Protection of mice from group A streptococcal infection by intranasal immunisation with a peptide vaccine that contains a conserved M protein B cell epitope and lacks a T cell autoepitope

Infection with group A streptococci (GAS) can lead to rheumatic fever (RF) and rheumatic heart disease (RHD) which are a major health concern particularly in indigenous populations worldwide, and especially in Australian Aboriginals. A primary route of GAS infection is via the upper respiratory tract, and therefore, a major goal of research is the development of a mucosal-based GAS vaccine, The majority of the research to date has focused on the GAS M protein since immunity to GAS is mediated by M protein type-specific opsonic antibodies. There are two major impediments to the development of a vaccine-the variability in M proteins and the potential for the induction of an autoimmune response. To develop a safe and broad-based vaccine, we have therefore focused on the GAS M protein conserved C-region, and have identified peptides, J8 and the closely related J8 peptide (J14), which may be important in protective immunity to GAS infection. Using a mucosal animal model system, our data have shown a high degree of throat GAS colonisation in B10.BR mice 24 h following intranasal immunisation with the mucosal adjuvant, cholera toxin B subunit (CTB), and/or diptheria toxoid (dT) carrier, or PBS alone, and challenge with the M1 GAS strain. However, GAS colonisation of the throat was significantly reduced following intranasal immunisation of mice with the vaccine candidate J8 conjugated to dT or J14-dT when administered with CTB. Moreover, J8-dT/CTB and J14-dT/CTB-immunised mice had a significantly higher survival when compared to CTB and PBS-immunised control mice. These data indicate that immunity to GAS infection can be evoked by intranasal immunisation with a GAS M protein C-region peptide vaccine that contains a protective B cell epitope and lacks a T cell autoepitope. (C) 2002 Published by Elsevier Science Ltd.

Differences in mouse strain influence leukocyte and immunoglobulin phenotype response to Porphyromonas gingivalis

This study examined the nature of the infiltrating cells in Porphyromonas gingivalis-induced lesions and immunoglobulins in the serum samples of BALB/c (H-2(d)), C57BL6 (H-2(b)), DBA/2J (H-2(d)) and CBA/CaH (H-2(k)) mice. Mice were immunized intraperitoneally with P. gingivalis outer membrane antigens or sham-immunized with phosphate-buffered saline followed by subcutaneous challenge with live organisms 1 week after the final immunization. The resulting skin abscesses were excised 7 days later, cryostat sections cut and an immunoperoxidase method used to detect the presence of CD4(+) and CD8(+) T-cell subsets, CD14(+) macrophages and CD19(+) B cells. Peroxidase positive neutrophils and IgG1- and IgG2a-producing plasma cells were also identified. Anti P. gingivalis IgG1 and IgG2a subclass antibodies were determined in serum obtained by cardiac puncture. Very few CD8(+) T cells and CD19(+) B cells were found in any of the lesions. The percentages of CD4(+) cells, CD14(+) cells and neutrophils were similar in lesions of immunized BALB/c and C57BL6 mice, with a trend towards a higher percentage of CD14(+) cells in sham-immunized mice. The percentage of CD14(+) cells was higher than that of CD4(+) cells in immunized compared with sham-immunized DBA/2J mice. The percentages of CD4(+) and CD14(+) cells predominated in immunized CBA/CaH mice and CD4(+) cells in sham-immunized CBA/CaH mice. The percentage of neutrophils in immunized CBA/CaH mice was significantly lower than that of CD14(+) cells and CD4(+) cells in sham-immunized mice. IgG1(+) plasma cells were more dominant than IgG2a(+) cells in immunized BALB/c, C57BL6 and DBA/2J mice, whereas IgG2a(+) plasma cells were more obvious in sham-immunized mice. IgG2a(+) plasma cells were predominant in immunized and sham-immunized CBA/CaH mice. In the serum, specific anti-P. gingivalis IgG2a antibody levels (Th1 response) were higher than IgG1 levels (Th2 response) in sham-immunized CBA/CaH and DBA/2J mice. In immunized BALB/c mice, IgG2a levels were lower than IgG1 levels, while IgG2a levels were higher in immunized C57BL6 mice. In conclusion, this study has shown differences in the proportion of infiltrating leukocytes and in the subclasses of immunoglobulin produced locally and systemically in response to P. gingivalis in different strains of mice, suggesting a degree of genetic control over the response to P. gingivalis.

