hepatitis C virus infections in Brazilian patients: association with genotypes, clinical parameters and response to long term alpha interferon therapy

The present study assessed the clinical significance of hepatitis C virus (HCV) genotypes and their influence on response to long term recombinant-interferon-alpha (r-IFN-a) therapy in Brazilian patients. One hundred and thirty samples from patients previously genotyped for the HCV and with histologically confirmed chronic hepatitis C (CH-C) were evaluated for clinical and epidemiological parameters (sex, age, time of HCV infection and transmission routes). No difference in disease activity, sex, age or mode and time of transmission were seen among patients infected with HCV types 1, 2 or 3. One hundred and thirteen of them were treated with 3 million units of r-IFN-a, 3 times a week for 12 months. Initial response (IR) was significantly better in patients with genotype 2 (100%) and 3 (46%) infections than in patients with genotype 1 (29%) (p < 0.005). Among subtypes, difference in IR was observed between 1b and 2 (p < 0.005), and between 1b and 3a (p < 0.05). Sustained response (SR) was observed in 12% for (sub)type 1a, 13% for 1b, 19% for 3a, and 40% for type 2; significant differences were found between 1b and 2 (p < 0.001), and between 1b and 3a (p < 0.05). Moreover, presence of cirrhosis was significantly associated with non response and response with relapse (p < 0.05). In conclusion, non-1 HCV genotype and lack of histological diagnosis of cirrhosis were the only baseline features associated with sustained response to treatment. These data indicate that HCV genotyping may have prognostic relevance in the responsiveness to r-IFN-a therapy in Brazilian patients with chronic HCV infection, as seen in other reports worldwide.

Adaptação cultural e contributo para a validação da escala petterns of activity measura - pain (POAM-P)

