A espectrometria de massas com plasma acoplado indutivamente com simples quadrupolo (q-ICP-MS) e cela de reação dinâmica (DRC-ICP-MS) foi avaliada para determinação seqüencial de Al, As, Cd, Co, Cu, Cr, Mn, Mo, Pb, Pt, Sb, Se, Sn, Te, Tl, V e Zn em sangue. Para as análises, amostras de sangue (200 µL) foram diluídas 1:50 em uma solução contendo 0,01% v/v Triton® X-100 e 0,5% v/v de ácido nítrico. As calibrações foram realizadas com ajuste de matriz, utilizando sangue ovino. Como gás de reação utilizou-se a NH3. O uso do DRC foi fundamental para a determinação de Cr e V. A escolha da melhor vazão do gás e a otimização do parâmetro de rejeição (RPq) foram estudadas utilizando sangue base ovino ou uma solução de matriz sintética (SMS), de acordo com a concentração do analito no sangue base. Os limites de detecção (3s/coeficiente angular) para determinação de 27Al, 75As, 111Cd, 59Co, 63Cu, 55Mn, 98Mo, 208Pb, 195Pt, 123Sb, 82Se, 120Sn, 130Te, 205Tl e 66Zn em sangue por q-ICP-MS foram 0,223; 0,014; 0,003; 0,011; 0,304; 0,009; 0,009; 0,003; 0,001; 0,005; 0,264; 0,006; 0,010; 0,001; 0,834 µg L-1, respectivamente, e para determinação de 52Cr e 51V em sangue por DRC-ICP-MS utilizando o gás de reação amônia os limites de detecção foram de 0,007 e 0,006 µg L-1. Para a determinação de 27Al, 63Cu, 65Cu, 64Zn e 66Zn em sangue por q-ICP-MS através da calibração com ajuste de matriz com SMS os limites de detecção foram 0,083; 0,090; 0,055; 0,281; 0,306 µg L-1, respectivamente. A validação do método foi realizada por meio da análise de materiais de referência do INSP-Canadá, NYSDOH-EUA e Seronorm-Noruega. / The use of inductively coupled plasma mass spectrometry with quadrupole (q-ICP-MS) and dynamic reaction cell (DRC-ICP-MS) was evaluated for sequential determination of Al, As, Cd, Co, Cu, Cr, Mn, Mo, Pb, Pt, Sb, Se, Sn, Te, Tl, V and Zn in whole blood by q-ICP-MS or DRC-ICP-MS methods. Prior to analysis, sample (200 µL) were diluted 1:50 v/v in a solution containing 0.01% v/v Triton® X-100 and 0.5% v/v nitric acid. For all elements, except Cr and V, the instrument was operated in q-ICP-MS mode. The use of DRC was only mandatory for Cr and V. NH3 was evaluated as the reaction gas. Selection of best flow rate of reaction gas and optimization of the quadrupole dynamic bandpass rejection parameter (RPq) were carried out, using base whole blood or synthetic matrix solution (SMS), in according with element base blood concentration. Method detection limits (3s/slope) for 27Al, 75As, 111Cd, 59Co, 63Cu, 55Mn, 98Mo, 208Pb, 195Pt, 123Sb, 82Se, 120Sn, 130Te, 205Tl and 66Zn determination in whole blood by q-ICP-MS against matrix matching calibration (base blood) were 0.223; 0.014; 0.003; 0.011; 0.304; 0.009; 0.009; 0.003; 0.001; 0.005; 0.264; 0.006; 0.010; 0.001; 0.834 µg L-1, respectively, and for 52Cr and 51V determination in whole blood by DRC-ICP-MS the detection limits were 0.007 e 0.006 µg L-1. For 27Al, 63Cu, 65Cu, 64Zn and 66Zn determination by q-ICP-MS through matrix matching calibration with SMS the detection limits were 0.083; 0.090; 0.055; 0.281; 0.306 µg L-1, respectively. Method validation was accomplished by the analysis of reference materials from INSP-Canada, NYSDOH-USA, Seronorm-Norway.