The t-SNARE syntaxin 4 is regulated during macrophage activation to function in membrane traffic and cytokine secretion

Activation of macrophages with lipopolysaccharide (LPS) induces the rapid synthesis and secretion of proinflammatory cytokines, such as tumor necrosis factor (TNFalpha), for priming the immune response [1, 2]. TNFalpha plays a key role in inflammatory disease [3]; yet, little is known of the intracellular trafficking events leading to its secretion. In order to identify molecules involved in this secretory pathway, we asked whether any of the known trafficking proteins are regulated by LPS. We found that the levels of SNARE proteins were rapidly and significantly up- or downregulated during macrophage activation. A subset of t-SNAREs (Syntaxin 4/SNAP23/Munc18c) known to control regulated exocytosis in other cell types [4, 5] was substantially increased by LPS in a temporal pattern coinciding with peak TNFalpha secretion. Syntaxin 4 formed a complex with Munc18c at the cell surface of macrophages. Functional studies involving the introduction of Syntaxin 4 cDNA or peptides into macrophages implicate this t-SNARE in a rate-limiting step of TNFalpha secretion and in membrane ruffling during macrophage activation. We conclude that in macrophages, SNAREs are regulated in order to accommodate the rapid onset of cytokine secretion and for membrane traffic associated with the phenotypic changes of immune activation. This represents a novel regulatory role for SNAREs in regulated secretion and in macrophage-mediated host defense.

alpha-conotoxins PnIA and [A10L] PnIA stabilize different states of the alpha 7-L247T nicotinic acetylcholine receptor

The effects of the native alpha-conotoxin PnIA, its synthetic derivative [ A10L] PnIA and alanine scan derivatives of [ A10L] PnIA were investigated on chick wild type alpha7 and alpha7-L247T mutant nicotinic acetylcholine receptors (nAChRs) expressed in Xenopus oocytes. PnIA and [A10L] PnIA inhibited acetylcholine (ACh)-activated currents at wtalpha7 receptors with IC50 values of 349 and 168 nM, respectively. Rates of onset of inhibition were similar for PnIA and [ A10L] PnIA; however, the rate of recovery was slower for [ A10L] PnIA, indicating that the increased potency of [ A10L] PnIA at alpha7 receptors is conveyed by its slower rate of dissociation from the receptors. All the alanine mutants of [ A10L] PnIA inhibited ACh-activated currents at wtalpha7 receptors. Insertion of an alanine residue between position 5 and 13 and at position 15 significantly reduced the ability of [ A10L] PnIA to inhibit ACh-evoked currents. PnIA inhibited the non-desensitizing ACh-activated currents at alpha7-L247T receptors with an IC50 194 nM. In contrast, [ A10L] PnIA and the alanine mutants potentiated the ACh-activated current alpha7-L247T receptors and in addition [ A10L] PnIA acted as an agonist. PnIA stabilized the receptor in a state that is non-conducting in both the wild type and mutant receptors, whereas [ A10L] PnIA stabilized a state that is non-conducting in the wild type receptor and conducting in the alpha7-L247T mutant. These data indicate that the change of a single amino acid side-chain, at position 10, is sufficient to change the toxin specificity for receptor states in the alpha7-L247T mutant.

Asymmetric contribution of α and β subunits to the activation of αβ heteromeric glycine receptors

This study investigated the role of beta subunits in the activation of alphabeta heteromeric glycine receptor (GlyR) chloride channels recombinantly expressed in HEK293 cells. The approach involved incorporating mutations into corresponding positions in alpha and beta subunits and comparing their effects on receptor function. Although cysteine-substitution mutations to residues in the N-terminal half of the alpha subunit M2-M3 loop dramatically impaired the gating efficacy, the same mutations exerted little effect when incorporated into corresponding positions of the beta subunit. Furthermore, although the alpha subunit M2-M3 loop cysteines were modified by a cysteine-specific reagent, the corresponding beta subunit cysteines showed no evidence of reactivity. These observations suggest structural or functional differences between alpha and beta subunit M2-M3 loops. In addition, a threonine-->leucine mutation at the 9' position in the beta subunit M2 pore-lining domain dramatically increased the glycine sensitivity. By analogy with the effects of the same mutation in other ligand-gated ion channels, it was concluded that the mutation affected the GlyR activation mechanism. This supports the idea that the GlyR beta subunit is involved in receptor gating. In conclusion, this study demonstrates that beta subunits contribute to the activation of the GlyR, but that their involvement in this process is significantly different to that of the alpha subunit.

