Enhancing the immunogenicity and modulating the fine epitope recognition of antisera to a helical group A streptococcal peptide vaccine candidate from the M protein using lipid-core peptide technology

A conserved helical peptide vaccine candidate from the M protein of group A streptococci, p145, has been described. Minimal epitopes within p145 have been defined and an epitope recognized by protective antibodies, but not by autoreactive T cells, has been identified. When administered to mice, p145 has low immunogenicity. Many boosts of peptide are required to achieve a high antibody titre (> 12 800). To attempt to overcome this low immunogenicity, lipid-core peptide technology was employed. Lipid-core peptides (LCP) consist of an oligomeric polylysine core, with multiple copies of the peptide of choice, conjugated to a series of lipoamino acids, which acts as an anchor for the antigen. Seven different LCP constructs based on the p145 peptide sequence were synthesized (LCP1-->LCP7) and the immunogenicity of the compounds examined. The most immunogenic constructs contained the longest alkyl side-chains. The number of lipoamino acids in the constructs affected the immunogenicity and spacing between the alkyl side-chains increased immunogenicity. An increase in immunogenicity (enzyme-linked immunosorbent assay (ELISA) titres) of up to 100-fold was demonstrated using this technology and some constructs without adjuvant were more immunogenic than p145 administered with complete Freund's adjuvant (CFA). The fine specificity of the induced antibody response differed for the different constructs but one construct, LCP4, induced antibodies of identical fine specificity to those found in endemic human serum. Opsonic activity of LCP4 antisera was more than double that of p145 antisera. These data show the potential for LCP technology to both enhance immunogenicity of complex peptides and to focus the immune response towards or away from critical epitopes.

The JK three-product cyclone - performance and potential applications

In spite of their wide application in comminution circuits, hydrocyclones have at least one significant disadvantage in that their operation inherently tends to return the fine denser liberated minerals to the grinding mill. This results in unnecessary overgrinding which adds to the milling cost and can adversely affect the efficiency of downstream processes. In an attempt to solve this problem, a three-product cyclone has been developed at the Julius Kruttschnitt Mineral Research Centre (JKMRC) to generate a second overflow in which the fine dense liberated minerals can be selectively concentrated for further treatment. In this paper, the design and operation of the three-product cyclone are described. The influence of the length of the second vortex finder on the performance of a 150-mm unit treating a mixture of magnetite and silica is investigated. Conventional cyclone tests were also conducted under similar conditions. Using the operational performance data of the three-product and conventional cyclones, it is shown that by optimising the length of the second vortex finder, the amount of fine dense mineral particles that reports to the three-product cyclone underflow can be reduced. In addition, the three-product cyclone can be used to generate middlings stream that may be more suitable for flash flotation than the conventional cyclone underflow, or alternatively, could be classified with a microscreen to separate the valuables from the gangue. At the same time, a fines stream having similar properties to those of the conventional overflow can be obtained. Hence, if the middlings stream was used as feed for flash flotation or microscreening, the fines stream could be used in lieu of the conventional overflow without compromising the feed requirements for the conventional flotation circuit. Some of the other potential applications of the new cyclone are described. (C) 2003 Elsevier Science B.V. All rights reserved.

Immunogenicity of recombinant HBsAg/HCV particles in mice pre-immunised with hepatitis B virus-specific vaccine

Due to their spatial structure virus-like particles (VLPs) generally induce effective immune responses. VLPs derived from the small envelope protein (HBsAg-S) of hepatitis B virus (HBV) comprise the HBV vaccine. Modified HBsAs-S VLPs, carrying the immunodominant hypervariable region (HVR1) of the hepatitis C virus (HCV) envelope protein E2 within the exposed 'a'-determinant region (HBsAg/HVR1-VLPs), elicited HVR1-specific antibodies in mice. A high percentage of the human population is positive for anti-HBsAg antibodies (anti-HBs), either through vaccination or natural infection. We, therefore, determined if pre-existing anti-HBs could influence immunisation with modified VLPs. Mice were immunised with a commercial HBV vaccine, monitored to ensure an anti-HBs response, then immunised with HBsAg/HVR1-VLPs. The resulting anti-HVR1 antibody titre was similar in mice with or without pre-existing anti-HBs. This suggests that HBsAg/HVR1-VLPs induce a primary immune response to HVR1 in anti-HBs positive mice and, hence, they may be used successfully in individuals already immunised with the HBV vaccine. (C) 2003 Elsevier Science Ltd. All rights reserved.