As chamadas club drugs compreendem um vasto grupo de substâncias frequentemente utilizadas em bares, festas e raves, com a finalidade de intensificar o contato social e a estimulação sensorial. Englobam desde substâncias sintéticas comumente conhecidas, como a anfetamina, a metanfetamina, o MDMA, até moléculas de surgimento mais recente, denominadas novas substâncias psicoativas. Isoladas ou associadas a outras drogas, é possível que sejam causa de morte per se, ou que predisponham o usuário a envolver-se em situações potencialmente fatais, sendo necessário que os órgãos de Perícia Criminal (Institutos Médico Legais e Institutos de Criminalística) estejam aptos a detectar e quantificar essas substâncias em amostras biológicas. O presente trabalho teve como objetivo desenvolver um método analítico para identificação e quantificação de club drugs em sangue total, utilizando cromatografia líquida acoplada a espectrometria de massas com analisador híbrido quadrupolotempo de voo (LC-QTOF). Após o desenvolvimento do método, este foi validado utilizando as diretrizes do guia de validação do Scientific Working Group for Forensic Toxicology (SWGTOX), sendo analisados de linearidade, limite de detecção, limite de quantificação, efeito matriz, precisão intradia, precisão interdia, exatidão e integridade de diluição, além de recuperação e eficiência do processo. O método desenvolvido compreendeu a determinação de MDA, MDMA, 2C-B, DOB, cetamina, mCPP, cocaína e cocaetileno. Amostras provenientes de casos reais de morte não natural, oriundas do Instituto Médico Legal Aristoclides Teixeira de Goiânia - GO foram analisadas pelo método desenvolvido. 56 casos foram selecionados, em sua maioria com histórico de morte por projétil de arma de fogo e acidente de transito. Das 56 amostras analisadas, 28,5% (n=16) foram positivas para cocaína e/ou cocaetileno. As demais substâncias pesquisadas não foram encontradas nas amostras. / Club drugs are a large group of substances consumed in pubs, parties and raves, aiming to intensify social contact and sensorial stimulation. The term comprises largely known substances such as amphetamine, methamphetamine, 3,4-methylenodioxymethamphetamine (MDMA), as well as so-called new psychoactive substances, which are synthetic drugs recently developed or recently introduced in drug market. Club drugs can be taken alone, combined with each other or, most frequently, with alcohol or other commonly abused drugs such as cocaine. In any of these situations, club drugs can possibly be the cause of death or potentialize the involvement of the user with crime and potentially fatal behavior. Thus, official organisms in charge of criminal investigation must be capable of identifying and quantifying these substances in biological samples. The present work aimed the development of an analytical method to identify and quantify club drugs in whole blood, using liquid chromatography - mass spectrometry with hybrid analyzer quadrupole - time of flight (LC-QTOF). After analytical development, the method was validated according to do Scientific Working Group for Forensic Toxicology (SWGTOX) guidelines, evaluating linearity, limit of detection, limit of quantification, matrix effect, precision, intermediate precision, bias and dilution integrity, besides recovery and process efficiency. The developed method comprised MDA, MDMA, 2C-B, DOB, ketamine, mCPP, cocaine and cocaethylene determination. Real samples related to non-natural deaths were collected at Institute of the Legal Medicine Aristoclides Teixeira, Goiânia, Goiás, Brazil, and analyzed by the developed method. 56 cases were selected, most of them related to fire gun injury and traffic events, 28,5% (n=16) of them being positive for cocaine and/or cocaethylene. None of the other drugs comprised in the analysis were detected in these samples.

<p>This study shows that herbs can be effectively screened for potiential bio-activity using in vitro methods. Further studies will be needed to better explore Artemisia afra&rsquo / s effect on immunoregulation, particularly long term effects of the herb on the immune system and its effect on other disease states.</p>