Modifying fish gelatin electrospun membranes for biomedical applications: cross- linking and swelling behavior

Development of suitable membranes is a fundamental requisite for tissue and biomedical engineering applications. This work presents fish gelatin random and aligned electrospun membranes cross-linked with glutaraldehyde (GA). It was observed that the fiber average diameter and the morphology is not influenced by the GA exposure time and presents fibers with an average diameter around 250 nm. Moreover, when the gelatin mats are immersed in a phosphate buffered saline solution (PBS), they can retain as much as 12 times its initial weight of solution almost instantaneously, but the material microstructure of the fiber mats changes from the characteristic fibrous to an almost spherical porous structure. Cross-linked gelatin electrospun fiber mats and films showed a water vapor permeability of 1.37 ± 0.02 and 0.13 ± 0.10 (g.mm)/(m2.h.kPa), respectively. Finally, the processing technique and cross-linking process does not inhibit MC-3T3-E1 cell adhesion. Preliminary cell culture results showed good cell adhesion and proliferation in the cross-linked random and aligned gelatin fiber mats.

Comparison of body fat content and distribution of familial amyloidotic polyneuropathy patients versus healthy subjects

The deposition of amyloid fibers at the peripheral nervous system can induce motor neuropathy in Familial Amiloidotic Polyneuropethy (FAP) patients. This produces progressive reductions in functional capacity. The only treatment for FAP is a liver transplant, followed by aggressive medication that can affect patients' metabolism. To our knowledge, there are no data on body fat distribution or comparison between healthy and FAP subjects, which may be important for clinical assessment and management of this disease. PURPOSE: To analyze body fat content and distribution between FAP patients and healthy subjects. METHODS: Body fat content and distribution were measured through Double Energy X-ray Densitometry (DXA) in two groups. Group 1 consisted of 43 Familial Amyloidotic Polyneuropathy patients (19 males, 32 + 8 Yrs, and 24 females, 37 + 5 yrs), who had liver transplant less than 2 months before. Group 2 consisted of 18 healthy subjects of similar age (8 males, 36 + 7 yrs, and 10 females, 39 + 5 yrs). RESULTS: Healthy subjects showed higher values than FAP patients for: BMI (24,2+2,3kg/m2 vs 22,3+3,8 kg/m2 respectively, p<0,05), % trunk BF (26,21+8,34kg vs 20,78+9,05kg respectively, p<0,05), % visceral BF (24,43+7,97% vs 19,21+9,30% respectively, p<0,05), % abdominal BF (26,63+8,51% vs 20,63+10,35% respectively, p<0,05) abdominal subcutaneous BF (0,533+0,421kg vs 0,353+0,257kg respectively, p=0,05), abdominal BF/BF ratio (0,09+0,02 vs 0,08+0,02 respectively, p<0,05) and abdominal BF/trunk BF ratio (0,19+0,03 vs 0,17+0,03 respectively, p<0,05). CONCLUSIONS: These results showed that FAP patients soon after liver transplantation exhibited a healthier body fat profile compared to controls. However, fat content and distribution varied widely in FAP subjects, suggesting an individualized approach for assessment and intervention rather than general guidelines. Future research is needed to investigate the long term consequences on body fat following liver transplant in this population.