Carboxy-fluorescein diacetate, succinimidyl ester labeled papillomavirus virus-like particles fluoresce after internalization and interact with heparan sulfate for binding and entry

Human papillomaviruses (HPVs) infect epithelial cells and are associated with genital carcinoma. Most epithelial cell lines express cell-surface glycosaminoglycans (GAGs) usually found attached to the protein core of proteoglycans. Our aim was to study how GAGs influenced HPV entry. Using a human keratinocyte cell line (HaCaT), preincubation of HPV virus-like particles (VLPs) with GAGs showed a dose-dependent inhibition of binding. The IC50 (50% inhibition) was only 0.5 mug/ml for heparin, 1 mug/ml for dextran sulfate, and 5-10 mug/ml for heparan sulfate from mucosal origin. Mutated chinese hamster ovary (CHO) cell lines lacking heparan sulfate or all GAGs were unable to bind HPV VLPs. Here we also report a method to study internalization by using VLPs labeled with carboxy-fluorescein diacetate, succinimidyl ester, a fluorochrome that is only activated after cell entry. Pretreatment of labeled HPV VLPs with heparin inhibited uptake, suggesting a primary interaction between HPV and cell-surface heparan sulfate. (C) 2003 Elsevier Science (USA). All rights reserved.

Ultra-sonografia intra-oral e transcutânea cervical no diagnóstico diferencial de celulite e abscessos periamigdalianos

OBJETIVOS: O objetivo deste estudo é determinar a especificidade, sensibilidade e a acurácia da ultra-sonografia (USG) intra-oral e transcutânea no diagnóstico de celulite e abscesso periamigdalianos. FORMA DE ESTUDO: Clínico Prospectivo. MATERIAL E MÉTODO: Trinta e nove pacientes foram atendidos no pronto-socorro de otorrinolaringologia do Hospital das Clínicas da Faculdade de Medicina da Universidade de São Paulo com diagnóstico clínico de celulite ou abscesso periamigdaliano. Em todos os pacientes, após a avaliação inicial, foram realizadas ultra-sonografias intra-oral e transcutânea. RESULTADOS: O USG intra-oral foi realizado em 35 casos e demonstrou sensibilidade de 95,2%, especificidade de 78,5% e a acurácia de 86,9%. A USG transcutânea foi factível em todos os 39 pacientes e diagnosticou abscesso periamigdaliano em 53,8% dos pacientes. A sensibilidade foi de 80%, a especificidade de 92,8% e a acurácia de 84,5%. CONCLUSÕES: O USG intra-oral foi bastante sensível no diagnóstico de abscessos periamigdalianos. O USG transcutâneo obteve especificidade superior ao intra-oral. Porém, quando o USG transcutâneo foi realizado em pacientes com trismo, este diagnosticou todos os abscessos periamigdalianos, já que se tratava de coleções grandes, comuns em pacientes com trismo. Estes exames tiveram acurácia semelhantes.

Ototoxicidade e otoproteção em orelha interna de cobaias utilizando gentamicina e amicacina: aspectos ultra-estruturais e funcionais

A ototoxicidade ainda é um desafio para medicina. A descoberta dos mecanismos endógenos autoprotetores das células ciliadas externas associados a métodos de avaliação funcional e ultra-estrutural das mesmas abriu nova perspectiva no entendimento e controle destes mecanismos. OBJETIVO: O trabalho objetivou determinar se subdoses de gentamicina protegia contra ototoxicidade da amicacina baseado nestes mecanismos e determinar se a amplitude das emissões otoacústicas teria correlação com grau de integridade das células ciliadas. MATERIAL E MÉTODO: Estudo experimental. Utilizando 31 cobaias, administrou-se soro fisiológico, gentamicina e amicacina, isoladamente e associadas, via intramuscular, por 12, 30 e 42 dias. Pesquisa de emissões otoacústicas foi realizada no início e final do experimento, comparado com estudo da integridade coclear, por microscopia eletrônica. RESULTADOS: Subdoses de gentamicina não protegeram a orelha interna contra toxicidade da amicacina; diminuições da amplitude das emissões otoacústicas apresentaram forte correlação com aumento de lesões das células ciliadas. CONCLUSÃO: Os achados contribuem para o entendimento dos mecanismos de ototoxicidade e otoproteção da orelha interna. A determinação da correlação entre amplitude de emissões e integridade celular tem grande importância no acompanhamento das lesões de células ciliadas, com possível aplicação no monitoramento de ototoxicidade por drogas em humanos.

