Neste estudo avaliamos a resposta imune humoral de indivíduos naturalmente expostos à malária em áreas endêmicas no Brasil. Os anticorpos IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgE e IgA anti-formas eritrocitárias de Plasmodium falciparum foram determinadas por ELISA. Anticorpos IgG, IgG1, IgG2 de alta avidez e IgG3 de baixa avidez predominaram nos indivíduos sem complicações de malária ou assintomáticos, enquanto anticorpos IgG4, IgE e IgM predominaram nos indivíduos com complicações clínicas por malária. Os resultados mostram que mesmo em regiões com transmissão instável de malária pode ser observado o desenvolvimento de imunidade protetora quando anticorpos apropriados são produzidos / In this study, we have evaluated the humoral immune response of individuals naturally exposed to malaria living in endemic areas of Brazil. We determined IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgE and IgA antibodies against Plasmodium falciparum blood stages by ELISA. We observed that the level of high avidity IgG, IgG1 and IgG2 and low avidity IgG3 antibodies were higher in asymptomatic individuals or with uncomplicated malaria, while IgG4, IgE and IgM antibodies were higher in individuals with complicated malaria. Taken together the results showed that even in unstable malaria regions it can be observed the development of protective immunity against malaria when appropriate antibodies are produced

A imunização materna com a vacina acelular para pertussis (dTpa) é uma intervenção adicional que visa fornecer proteção aos recém-nascidos (RN). No entanto, tem sido relatado que altos níveis de anticorpos adquiridos por transferência placentária podem afetar adversamente a resposta imune desses RN após a imunização ativa, devido ao mascaramento antigênico. Neste estudo, avaliamos a aquisição passiva neonatal de anticorpos específicos para pertussis e sua influência na resposta imune celular dos neonatos. A casuística foi composta por gestantes vacinadas com a vacina dTpa (grupo caso, n=66) ou por gestantes que não receberam a vacina (grupo controle, n=101). As concentrações de anticorpos IgG séricos específicos para Bordetella pertussis total (Bp), toxina pertussis (PT), hemaglutinina filamentosa (FHA) e pertactina (PRN) foram quantificadas em soro materno e de cordão umbilical de seu respectivo RN, e as concentrações de anticorpos IgA específicos para Bp e PT foram dosadas nas amostras de colostro por meio de ensaio imunoenzimático. A responsividade dos linfócitos do sangue neonatal foi avaliada após estimulação ex vivo com Bp inativada por citometria de fluxo com o intuito de detectar a proliferação, produção de citocinas e fenótipo de ativação dos linfócitos T em um contexto de altas concentrações de IgG específicas adquiridas após a vacinação materna. As concentrações de anticorpos IgG anti-Bp, PT, FHA e PRN foram maiores nas amostras de soro materno e de cordão umbilical do grupo caso quando comparadas ao grupo controle (p < 0,0001), com índices de correlação positivos em ambos os grupos para todos os antígenos estudados (p < 0,0001). As vacinações realizadas entre 26 e 31 semanas de gestação foram associadas com as melhores taxas de transferência placentária, embora índices significativamente menores foram detectados no grupo caso (p < 0,01). As concentrações de anticorpos IgA anti-Bp e anti-PT no colostro não foram afetadas pelo estado vacinal da parturiente. Os ensaios de cultura celular revelaram que os RN responderam ao estímulo com Bp, com maior expressão de CD40L, CD69 e proliferação de células T CD4, em comparação com células não estimuladas. Também foi observada uma menor resposta Th1, enquanto a resposta Th2 foi preservada, em comparação com os adultos, mas sem diferenças entre os grupos de neonatos em nenhum dos parâmetros estudados. Nossos resultados indicam que níveis mais altos de anticorpos IgG específicos para B. pertussis no soro dos RN após a vacinação materna não afetam a resposta imune neonatal mediada por células / Maternal immunization with pertussis acellular vaccine (Tdap) is an additional intervention that provides protection to newborns. However, it has been reported that high antibody levels acquired via placental transfer may adversely affect the immune response of newborns after active immunization due to epitope masking. In this study, we evaluated neonatal passive acquisition of pertussis-specific antibodies and their influence on the neonatal cell-mediated immune response. The sample consisted of pregnant women vaccinated with the Tdap vaccine (case group, n=66) or pregnant