Development and biological evaluation of API-ILs based on anti-bacterial and anti-fungal drugs

In recent years Ionic Liquids (ILs) are being applied in life sciences. ILs are being produce with active pharmaceutical drugs (API) as they can reduce polymorphism and drug solubility problems [1] Also ILs are being applied as a drug delivery device in innovative therapies What is appealing in ILs is the ILs building up platform, the counter-ion can be carefully chosen in order to avoid undesirable side effects or to give innovative therapies in which two active ions are paired. This work shows ILs based on ampicillin (an anti-bacterial agent) and ILs based on Amphotericin B. Also we show studies that indicate that ILs based on Ampicillin could reverse resistance in some bacteria. The ILs produced in this work were synthetized by the neutralization method described in Ferraz et. al. [2] Ampicillin anion was combined with the following organic cations 1-ethyl-3-methylimidazolium, [EMIM]; 1-hydroxy-ethyl-3-methylimidazolium, [C2OHMIM]; choline, [cholin]; tetraethylammonium, [TEA]; cetylpyridinium, [C16pyr] and trihexyltetradecylphosphonium, [P6,6,6,14]. Amphotericin B was combined with [C16pyr], [cholin] and 1-metohyethyl-3-methylimidazolium, [C3OMIM]. The ILs-APIs based on ampicillin[2] were tested against sensitive Gram-negative bacteria Escherichia coli ATCC 25922 and Klebsiella pneumonia (clinical isolated), as well as on Gram positive Staphylococcus Aureus ATCC 25923, Staphylococcus epidermidis and Enterococcus faecalis. The arising resistance developed by bacteria to antibiotics is a serious public health threat and needs new and urgent measures. We study the bacterial activity of these compounds against a panel of resistant bacteria (clinical isolated strains): E. coli CTX M9, E. coli TEM CTX M9, E. coli TEM1, E. coli CTX M2, E. coli AmpC Mox2. In this work we demonstrate that is possible to produce ILs from anti-bacterial and anti-fungal compounds. We show here that the new ILs can reverse the bacteria resistance. With the careful choice of the organic cation, it is possible to create important biological and physic-chemical properties. This work also shows that the ion-pair is fundamental in ampicillin mechanism of action.

Nutritional status influences generic and disease-specific quality of life measures in haemodialysis patients

Background: Poor nutritional status and worse health-related quality of life (QoL) have been reported in haemodialysis (HD) patients. The utilization of generic and disease specific QoL questionnaires in the same population may provide a better understanding of the significance of nutrition in QoL dimensions. Objective: To assess nutritional status by easy to use parameters and to evaluate the potential relationship with QoL measured by generic and disease specific questionnaires. Methods: Nutritional status was assessed by subjective global assessment adapted to renal patients (SGA), body mass index (BMI), nutritional intake and appetite. QoL was assessed by the generic EuroQoL and disease specific Kidney Disease Quality of Life-Short Form (KDQoL-SF) questionnaires. Results: The study comprised 130 patients of both genders, mean age 62.7 ± 14.7 years. The prevalence of undernutrition ranged from 3.1% by BMI ≤ 18.5 kg/m2 to 75.4% for patients below energy and protein intake recommendations. With the exception of BMI classification, undernourished patients had worse scores in nearly all QoL dimensions (EuroQoL and KDQoL-SF), a pattern which was dominantly maintained when adjusted for demographics and disease-related variables. Overweight/obese patients (BMI ≥ 25) also had worse scores in some QoL dimensions, but after adjustment the pattern was maintained only in the symptoms and problems dimension of KDQoL-SF (p = 0.011). Conclusion: Our study reveals that even in mildly undernourished HD patients, nutritional status has a significant impact in several QoL dimensions. The questionnaires used provided different, almost complementary perspectives, yet for daily practice EuroQoL is simpler. Assuring a good nutritional status, may positively influence QoL.

Association of FTO and PPARG polymorphisms with obesity in Portuguese women

Purpose: We evaluated the association between risk of obesity in the Portuguese population and two obesity-related single-nucleotide gene polymorphisms: fat-mass and obesity-associated (FTO) rs9939609 and peroxisome proliferator-activated receptor gamma (PPARG) rs1801282. Patients and methods: A total of 194 Portuguese premenopausal female Caucasians aged between 18 and 50 years (95 with body mass index [BMI] ≥30 g/m2, 99 controls with BMI 18.5–24.9 kg/m2) participated in this study. The association of the single-nucleotide polymorphisms with obesity was determined by odds ratio calculation with 95% confidence intervals. Results: Significant differences in allelic expression of FTO rs9939609 (P<0.05) were found between control and case groups, indicating a 2.5-higher risk for obesity in the presence of both risk alleles when comparing the control group with the entire obese group. A fourfold-higher risk was found for subjects with class III obesity compared to those with classes I and II. No significant differences in BMI were found between the control and case groups for PPARG rs1801282 (P>0.05). Conclusion: For the first time, a study involving an adult Portuguese population shows that individuals harboring both risk alleles in the FTO gene locus are at higher risk for obesity, which is in agreement to what has been reported for other European populations.