Durability performance of concrete made with fine recycled concrete aggregates

Fine recycled aggregates are seen as the last choice in recycling for concrete production. Many references quote their detrimental influence on the most important characteristics of concrete: compressive and tensile strength; modulus of elasticity; water absorption; shrinkage: carbonation and chloride penetration. These two last characteristics are fundamental in terms of the long-term durability of reinforced or prestressed concrete. In the experimental research carried out at IST, part of which has already been published, different concrete mixes (with increasing rates of substitution of fine natural aggregates sand - with fine recycled aggregates from crushed concrete) were prepared and tested. The results were then compared with those for a reference concrete with exactly the same composition and grading curve, but with no recycled aggregates. This paper presents the main results of this research for water absorption by immersion and capillarity, chloride penetration (by means of the chloride migration coefficient), and carbonation resistance, drawing some conclusions on the feasibility of using this type of aggregate in structural concrete, while taking into account any ensuing obvious positive environmental impact.

Environmental life cycle assessment of concrete made with fine recycled concrete aggregates

The majority of worldwide structures use concrete as its main material. This happens because concrete is economically feasible, due to its undemanding production technology and case Of use. However, it is widely recognized that concrete production has a strong environmental impact in the planet. Natural aggregates use is one of the most important problems of concrete production nowadays, since they are obtained from limited, and in some countries scarce, resources. In Portugal, although there are enough stone quarries to cover coarse aggregates needs for several more years, Supplies of fine aggregates are becoming scarcer, especially in the northern part of the country. On the other hand, as concrete structures' life cycle comes to an end, an urgent need emerges to establish technically and economically viable solutions for demolition debris, other than for use as road base and quarry fill. This paper presents a partial life cycle assessment (LCA) of concrete made with fine recycled concrete aggregates performed with EcoConcrete tool. EcoConcrete is a tailor-made, interactive, learning and communications tool promoted by the Joint Project Group (JPG) on the LCA of concrete, to qualify and quantify the overall environment impact of concrete products. It consists of an interactive Excel-spreadsheet in which several environmental inputs (material quantities, distances from origin to production Site, production processes) and outputs (material, energy, emissions to air, water, soil or waste) are collected in a life cycle inventory, and are then processed to determine the environmental impact (assessment) of the analysed concrete, in terms of ozone layer depletion, smog or "greenhouse" effect.

Fraccionamento do efluente da indústria da cortiça por ultra-nanofiltração: caracterização e estudo da sua biodegradabilidade

O efluente da Indústria da Cortiça apresenta elevados teores de carga orgânica e de compostos fenólicos, nomeadamente teninos, com baixa biodegradabilidade e significativa toxicidade, o que dificulta o seu tratamento. O objectivo deste trabalho é o estudo da biodegradabilidade de diferentes fracções do efluente da cortiça, na perspectiva de contribuir para viabilizar o seu tratamento. Biológico, em simultâneo com a sua valorização, pela recuperação do seu conteúdo em taninos. O fraccionamento foi realizado através de processos de separação por membranas, nomeadamente por ultrafiltração e nanofiltração, de Limite de Exclusão Molecular (MWCO) compreendido entre 0,125-91kDa. O efluente e as fracções permeadas obtidas foram analisadas em termos de conteúdo orgânico (COT, CBO, CQO), fenóis, taninos, cor, pH e condutividade. A caracterização do efluente revelou um elevado conteúdo orgânico (2285-2604 mg de CQO/L, 670,5-1056,8 mg C/L, 1000-1225 mg CBO/L), biodegradabilidade relativamente baixa (0,44-0,47 para CBO/CQO; k de 0,25-0,24 d-1) e elevado conteúdo de fenóis (0,36-0,41 g de ácido tânico/L) e de taninos (0,25-0,27 g ácido tânico/L). O fraccionamento do efluente permitiu concluir uma diminuição do seu conteúdo poluente nos permeados com a diminuição do MWCO das membranas utilizadas. A diminuição simultânea de matéria orgânica e fenóis foi particularmente significativa no permeado da membrana de MWCO de 3,8 kDa que, por outro lado, apresenta uma melhoria significativa para o índice de biodegradabilidade e k (0,83;0,38-0,44 d-1), o que significa que este permeado apresenta já viabilidade para sofrer um processo biológico para remoção de matéria orgânica e o concentrado apresenta um elevado potencial para recuperação de taninos. Nas fracções de MWCO inferior a 3,8 kDa, a diminuição de matéria orgânica é muito significativa, com um aumento da sua fracção biodegradável. Embora os resultados não sejam totalmente conclusivos em termos de biodegradabilidade, esta fracção apresenta já características adequadas para reutilização como água de processo.