women who received no vaccine (control group n=101). Whole-cell Bordetella pertussis (Bp), pertussis toxin (PT), filamentous hemagglutinin (FHA) and pertactin (PRN)-specific serum IgG concentrations were quantified in paired maternal-cord sera, and Bp- and PT-specific IgA concentrations were evaluated in colostrum samples by immunoenzymatic assay. Ex vivo neonatal blood lymphocyte responsiveness after inactivated Bp stimulation was assessed using flow cytometry to detect the proliferation, cytokine production and activation phenotype of T lymphocytes in the context of high specific IgG concentrations acquired after maternal vaccination. Anti-Bp, PT, FHA and PRN IgG antibody concentrations in maternal and cord serum samples from case group were higher than those in control group (p < 0.0001), with positive correlation indexes in both groups for all pertussis antigens (p < 0.0001). Vaccinations performed between 26 and 31 gestation weeks were associated with the best placental transfer ratios, although significantly lower ratios were detected in case group (p < 0.01). Anti-Bp and anti-PT IgA concentrations in colostrum were not affected by vaccine status. Cell culture assays revealed that newborns responded to Bp stimulation with higher expression of CD40L CD69 and CD4+ T cell proliferation compared to unstimulated cells. It was also observed a lower Th1 response, while a preserved Th2 response compared to adults, but there were no differences between neonatal groups for any of the studied parameters. Our results indicate that higher pertussis-specific IgG levels in newborn sera after maternal vaccination do not affect the neonatal cell-mediated immune response

A imunização materna com a vacina acelular para pertussis (dTpa) é uma intervenção adicional que visa fornecer proteção aos recém-nascidos (RN). No entanto, tem sido relatado que altos níveis de anticorpos adquiridos por transferência placentária podem afetar adversamente a resposta imune desses RN após a imunização ativa, devido ao mascaramento antigênico. Neste estudo, avaliamos a aquisição passiva neonatal de anticorpos específicos para pertussis e sua influência na resposta imune celular dos neonatos. A casuística foi composta por gestantes vacinadas com a vacina dTpa (grupo caso, n=66) ou por gestantes que não receberam a vacina (grupo controle, n=101). As concentrações de anticorpos IgG séricos específicos para Bordetella pertussis total (Bp), toxina pertussis (PT), hemaglutinina filamentosa (FHA) e pertactina (PRN) foram quantificadas em soro materno e de cordão umbilical de seu respectivo RN, e as concentrações de anticorpos IgA específicos para Bp e PT foram dosadas nas amostras de colostro por meio de ensaio imunoenzimático. A responsividade dos linfócitos do sangue neonatal foi avaliada após estimulação ex vivo com Bp inativada por citometria de fluxo com o intuito de detectar a proliferação, produção de citocinas e fenótipo de ativação dos linfócitos T em um contexto de altas concentrações de IgG específicas adquiridas após a vacinação materna. As concentrações de anticorpos IgG anti-Bp, PT, FHA e PRN foram maiores nas amostras de soro materno e de cordão umbilical do grupo caso quando comparadas ao grupo controle (p < 0,0001), com índices de correlação positivos em ambos os grupos para todos os antígenos estudados (p < 0,0001). As vacinações realizadas entre 26 e 31 semanas de gestação foram associadas com as melhores taxas de transferência placentária, embora índices significativamente menores foram detectados no grupo caso (p < 0,01). As concentrações de anticorpos IgA anti-Bp e anti-PT no colostro não foram afetadas pelo estado vacinal da parturiente. Os ensaios de cultura celular revelaram que os RN responderam ao estímulo com Bp, com maior expressão de CD40L, CD69 e proliferação de células T CD4, em comparação com células não estimuladas. Também foi observada uma menor resposta Th1, enquanto a resposta Th2 foi preservada, em comparação com os adultos, mas sem diferenças entre os grupos de neonatos em nenhum dos parâmetros estudados. Nossos resultados indicam que níveis mais altos de anticorpos IgG específicos para B. pertussis no soro dos RN após a vacinação materna não afetam a resposta imune neonatal mediada por células / Maternal immunization with pertussis acellular vaccine (Tdap) is an additional intervention that provides protection to newborns. However, it has been reported that high antibody levels acquired via placental transfer may adversely affect the immune response of newborns after active immunization due to epitope masking. In this