Gelenkknorpel ist ein gefäßloses, hoch spezialisiertes Gewebe mit nur sehr begrenzter Regenerationsfähigkeit. Entstandene Läsionen werden bei natürlicher Heilung durch min-derwertigen Faserknorpel gefüllt. Ein vielversprechender Therapieansatz kommt aus dem Gebiet des Tissue Engineering. Dabei werden isolierte Chondrozyten in vitro vermehrt und anschließend in den Defekt eingebracht. In den letzten Jahren ist hier die 3 D-Kultivierung in patientenspezifischen Biomaterialien zunehmend in den Fokus der Forschung geraten. Ziel der hier vorgestellten Studie war es, die Tauglichkeit von Vollblut und Blutplasma als Aus-gangsmaterial für MACTs aufgrund makroskopischer Eigenschaften, Zellzahlentwicklung im Konstrukt, Zellvitalität und Syntheseleistung charakteristischer EZM-Marker zu untersuchen. Es wurde für diese Studie Knorpel aus den Fesselgelenken vier geschlachteter Pferde (2-16 Jahre) entnommen, mechanisch zerkleinert und anschließend mit Kollagenase A verdaut. Von einem 6 jährigen klinikeigenen Wallach wurde Vollblut in Citratröhrchen gewonnen, ein Teil zu Plasma weiterverarbeitet und beides bei -80 °C bis zur weiteren Verwendung Schock gefroren. Zur Herstellung der walzenförmigen Konstrukte mit den Maßen 9,6 cm2 x 4,7 mm, wurden 4,5 ml Vollblut bzw. Plasma mit je 3x106 Chondrozyten suspensiert und durch Zuga-be von CaCl2 zur Koagulation gebracht. Der Kultivierungszeitraum betrug 28 Tage in DMEM, angereichert mit 10% allogenem Serum und 1% Antibiotika. Die Konstrukte wurden an den Tagen 1, 14 und 28 auf Zellzahl, -vitalität und mithilfe qRT-PCR auf hyaline Knorpelmarker wie Kollagen Typ II und Aggrekan untersucht. Zudem wurden histologische und immunhisto-chemische Präparate der Konstrukte angefertigt. Die Zellvitalität betrug sowohl in den VB-, als auch in den BP-Konstrukten ≥95% bei steigen-der Zellzahl (bis zu 5x106 im Vollblutkonstrukt). Die MACTs beider Ausgangsmaterialien schrumpften auf eine Größe von 2 cm² x 2 mm. Histologisch konnten in beiden Konstruktar-ten mit der Alzianblaufärbung sGAG belegt werden. Darüber hinaus wurde der sGAG Gehalt mit dem DMMB Assay quantitativ ermittelt. Aggrekan, C-4-S, C-6-S und COMP wurden als wichtige Bestandteile der EZM immunhistochemisch angefärbt und waren ebenfalls in beiden Konstruktarten nachweisbar. Mit der qRT PCR konnte die Genexpression von Aggrekan, Kol II und Kol I über den zeitlichen Verlauf ermittelt werden. Es stellte sich heraus, dass sich sowohl die Genexpression von Aggrekan als auch die von Kol II, den beiden Indikatorprotei-nen EZMs des hyalinen Gelenkknorpels über den Kultivierungszeitraum absenkten. Dies deutet auf eine Dedifferenzierung der Chondrozyten hin. Die Expression von Kol I dagegen stieg um ein Vielfaches an. Auch das kennzeichnet eine Dedifferenzierung. Zieht man ande-re Studien heran, so ist die festgestellte Umstellung der Genexpression aber vergleichsweise niedrig, eine Dedifferenzierung weg vom chondrogenen Phänotyp in der hier vorliegenden Arbeit also weniger stark ausgebildet. Biokompatibilität mit und Abbaubarkeit im Empfängerorganismus konnten in dieser in vitro Untersuchung nicht evaluiert werden. Zusammenfassend kann man festhalten, dass VB und BP als Ausgangsbiomaterialien zur Herstellung von MACT geeignet sind. Inwieweit es gelingen wird, den chondrogenen Phäno-typ beispielweise durch mechanische Stimulation der eingesäten Zellen stärker zu erhalten, muss in folgenden Studien geklärt werden. Ebenfalls weiterer Forschungsbedarf ist bei den Eigenschaften Elastizität und Steifheit gegeben. Grundsätzlich gilt, dass MACTs auf VB- und BP-Basis einen einfachen, kostengünstigen und patientenspezifisch herstellbaren Therapie-ansatz für die Behandlung von Knorpeldefekten darstellen. / Articular cartilage is a vessel-free, highly specialized tissue with only very limited regenera-tive capacity. Resulting lesions are filled with inferior fibrocartilage by natural healing. A promising therapeutic approach comes from the field of tissue engineering. Therefore iso-lated chondrocytes are expanded in vitro and then placed into the defect. In the last few years the 3-D culturing in patient-specific biomaterials has come increasingly into the focus of research. Purpose of the present study was to evaluate the suitability of whole blood and blood plasma as a basic material for MACT based on macroscopic properties, development of cell number in the construct, cell viability and synthetic performance of characteristic markers. For this study cartilage from the four fetlock joints of slaughtered horses (2-16 years) were removed, crushed mechanically and then digested with Collagenase A. Whole blood was obtained in citrate tubes of a 6 year old gelding owned by the clinic, finished part to both plasma and shock frozen at -80°C until further use. To prepare the cylindrical constructs, measuring 9.6 cm2 x 4.7 mm, 4.5 ml of whole blood or plasma, each with 3x106 chondro-cytes were suspended and coagulated by the addition of CaCl2. The cultivation period was 28 days in DMEM supplemented with 10% allogenic serum and 1% antibiotics. The con-structs were evaluated on days 1, 14 and 28 on cell number, viability, and using qRT-PCR examination for hyaline cartilage markers such as collagen type II and aggrecan. In addition, histological and immunohistochemical preparations of the constructs were made. The cell vitality was in the WB, as well as in the BP constructs ≥ 95% with increasing cell number (up to 5x106 in whole blood construct). The MACTs of both basic materials shrink to a size of 2 cm x 2 mm². Histologically sGAG could be verified in both construct species by Alzianblau staining. In addition, the GAG content was determined with the DMMB assay quantitatively. Aggrecan, chondroitin-4-sulphate, chondroitin-6-sulphate and COMP as major components of the ECM were stained immunohistochemically and were also detectable in both types of constructs. Aggrecan, collagen II and collagen I were determined on the time course by qRT PCR gene expression. It turned out that the gene expressions of both, aggre-can and of collagen II, the two ECM protein indicators of hyaline cartilage lowered over the cultivation period. This indicates a dedifferentiation of chondrocytes. The expression of colla-gen I on the other hand increased to a multiple, also featuring a dedifferentiation. If one approached other studies, the observed change in gene expression is comparatively low. In the present work the dedifferentiation from the chondrogenic phenotype is less distinctive. Biocompatibility and biodegradability in the recipient organism could not be evaluated in this in vitro investigation. In summary, one should notice that VB and BP are suitable basic materials for the production of MACT. It has to be clarified by following studies, to what extent the chondrogenic pheno-type can be strengthened, for example by mechanical stimulation of cells sown. Also further research is needed on the given properties, e.g. elasticity and stiffness. Generally MACT based on WB and BP- is a simple, inexpensive and patient-specific produced therapeutic approach for the treatment of cartilage defects.