Magnesium, insulin resistance and body composition in healthy postmenopausal women

Objective: This study was conducted to determine the association between magnesium (Mg), body composition and insulin resistance in 136 sedentary postmenopausal women, 50 to 77 years of age. Methods: Diabetics, hypertensives and women on hormonal replacement therapy were excluded and the remaining 74 were divided according to BMI≥25 (obese: OG) and BMI<25 kg/m2 (non-obese: NOG). Nutritional data disclosed that intakes were high for protein and saturated fat, low for carbohydrates, polyunsaturated fat and Mg and normal for the other nutrients, according to recommended dietary allowances (RDA). Mg values in red blood cells (RBC-Mg) and plasma (P-Mg), were determined, as were fasting glucose, and insulin levels, Homeostasis Model Assessment (HOMA), body mass index (BMI), body fat percent (BF %), abdominal fat (AF) and free fat mass (FFM). Results: RBC-Mg values were low in both groups when compared with normal values. There were significant differences in body composition parameters, HOMA and insulin levels, with higher basal insulin levels in OG. RBC-Mg was directly correlated with insulin, HOMA and FFM in both groups, according to Pearson correlations. HOMA in OG was also directly correlated with BMI, FFM and AF. In NOG, HOMA was only correlated with FFM. The low RBC-Mg levels observed were probably due to low Mg intake and to deregulation of factors that control Mg homeostasis during menopause. Conclusions: Both Mg deficit and obesity may independently lead to a higher risk for insulin resistance and cardiovascular disease.

Sei whale (Balaenoptera borealis) ecology and management in the North Atlantic

Tese de Doutoramento em Ciências do Mar, especialidade em Ecologia Marinha.

Synthesis, structural characterization and leishmanicidal activity evaluation of ferrocenyl N-heterocyclic compounds

Agência Financiadora: FCT - PTDC/QUI/72656/2006 ; SFRH/BPD/27454/2006; SFRH/BPD/44082/2008; SFRH/BPD/41138/2007

Association between tooth loss and obesity in Brazilian adults: a population-based study

OBJECTIVE: To examine the association between tooth loss and general and central obesity among adults. METHODS: Population-based cross-sectional study with 1,720 adults aged 20 to 59 years from Florianópolis, Southern Brazil. Home interviews were performed and anthropometric measures were taken. Information on sociodemographic data, self-reported diabetes, self-reported number of teeth, central obesity (waist circumference [WC] > 88 cm in women and > 102 cm in men) and general obesity (body mass index [BMI] ≥ 30 kg/m²) was collected. We used multivariable Poisson regression models to assess the association between general and central obesity and tooth loss after controlling for confounders. We also performed simple and multiple linear regressions by using BMI and WC as continuous variables. Interaction between age and tooth loss was also assessed. RESULTS: The mean BMI was 25.9 kg/m² (95%CI 25.6;26.2) in men and 25.4 kg/m2 (95%CI 25.0;25.7) in women. The mean WC was 79.3 cm (95%CI 78.4;80.1) in men and 88.4 cm (95%CI 87.6;89.2) in women. A positive association was found between the presence of less than 10 teeth in at least one arch and increased mean BMI and WC after adjusting for education level, self-reported diabetes, gender and monthly per capita income. However, this association was lost when the variable age was included in the model. The prevalence of general obesity was 50% higher in those with less than 10 teeth in at least one arch when compared with those with 10 or more teeth in both arches after adjusting for education level, self-reported diabetes and monthly per capita family income. However, the statistical significance was lost after controlling for age. CONCLUSIONS: Obesity was associated with number of teeth, though it depended on the participants' age groups.