study, we evaluated neonatal passive acquisition of pertussis-specific antibodies and their influence on the neonatal cell-mediated immune response. The sample consisted of pregnant women vaccinated with the Tdap vaccine (case group, n=66) or pregnant women who received no vaccine (control group n=101). Whole-cell Bordetella pertussis (Bp), pertussis toxin (PT), filamentous hemagglutinin (FHA) and pertactin (PRN)-specific serum IgG concentrations were quantified in paired maternal-cord sera, and Bp- and PT-specific IgA concentrations were evaluated in colostrum samples by immunoenzymatic assay. Ex vivo neonatal blood lymphocyte responsiveness after inactivated Bp stimulation was assessed using flow cytometry to detect the proliferation, cytokine production and activation phenotype of T lymphocytes in the context of high specific IgG concentrations acquired after maternal vaccination. Anti-Bp, PT, FHA and PRN IgG antibody concentrations in maternal and cord serum samples from case group were higher than those in control group (p < 0.0001), with positive correlation indexes in both groups for all pertussis antigens (p < 0.0001). Vaccinations performed between 26 and 31 gestation weeks were associated with the best placental transfer ratios, although significantly lower ratios were detected in case group (p < 0.01). Anti-Bp and anti-PT IgA concentrations in colostrum were not affected by vaccine status. Cell culture assays revealed that newborns responded to Bp stimulation with higher expression of CD40L CD69 and CD4+ T cell proliferation compared to unstimulated cells. It was also observed a lower Th1 response, while a preserved Th2 response compared to adults, but there were no differences between neonatal groups for any of the studied parameters. Our results indicate that higher pertussis-specific IgG levels in newborn sera after maternal vaccination do not affect the neonatal cell-mediated immune response

In this thesis some multivariate spectroscopic methods for the analysis of solutions are proposed. Spectroscopy and multivariate data analysis form a powerful combination for obtaining both quantitative and qualitative information and it is shown how spectroscopic techniques in combination with chemometric data evaluation can be used to obtain rapid, simple and efficient analytical methods. These spectroscopic methods consisting of spectroscopic analysis, a high level of automation and chemometric data evaluation can lead to analytical methods with a high analytical capacity, and for these methods, the term high-capacity analysis (HCA) is suggested. It is further shown how chemometric evaluation of the multivariate data in chromatographic analyses decreases the need for baseline separation. The thesis is based on six papers and the chemometric tools used are experimental design, principal component analysis (PCA), soft independent modelling of class analogy (SIMCA), partial least squares regression (PLS) and parallel factor analysis (PARAFAC). The analytical techniques utilised are scanning ultraviolet-visible (UV-Vis) spectroscopy, diode array detection (DAD) used in non-column chromatographic diode array UV spectroscopy, high-performance liquid chromatography with diode array detection (HPLC-DAD) and fluorescence spectroscopy. The methods proposed are exemplified in the analysis of pharmaceutical solutions and serum proteins. In Paper I a method is proposed for the determination of the content and identity of the active compound in pharmaceutical solutions by means of UV-Vis spectroscopy, orthogonal signal correction and multivariate calibration with PLS and SIMCA classification. Paper II proposes a new method for the rapid determination of pharmaceutical solutions by the use of non-column chromatographic diode array UV spectroscopy, i.e. a conventional HPLC-DAD system without any chromatographic column connected. In Paper III an investigation is made of the ability of a control sample, of known content and identity to diagnose and correct errors in multivariate predictions something that together with use of multivariate residuals can make it possible to use the same calibration model over time. In Paper IV a method is proposed for simultaneous determination of serum proteins with fluorescence spectroscopy and multivariate calibration. Paper V proposes a method for the determination of chromatographic peak purity by means of PCA of HPLC-DAD data. In Paper VI PARAFAC is applied for the decomposition of DAD data of some partially separated peaks into the pure chromatographic, spectral and concentration profiles.