<p>This study shows that herbs can be effectively screened for potiential bio-activity using in vitro methods. Further studies will be needed to better explore Artemisia afra&rsquo / s effect on immunoregulation, particularly long term effects of the herb on the immune system and its effect on other disease states.</p>

Chronic, noncommunicable diseases such as chronic fatigue syndrome (also known as myalgic encephalomyelitis) are rapidly becoming a worldwide epidemic that profoundly affects public health and productivity. Chronic fatigue syndrome (CFS) is characterised by severe and debilitating fatigue and although its etiology is still unknown, recent studies have found considerable evidence that mitochondrial dysfunction and oxidative stress might be responsible for the underlying energy deficit in these patients. Adenine and pyridine nucleotides could be used as potential biomarkers for energy related disorders such as chronic fatigue syndrome because of their various functions in the energy and redox pathways. The first part of this study focussed on developing a liquid chromatography electrosprayionisation tandem mass spectrometry (LC-ESI-MS/MS) method for the quantification of these nucleotides in blood samples. Due to the instability of nucleotides in biological matrices it was also necessary to find a suitable extraction method that would be able to stop enzymatic activity via protein precipitation. Out of the four extraction methods investigated during this study, deproteinisation of whole blood samples with perchloric acid produced the highest nucleotide abundances. Although nucleotide standards were found to be stable in perchloric acid, nucleotide levels in blood samples were not stabilised by addition of perchloric acid. The second part of this study consisted of measuring the nucleotide levels in blood samples of controls and possible CFS patients in order to test the proof of concept of the new LCESI- MS/MS method. Despite changes in the nucleotide levels due to perchloric acid and problems with nucleotide instability, it was still possible to distinguish between the two groups based on the results obtained with the new LC-ESI-MS/MS method. The newly developed LC-ESI-MS/MS method proved to be reliable and adequate for nucleotide quantification in whole blood samples, thus the aim of this study was achieved. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2014