Size at birth and blood pressure in young adults: findings from a Brazilian birth cohort study

^len^lpt^aOBJETIVO: Descrever a prevalência de pressão arterial limítrofe (PAL) e hipertensão (HT) entre adultos jovens e avaliar a associação entre tamanho ao nascer e PAL/HT. MÉTODOS: Dados foram coletados do primeiro estudo brasileiro de coorte de nascimentos em Ribeirão Preto (sudeste do Brasil), iniciado em 1978/79. De 6.827 recém-nascidos de parto único hospitalar, 2.060 foram avaliados aos 23/25 anos. Foram realizadas coleta de sangue, avaliação antropométrica e obtidas informações sobre ocupação, escolaridade, hábitos de vida e doenças crônicas. Pressão arterial (PA) foi classificada em: 1) PAL: PA sistólica (PAS) ≥ 130 e < 140 mm Hg e/ou PA diastólica (PAD) ≥ 85 e < 90 mmHg; 2) HT: PAS ≥ 140 e/ou PAD ≥ 90 mm Hg. Foi aplicado modelo de regressão logística politômica. RESULTADOS: A prevalência de PAL foi de 13,5% (homens 23,2%) e a de HT, 9,5% (homens 17,7%). PAL foi independentemente associada com sexo masculino (RR 8,84; IC95%: 6,09;12,82), comprimento ao nascer ≥ 50 cm (RR 1,97; 1,04; 3,73), índice de massa corporal (IMC) ≥ 30 kg/m² (RR 3,23; 2,02; 5,15) e circunferência de cintura alterada (RR 1,61; 1,13;2,29), enquanto HT associou-se com sexo masculino (RR 15,18; 8,92;25,81), IMC ≥ 30 kg/m² (RR 3,68; 2,23;6,06), circunferência de cintura alterada (RR 2,68; 1,77;4,05) e glicemia elevada (RR 2,55; 1,27;5,10), mas não com comprimento ao nascer. CONCLUSÕES: As prevalências de PAL e HT entre os adultos jovens dessa coorte foram maiores em homens que em mulheres. Maior comprimento ao nascer foi associado com PAL, mas não com HT, enquanto peso ao nascer não foi associado com PAL ou HT. Fatores de risco do adulto explicaram a maioria dos aumentos de PAL ou HT.^les^aOBJETIVO: Describir la prevalencia de presión arterial limítrofe (PAL) e hipertensión (HT) entre adultos jóvenes y evaluar la asociación entre tamaño al nacer y PAL/HT. MÉTODOS: : Los datos fueron colectados en el primer estudio de cohorte de nacimientos brasileño en Ribeirao Preto (sureste de Brasil), iniciado en 1978/79. De 6.827 recién nacidos de parto único hospitalario, 2.060 fueron evaluados a los 23/25 años. Se realizaron colecta de sangre, evaluación antropométrica y obtenidas informaciones sobre ocupación, escolaridad, hábitos de vida y enfermedades crónicas. Presión arterial (PA) fue clasificada en: 1) PAL: PA sistólica (PAS) ≥ 130 y < 140 mm Hg y/o PA diastólica (PAD) ≥ 85 y < 90 mm Hg; 2) HT: PAS ≥ 140 y/o PAD ≥ 90 mm Hg. Se aplicó modelo de regresión logística politómica. RESULTADOS: La prevalencia de PAL fue de 13,5% (hombres 23,2%) y la de HT, 9,5% (hombres 17,7%). PAL fue independientemente asociada con sexo masculino (Riesgo Relativo - RR) 8,84; 95%IC: 6,09;12,82), estatura al nacer ≥ 50 cm (RR 1,97; 1,04; 3,73), índice de masa corporal (IMC) ≥ 30 kg/m2 (RR 3,23; 2,02; 5,15) y circunferencia de cintura alterada (RR 1,61; 1,13;2,29), mientras el HT se asoció con sexo masculino (RR 15,18; 8,92;25,81), IMC ≥ 30 kg/m2 (RR 3,68; 2,23;6,06), circunferencia de cintura alterada (RR 2,68; 1,77;4,05) y glicemia elevada (RR 2,55; 1,27;5,10), pero no con estatura al nacer. CONCLUSIONES: Las prevalencias de PAL y HT entre los adultos jóvenes de la cohorte fueron mayores en hombres que en mujeres. Mayor estatura al nacer fue asociado con PAL, pero no con HT, mientras que el peso al nacer no estuvo asociado con PAL o HT. Factores de riesgo de adulto explicaron la mayoría de los aumentos de PAL o HT.