A alergia ao látex representa um importante problema de saúde em pacientes denominados de risco e está relacionada com a ocorrência de diversas manifestações clínicas, inclusive reações potencialmente fatais. O estudo de fatores associados à sensibilização e alergia ao látex é fundamental para o estabelecimento de medidas eficazes quanto à prevenção, tratamento e conhecimento do prognóstico. O principal grupo de risco para a alergia ao látex são os pacientes com defeito de fechamento do tubo neural. O estudo atual teve como objetivo principal identificar fatores clínicos e sorológicos associados à sensibilização e alergia ao látex em pacientes com defeito de fechamento do tubo neural. A pesquisa também analisou concentrações de corte de IgE específica sérica para látex e alérgenos do látex que pudessem identificar pacientes sensibilizados e alérgicos ao látex e avaliou o comportamento da IgG4 específica para látex como fator de proteção associado à ausência de sintomas. Com o intuito de responder estas perguntas, foi realizado um estudo transversal tipo coorte retrospectiva com 400 pacientes com defeito de fechamento do tubo neural, entre 0 e 18 anos, que estavam em seguimento na Associação de Assistência à Criança Deficiente - AACD. Após responderem a questionário específico, os pacientes foram submetidos à coleta de sangue periférico para a detecção dos níveis séricos de IgE total, IgE e IgG4 séricas específicas para látex, IgE sérica específica para rHevb1, 3, 5, 6.01, 6.02, 8, 9 e 11, Dermatophagoides pteronyssinus, Blomia tropicalis, abacate, banana, castanha, mamão, batata, através da metodologia ImmunoCap®. De acordo com a história clínica e o valor da IgE sérica específica para látex, os pacientes foram classificados em quatro grupos: sensibilizados asintomáticos ao látex, alérgicos ao látex, sintomáticos sem sensibilização e controle negativo. A prevalência total de sensibilização ao látex nesta amostra de pacientes foi de 33,2%, sendo 12,2% alérgicos e 21,0% sensibilizados assintomáticos ao látex. Os sintomas cutâneos foram a manifestação clínica mais comum de alergia ao látex (79,6%), sendo a urticária de contato a mais prevalente (67,3%). Houve episódios de anafilaxia associados à exposição ao látex em 21 pacientes (5,2%). Níveis de IgE sérica específica para látex de 0,77 kUA/L foram capazes de diferenciar com boa acurácia os pacientes alérgicos dos demais. Na comparação entre os grupos controle negativo e alérgico, sensibilizado e alérgico, houve diferença entre as variáveis clínicas, cirúrgicas e laboratoriais que estiveram associadas à alergia ao latex. A razão entre IgG4/IgE séricas específicas para látex foi estatisticamente maior no grupo controle negativo. A análise multivariada mostrou na comparação entre o grupo controle negativo e alérgico que a presença de IgE sérica específica para rHevb1 e IgE rHevb5 estão associadas com alergia. Na comparação entre o grupo sensibilizado e alérgico, houve associação de alergia com a presença de IgE sérica específica para rHevb5 e com escore clínico >= 40%. Por outro lado, a IgG4 sérica específica para látex esteve associada à ausência de sintomas no grupo sensibilizado / Latex allergy is an important health problem in patients at risk groups and it is associated with the occurrence of many clinical manifestations, including potentially fatal reactions. The study of factors associated with latex sensitization and allergy is crucial to establish effective measures of prevention, treatment and prognostic. The main risk group for latex allergy is patients with neural tube defects. The objective of this study was to identify the clinical and serologic factors associated with sensitization and allergy to latex. Levels of specific IgE to latex and latex allergens associated with allergy, as well as levels of specific IgG4 associated with clinical tolerance, were established, and its accuracy and cutoff values were calculated. The profile of clinical manifestations, including anaphylaxis, was also characterized. This study was a retrospective cross-sectional cohort of 400 patients with neural tube defects, between 0 and 18 years, who were being followed at the Association for Assistance of Disabled Children - AACD. After answering a specific questionnaire, patients were submitted to blood draw for the detection of total IgE, specific IgG4 to latex, and specific IgE to latex, rHevb (1, 3, 5, 6.01, 6.02, 8, 9, 11), Dermatophagoides pteronyssinus, Blomia tropicalis, avocado, banana, cashew, papaya, and potato, by ImmunoCAP ® methodology. According to the history and value of specific IgE to latex, patients were classified into four groups: sensitized asymptomatic patients, allergic patients, symptomatic patients and negative control. The overall prevalence of latex sensitization in this sample of patients was 33.2%, with 12.2% of allergic and 21.0% of sensitized patients. The skin symptoms were the most common clinical manifestation of latex allergy (79.6%), and contact urticaria was the most prevalent (67.3%). Anaphylaxis after latex exposure was observed in 21 patients (5.2%). Levels of specific IgE to latex of 0.77 kUA/L were able to differentiate allergic patients, with good accuracy. Comparing the negative control and allergic groups, the sensitized and allergic groups they had differents clinical, surgical and laboratory factors associated with allergy to latex. The ratio serum specific IgG4/IgE to latex was statistically higher in the negative control group. Multivariate analysis showed, in the comparison between the negative control and allergic groups, that the presence of serum specific IgE to rHevb1 and rHevb5 were associated with allergy. Comparing the sensitized and allergy groups, allergy was associated with the presence of serum specific IgE to rHevb5 and clinical score >= 40%. Moreover, serum specific IgG4 to latex was associated with lack of symptoms in the sensitized group.