RESUMO: A dor lombar crónica (DLC) é uma das condições clínicas mais comuns e com elevados custos socioeconómicos no mundo ocidental. Estudos recentes indicam que os utentes com DLC apresentam diferentes padrões de atividade que influenciam os níveis de incapacidade funcional. Contudo, a evidência acerca destas associações é, ainda, limitada e inconclusiva. Em Portugal, não existe, do nosso conhecimento, nenhuma escala validada para a população portuguesa que meça estes padrões de atividade em utentes com DLC. Objetivos: Adaptar culturalmente a escala Patterns of Activity Measure – Pain (POAM-P) para a população portuguesa com dor lombar crónica inespecífica (DLCI) e contribuir para a sua validação. Metodologia: A versão original (inglesa) do POAM-P foi traduzida e adaptada para a língua portuguesa (POAM-P-VP) através de uma equipa multidisciplinar que incluiu tradutores, retrotradutores (cegos e independentes), peritos de diferentes áreas e utentes com DLCI, de acordo com as recomendações de linhas orientadoras atuais para este processo. A análise factorial e das propriedades psicométricas da POAM-P-VP contou com uma amostra de 132 utentes. A consistência interna foi analisada através do coeficiente alpha de Cronbach (α) e para a análise da fiabilidade teste-reteste recorreu-se ao coeficiente de correlação intraclasse (ICC:2,1). A análise da validade de construto convergente e discriminativa das componentes da POAM-P-VP foi conseguida através da aplicação da versão portuguesa da escala Tampa Scale of Kinesiophobia (TSK-13-VP), e recorrendo ao cálculo do coeficiente de Spearman. Todos os cálculos estatísticos foram realizados no software IBM SPSS Statistics (versão 20). Resultados: A análise factorial permitiu identificar três componentes da POAM-P-VP (evitamento, persistência excessiva e persistência consistente com a dor), sendo estruturalmente diferentes das subescalas do POAM-P original. Estas componentes apresentaram uma consistência interna boa a elevada. As componentes 1 e 2 apresentaram uma fiabilidade teste-reteste moderada a excelente, e a componente 3 uma fiabilidade teste-reteste pobre, limitando o seu poder de uso na prática clínica e em investigação. Relativamente à validade de construto, nenhuma das hipóteses estabelecidas no estudo apriori foram verificadas, não podendo aferir acerca da relação dos padrões de atividade com a cinesiofobia, medida pelo TSK-13-VP. Porém, a componente de evitamento da POAM-P-VP parece medir conteúdos partilhados com a TSK-13-VP (rs = 0.15, p<0.048). Conclusão: A adaptação e contributo para a validação da versão portuguesa da escala POAM-P constituiu um ponto de partida para a existência de um instrumento de medição de padrões de atividade de utentes portugueses com DLC, requerendo mais estudos para a sua validação. Apesar de algumas limitações, considera-se que este estudo é de grande importância para os fisioterapeutas e investigadores que buscam um maior conhecimento e efetividade das abordagens de intervenção em utentes com dor lombar crónica.-------------- ABSTRACT: Chronic low back pain (CLBP) is one of the most common clinical conditions as well as one with high economical costs within western countries. Recent studies have shown that patients with LBP present different patterns of activity which influence their levels of functional capacity. However, evidence on these associations is still limited and inconclusive. To our knowledge, there is in Portugal no valid scale for measuring these patterns of activity in CLBP patients. Purpose: Culturally adapt the Patterns of Activity Measure – Pain (POAM-P) scale to the Portuguese population with non-specific chronic low back pain (NSLBP) and contribute to its validation. Method: The original English version of POAM-P was blindly and independently translated, back translated and adapted to the Portuguese language (POAM-P-VP) by a multidisciplinary team of translators, experts from different fields, and patients with NSLBP, according to established guidelines for this process. Factorial and psychometric properties’ analysis of POAM-P-VP comprised a sample of 132 patients. The internal consistency was analyzed based on Cronbach's alpha-coefficient (α) and for test-retest reliability analysis the Intraclass Correlation Coefficient (ICC) was used. The analysis of convergent and discriminant construct validity of POAM-P-VP components was achieved through the use of the Portuguese version of the Tampa Scale of Kinesiophobia (TSK-13-VP), using the Spearman coefficient calculation. All statistical calculations were performed using IBM SPSS Statistics software (v.20). Results: The factor analysis allowed for the identification of three components of POAM-P-VP (avoidance, excessive persistence and pain-contingent persistence), structurally different from the original POAM-P subscales. These components demonstrated a good to high level of internal consistency. Components 1 and 2 demonstrated moderate to excellent test-retest reliability, whereas component 3 presented low test-retest reliability thus limiting its clinical and investigative use. With regard to construct validity, none of the previously established hypothesis was verified, therefore not making it possible to assess the relation between activity patterns and kinesiophobia, measured by TSK-13-VP. However, the avoidance component of POAM-P-VP seems to share measurable contents with TSK-13-VP (rs = 0.15, p<0.048). Conclusion: The adaptation and contribution to the validation of the Portuguese version of POAM-P scale, sets a starting point to the existence of a useful instrument for measuring activity patterns in Portuguese CLBP patients, requiring further studies towards its validation. Despite some limitations, this study is considered of high importance to physiotherapists as well as investigators in search of deeper knowledge and effective practical approaches on chronic low back pain patients.

Chemical composition and acaricidal activity of the leaf and fruit essential oils of Protium heptaphyllum (Aubl.) Marchand (Burseraceae)

Essential oils from leaves and fruits of Protiumheptaphyllum collected in Tamandaré beach Pernambuco/Brazil were analysed by GC/MS and tested for toxicity and repellent effect against the two spotted spider mite (Tetranychus urticae). The major constituent identified in the fruits was alpha-terpinene (47.6 %) whereas oil from leaf contained mainly sesquiterpenes such as 9-epi-caryophyllene (21.4 %), trans-isolongifolanone (10.7 %) and 14-hydroxi-9-epi-caryophyllene (16.7 %). The fruit oil was found to be more effective against the mite when compared to the leaf oil. Both showed mortality properties and oviposition deterrence in higher concentration (10 µl.l-1 air), but only the essential oil from fruits induced repellence on T. urticae.