Optimização do processo de extracção por ultra-sons

Neste trabalho foi estudado o método de preparação de amostras baseado numa extracção ácida assistida por ultra-sons para a quantificação de metais pesados (Pb, Ni, Cd e Cr), em amostras de solo. Usou-se a espectrometria de absorção atómica com câmara de grafite (GFAAS) para quantificar os metais nestas amostras recolhidas em diferentes locais da cidade de Lisboa. A optimização dos parâmetros que influenciam a extracção dos metais por ultra-sons foi realizada a cinco variáveis: quantidade de amostra, volume de extractor (água-régia), tempo de sonificação, potência de sonificação e ciclo de sonificação. A eficiência do método foi testada por comparação dos resultados com os obtidos pela digestão ácida “tradicional” em banho de areia. A optimização do processo foi realizada para o metal chumbo. Os resultados analíticos obtidos por ambos os métodos apresentaram diferenças significativas. A extracção de chumbo das amostras, por ultra-sons, apresentou uma eficiência entre 65 a 78% quando comparada com o método “tradicional”. A recuperação de cádmio apresentou percentagens entre 36 e 90%. No caso do níquel e crómio, concluiu-se haver necessidade de optimizar o processo para cada um dos metais, pois apresentaram uma baixa taxa de recuperação.

Valores de predição da avaliação neurológica e ultra-sonográfica cerebral neonatal em relação ao desenvolvimento de prematuros de muito baixo peso

OBJETIVO: Verificar os valores de predição de dois exames (neurológico e ultra-sonográfico) realizados no período neonatal, em relação ao desenvolvimento neuromotor e cognitivo de prematuros. MÉTODO: Foram estudados 70 prematuros com peso de nascimento inferior a 1.500g. A média do peso de nascimento e idade gestacional foram, respectivamente, 1.185g (DP:180) e 32,2 semanas (DP:1,4). Foi realizada avaliação neurológica pelo método de Dubowitz e Dubowitz e ultra-sonografia cerebral por volta de 40 semanas de idade corrigida. Após a alta do berçário estas crianças foram acompanhadas no ambulatório de seguimento. RESULTADOS: Com idade média de 21 meses (DP: 4,9) corrigidos para a prematuridade, 25,7% das crianças apresentaram comprometimento neuromotor, e 20,3%, alteração cognitiva. O exame neurológico neonatal foi mais sensível que específico em relação à alteração neuromotora (sensibilidade:77,7%, especificidade:57,6%), e cognitiva (sensibilidade: 78,5%, especificidade: 56,4%), e apresentou baixo valor de predição tanto para alteração neuromotora (38,9%) quanto cognitiva (31,4%). A ultra-sonografia apresentou elevada especificidade tanto para o desenvolvimento neuromotor (92,3%) quanto cognitivo (89,1%). O valor preditivo da ultra-sonografia foi satisfatório para anormalidades neuromotoras (69,2%), porém baixo para as alterações cognitivas (50,0%). CONCLUSÃO: A associação dos dois exames com resultados anormais apresentou valores de predição mais elevados para as anormalidades nas duas áreas do desenvolvimento.