Os efeitos protetores da imunidade humoral são medidas por uma família de glicoproteínas chamadas anticorpos ou imunoglobulinas. As preparações de imunoglobulina G (IgG) utilizadas em nosso país são importantes. No Brasil, a primeira preparação de IgG foi obtida na Fundação Pró-Sangue Hemocentro de São Paulo em 1993. O presente estudo avaliou preparações de IgG obtidas de misturas de plasma humano de variadas procedências, inclusive a preparação obtida no Brasil. Foram avaliados os seguintes parâmetros: concentração protéica, distribuição das subclasses da IgG, atividade de anticorpos específicos e segurança quanto a agentes patogênicos transmissíveis pelo sangue. Em algumas preparações, a concentração protéica de IgG e a distribuição das suas subclasses estavam fora das especificações. As preparações apresentaram atividade de anticorpos específicos contra os vírus das hepatites A e B, do herpes simples, da rubéola, citomegalovírus; contra a bactéria Streptococcus pyogenes &#946;-hemolítico do grupo A e contra o parasita Toxoplasma gondii. A qualidade de matéria-prima utilizada em algumas das preparações de IgG não foi adequada em função de reações positivas para anticorpos contra alguns agentes infecciosos, tais como HTLV I/II, HAV, HBV, HCV e Treponema pallidum. Esse estudo também mostrou a necessidade de se implantar urgente um programa abrangente para avalição das preparações de IgG a serem consumidas pela população brasileira. / A family of glicoproteins, which are called antibodies or immunoglobulins (IgG), mediates the protective effects of humoral immunity. In Brazil, the IgG for intravenous use are imported from other countries. The first Brazilian immunoglobulin G for therapheutic use was obtained from human plasma at the Fundação Pró-Sangue Hemocentro de São Paulo. The present study was carried out to evaluate different preparations of IgG, human plasmad-derived, include the preparation from Brazil. The protein concentration, IgG subclass distribution, specific antibody activities and safety regarding the main blood transmitted infectious diseases were analyzed. In some preparations, IgG protein concentration and subclass distribution were different from their specifications. Some preparations showed specific antibody activity against the following antigens: A and B hepatitis virus, rubella, herpes simplex virus, citomegalovirus, measles virus, Streptococcus pyogenes &#946;-hemolytic group A and Toxoplasma gondii. The presence of antibodies against antigens such as HTLV I/II, HAV, HBV, HCV and Treponema pallidum has compromissed the quality guaranty of the material-source (plasma) used in some preparations. This study has also showed that a complete and effective program for the quality evaluation of IgG preparations used in Brazil is needed and should be urgently established

Neste estudo avaliamos a resposta imune humoral de indivíduos naturalmente expostos à malária em áreas endêmicas no Brasil. Os anticorpos IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgE e IgA anti-formas eritrocitárias de Plasmodium falciparum foram determinadas por ELISA. Anticorpos IgG, IgG1, IgG2 de alta avidez e IgG3 de baixa avidez predominaram nos indivíduos sem complicações de malária ou assintomáticos, enquanto anticorpos IgG4, IgE e IgM predominaram nos indivíduos com complicações clínicas por malária. Os resultados mostram que mesmo em regiões com transmissão instável de malária pode ser observado o desenvolvimento de imunidade protetora quando anticorpos apropriados são produzidos / In this study, we have evaluated the humoral immune response of individuals naturally exposed to malaria living in endemic areas of Brazil. We determined IgG, IgG1, IgG2, IgG3, IgG4, IgM, IgE and IgA antibodies against Plasmodium falciparum blood stages by ELISA. We observed that the level of high avidity IgG, IgG1 and IgG2 and low avidity IgG3 antibodies were higher in asymptomatic individuals or with uncomplicated malaria, while IgG4, IgE and IgM antibodies were higher in individuals with complicated malaria. Taken together the results showed that even in unstable malaria regions it can be observed the development of protective immunity against malaria when appropriate antibodies are produced

Intimina é uma adesina de natureza protéica das bactérias diarreiogênicas Escherichia coli enteropatogenica (EPEC) e enterohemorrágica (EHEC), capazes de induzir a lesão \'attaching e effacing\' em enterócitos. Os principais subtipos de intiminas de EPEC e EHEC prevalentes no Brasil são &#945;, &#946; e &#947;. Nosso objetivo foi investigar a transferência de anticorpos maternos anti-intiminas aos recém-nascidos de mães saudáveis de São Paulo, Brasil. Foram pesquisados anticorpos SIgA no colostro e IgG no soro de 50 mulheres saudáveis e no soro de cordão umbilical de seus recém-nascidos, por ELISA utilizando como antígeno proteínas recombinantes purificadas das regiões conservadas e variáveis de intiminas &#945;, &#946; e &#947;. As concentrações de anticorpos no colostro foram superiores quando comparadas com as concentrações do soro para todos os tipos de intiminas. Não se observaram diferenças estatísticas entre as concentrações de anticorpos reativos com as diferentes intiminas nas amostras de colostro. As concentrações de anticorpos reativos com a região conservada da intimina foram significativamente mais elevadas em comparação com as regiões variáveis no soro dos grupos de mães e de recém-nascidos. Houve alta correlação entre todos os anticorpos anti-intiminas nas amostras de colostro. Comparando-se as concentrações de anticorpos séricos, os coeficientes foram maiores entre anti-&#945; e anti-&#946; que entre os outros pares. Nossos resultados confirmam a transferência de anticorpos maternos para o recém-nascido pela placenta e pelo aleitamento materno e reforça o efeito protetor da amamentação contra infecção por EPEC. / Intimin is a proteic adhesin of enteropatogenic (EPEC) and enterohemorragic (EHEC) Escherichia coli, capable of inducing attachment and effacement lesion in enterocytes. The main subtypes of intimins of EPEC and EHEC prevalent in Brazil are &#945;, &#946; and &#947;. Our aim is to investigate the transference of maternal anti-intimin antibodies to the newborns of healthy mothers from Sao Paulo, Brazil. IgG and SIgA antibodies were determined in sera and colostrum from 50 healthy women and cord sera from their newborns, by ELISA using as antigens purified recombinant proteins, conserved and variable regions of &#945;, &#946; and &#947; intimins. The IgA antibody concentrations of colostrum are higher than IgG antibodies in serum for all intimins and there were no statistical differences between them in colostrum samples. The concentrations of antibodies reactive with the conserved region of intimin are significantly higher compared to the variable regions in the sera groups, mothers and newborns. There were high correlation coefficients between all the anti-intimins antibodies in colostrum samples. In the comparison of the seric antibody concentrations, the coefficients were higher between anti-&#945; and anti-&#946; than all the other pairs. Our results confirm the transference of maternal antibodies to the newborns by placenta and breastfeeding and reinforce the high protection effect of breastfeeding against EPEC infection.

Safety concerns arising from cytotoxic behavior of nanoparticles (NPs) in complex biological environment remain the main problem limiting NPs application in biomedicine. In this study, we have investigated cytotoxicity of NPs with different composition, shape and size, namely SiO2 NPs (SiNPs, 7-14 nm), superparamagnetic iron oxide NPs (SPIONs, 8 nm) and carboxylated multiwalled carbon nanotubes (CNTCOOHs, diameter: 60-100 nm, length: 1-2 μm). Cytotoxicity was evaluated with newly designed screening assay capable to simultaneously assess activity of cell dehydrogenases, activity of lactate dehydrogenase (LDH) released from cells into environment and number of intact cell nuclei and apoptotic bodies in human umbilical vein endothelial cell (HUVEC) culture growing in the very same well of the 96-well plate. Aforementioned attributes were subsequently utilized to obtain information about cell viability and necrotic and apoptotic aspects of cell death. Results from this "three-in-one" cell death screening (CDS) assay showed that SiNPs and CNTCOOHs evoked pronounced cytotoxic effect demonstrated as decrease of cell viability and development of apoptotic bodies formation. In contrast to this, SPIONs induced only mild cytotoxicity. Moreover, SiNPs impaired cell membrane leading to increased LDH release...