Assessment of exposure to airborne ultrafine particles in the urban environment of Lisbon, Portugal

The aim of this study was the assessment of exposure to ultrafine in the urban environment of Lisbon, Portugal, due to automobile traffic, and consisted of the determination of deposited alveolar surface area in an avenue leading to the town center during late spring. This study revealed differentiated patterns for weekdays and weekends, which could be related with the fluxes of automobile traffic. During a typical week, ultrafine particles alveolar deposited surface area varied between 35.0 and 89.2 μm2/cm3, which is comparable with levels reported for other towns such in Germany and the United States. These measurements were also complemented by measuring the electrical mobility diameter (varying from 18.3 to 128.3 nm) and number of particles that showed higher values than those previously reported for Madrid and Brisbane. Also, electron microscopy showed that the collected particles were composed of carbonaceous agglomerates, typical of particles emitted by the exhaustion of diesel vehicles. Implications: The approach of this study considers the measurement of surface deposited alveolar area of particles in the outdoor urban environment of Lisbon, Portugal. This type of measurements has not been done so far. Only particulate matter with aerodynamic diameters <2.5 (PM2.5) and >10 (PM10) μm have been measured in outdoor environments and the levels found cannot be found responsible for all the observed health effects. Therefore, the exposure to nano- and ultrafine particles has not been assessed systematically, and several authors consider this as a real knowledge gap and claim for data such as these that will allow for deriving better and more comprehensive epidemiologic studies. Nanoparticle surface area monitor (NSAM) equipments are recent ones and their use has been limited to indoor atmospheres. However, as this study shows, NSAM is a very powerful tool for outdoor environments also. As most lung diseases are, in fact, related to deposition of the alveolar region of the lung, the metric used in this study is the ideal one.

Comparison of indoor and outdoor fungi and particles in poultry units

A descriptive study was developed in order to compare indoor and outdoor air contamination caused by fungi and particles in seven poultry units. Twenty eight air samples of 25 litters were collected through the impaction method on malt extract agar. Air sampling and particles concentration measurement were done in the interior and also outside premises of the poultries’ pavilions. Regarding the fungal load in the air, indoor concentration of mold was higher than outside air in six poultry units. Twenty eight species / genera of fungi were identified indoor, being Scopulariopsis brevicaulis (40.5%) the most commonly isolated species and Rhizopus sp. (30.0%) the most commonly isolated genus. Concerning outdoor, eighteen species/genera of fungi were isolated, being Scopulariopsis brevicaulis (62.6%) also the most isolated. All the poultry farms analyzed presented indoor fungi different from the ones identified outdoors. Regarding particles’ contamination, PM2.5, PM5.0 and PM10 had a statistically significant difference (Mann-Whitney U test) between the inside and outside of the pavilions, with the inside more contaminated (p=.006; p=.005; p=.005, respectively). The analyzed poultry units are potential reservoirs of substantial amounts of fungi and particles and could therefore free them in the atmospheric air. The developed study showed that indoor air was more contaminated than outdoors, and this can result in emission of potentially pathogenic fungi and particles via aerosols from poultry units to the environment, which may post a considerable risk to public health and contribute to environmental pollution.

Phase diagrams of particles with dissimilar patches: X-junctions and Y-junctions

We use Wertheim's first-order perturbation theory to investigate the phase behaviour and the structure of coexisting fluid phases for a model of patchy particles with dissimilar patches (two patches of type A and f(B) patches of type B). A patch of type alpha = {A, B} can bond to a patch of type beta = {A, B} in a volume nu(alpha beta), thereby decreasing the internal energy by epsilon(alpha beta). We analyse the range of model parameters where AB bonds, or Y-junctions, are energetically disfavoured (epsilon(AB) < epsilon(AA)/2) but entropically favoured (nu(AB) >> nu(alpha alpha)), and BB bonds, or X-junctions, are energetically favoured (epsilon(BB) > 0). We show that, for low values of epsilon(BB)/epsilon(AA), the phase diagram has three different regions: (i) close to the critical temperature a low-density liquid composed of long chains and rich in Y-junctions coexists with a vapour of chains; (ii) at intermediate temperatures there is coexistence between a vapour of short chains and a liquid of very long chains with X-and Y-junctions; (iii) at low temperatures an ideal gas coexists with a high-density liquid with all possible AA and BB bonds formed. It is also shown that in region (i) the liquid binodal is reentrant (its density decreases with decreasing temperature) for the lower values of epsilon(BB)/epsilon(AA). The existence of these three regions is a consequence of the competition between the formation of X- and Y-junctions: X-junctions are energetically favoured and thus dominate at low temperatures, whereas Y-junctions are entropically favoured and dominate at higher temperatures.