Koffein (1,3,7-Trimethylxanthin) und Coprostanol (5beta-cholestan-3beta-ol) wurden im Berliner Oberflächenwasser nachgewiesen. Ihre Konzentrationen korrelierten mit dem Verunreinigungsgrad der Proben, was nahelegt, dass sie sich als Marker für menschliche Aktivität eignen. Bemerkenswerterweise wurde Koffein in jeder einzelnen Oberflächenwasserprobe oberhalb der Bestimmungsgrenze von 0,025 µg/L gefunden. Um Oberflächenwasserproben in größeren Serien zu untersuchen, war die Entwicklung zweier neuer Methoden erforderlich: ein Immunoassay, basierend auf einem monoklonalen Antikörper für Koffein und eine dispersive flüssig-flüssig Mikroextraktionsmethode (DLLME), gefolgt von Flüssigkeitschromatographie gekoppelt mit Tandem-Massenspektrometrie (LC-MS/MS) für Coprostanol. Der entwickelte Koffein-Immunoassay zeigt die beste je erhaltene Nachweisgrenze für Koffein (0,001 µg/L), erlaubt Hochdurchsatz-Analysen und erfordert keine Probenvorbereitung. Der Assay wurde auch erfolgreich für die Messung von Koffein in Getränken, Haarwaschmitteln, Koffeintabletten und menschlichem Speichel angewendet. Antikörper gegen Coprostanol sind nicht kommerziell erhältlich. Eine neue Strategie Anti-Coprostanol-Antikörper zu generieren wurde erarbeitet, die eine analoge Verbindung – Isolithocholsäure (ILA) – als Hapten verwendet, mit der eine Gruppe von Mäusen immunisiert wurde. Ein polyklonales Anti-ILA-Serum wurde produziert, welches Coprostanol bindet, aber die niedrige Affinität erlaubte nicht den Aufbau eines Immunoassays, der die Messung von Umweltkonzentrationen des Anayten (im Bereich ng/L) zulässt. Spezifische Anti-ILA-Immunglobuline G wurden auch in den Faeces der Mäuse gefunden. Coprostanol wurde in den Wasserproben durch die Verwendung einer neuentwickelten LC-MS/MS-Methode unter APCI-Ionisation (atmospheric pressure chemical ionisation) gemessen. Konzentrationen oberhalb von 0,1 µg/L wurden nach Voranreicherung der Probe mittels DLLME bestimmt. / Caffeine (1,3,7-trimethylxanthine) and coprostanol (5beta-cholestan-3beta-ol) were detected in samples of Berlin’s surface water. Their concentrations correlated with the contamination status of the samples, suggesting their usefulness as markers of human activity. Remarkably, caffeine concentrations were always well above the limit of quantitation of 0.025 µg/L. In order to screen surface water samples in larger series, the development of two novel methods was required: a monoclonal antibody-based immunoassay for caffeine and a dispersive liquid-liquid microextraction (DLLME) method, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS) for coprostanol. The caffeine immunoassay developed shows the best analytical limit of detection (LOD) obtained so far for caffeine (0.001 µg/L), allows high-throughput analysis, and does not require sample pre-treatment. The assay was also successfully employed to measure caffeine in beverages, shampoos, caffeine tab-lets, and human saliva. Antibodies to coprostanol are not commercially available. A new strategy to generate anti-coprostanol antibodies was elaborated using an analogous com-pound as hapten – isolithocholic acid (ILA) – and immunizing a group of mice. A polyclonal anti-ILA serum was produced, which binds coprostanol but the low affinity did not permit setting up an immunoassay to measure environmental concentrations of the analyte (in the range of ng/L). Specific anti-ILA immunoglobulin G were also found in the faeces of the immunized mice. Coprostanol was quantified in the water samples using a newly developed LC-MS/MS method using atmospheric pressure chemical ionisation (APCI). Concentrations above 0.1 µg/L were determined after sample preconcentration using DLLME. This extraction method also proved to be successful for enrichment of coprostanol-related compounds such as cholesterol, cholestanol, cholestanone, ergosterol, and stigmasterol.