Magister Scientiae - MSc (Medical BioSciences) / In the past three decades there has been a huge increase in the use of herbal medicine globally. The active principles of these herbal medicines are mostly unknown with supportive evidence for safety and efficacy very rare. SeptilinTM is a phytopharmaceutical formulation which is recommended for the treatment and management of various infections. It has been claimed to have immunomodulatory actions that potentiates the body's immune response. The immunomodulatory activity of SeptilinTM has not been well investigated via appropriate in vitro models. Therefore this study was undertaken to investigate the in vitro effects of SeptilinTM on biomarkers of specific immune pathways by using WBC. Stimulated and unstimulated WBC were incubated with the product. Enzyme linked immunosorbent assays were used to screen for IL-6, IL-10, and IFN? as biomarkers for inflammation, humoral immunity, and cell mediated immunity, respectively. Results show that the presence of SeptilinTM in LPS stimulated WBC has no effect on the release of IL-6 and IFN? production but stimulated IL-10 production. SeptilinTM in unstimulated WBC has no effect on the release of IL-10 and IFN? production but stimulatory effects on IL-6 production.

No description available.

O agregado proteico P-MAPA apresentou potencial imunomodulatório em diversos estudos, mas a sua ação sobre o sistema complemento e receptores do tipo Toll (TLRs) não é, ainda, conhecida. Neste estudo o P-MAPA promoveu ativação das vias clássica e alternativa do sistema complemento e produção de C3a e C5a. Utilizando um modelo ex vivo de sangue total humano, o composto promoveu aumento da expressão de CD11b e CD14, diminuição da expressão de C5aR, TLR2 e TLR4, em leucócitos de sangue periférico e também quando combinado com LPS, porém não promoveu alterações na expressão de C3aR. O P-MAPA induziu redução de IFN-g no plasma, aumento da produção de TNF-&alpha;, IL-8, IL-12 e peróxinitrito, mas não induziu produção de superóxido, IL-6, IL-1&beta;, TGF-&beta; ou IL-10. Por meio de testes in vivo, foi possível determinar a dose letal do P-MAPA. Em conjunto, os dados obtidos mostram que o P-MAPA apresenta ação pró-inflamatória em modelo ex vivo de sangue total humano e que o tratamento combinado com LPS leva a uma amplificação dos seus efeitos. / P-MAPA, a protein aggregate has been described as a promising immunomodulator, however, its role on the complement system and Toll-like receptors (TLRs) is unknown. In the study, P-MAPA has promoted activation of the complement\'s classical and alternative pathways and the production of C3a and C5a. Using an ex vivo model of human whole blood, the compound promoted increase of CD11b and CD14 expression, decrease of C5aR, TLR2 and TLR4, in peripheral blood leucocytes and when combined with LPS, but did not change C3aR expression. P-MAPA promoted reduction of IFN-g in plasma, increased production of TNF-&alpha;, IL-8, IL-12 and peroxynitrite, but did not induce the production of superoxide, IL-6, IL-1&beta;, TGF-&beta; or IL-10. Through in vivo tests, we were able to determine a lethal dose for P-MAPA. Altogether, our data indicate that P-MAPA has proinflammatory action in ex vivo model of human whole blood and that the treatment combined with LPS leads to amplification of its effects.

A espectrometria de massas com plasma acoplado indutivamente com simples quadrupolo (q-ICP-MS) e cela de reação dinâmica (DRC-ICP-MS) foi avaliada para determinação seqüencial de Al, As, Cd, Co, Cu, Cr, Mn, Mo, Pb, Pt, Sb, Se, Sn, Te, Tl, V e Zn em sangue. Para as análises, amostras de sangue (200 µL) foram diluídas 1:50 em uma solução contendo 0,01% v/v Triton® X-100 e 0,5% v/v de ácido nítrico. As calibrações foram realizadas com ajuste de matriz, utilizando sangue ovino. Como gás de reação utilizou-se a NH3. O uso do DRC foi fundamental para a determinação de Cr e V. A escolha da melhor vazão do gás e a otimização do parâmetro de rejeição (RPq) foram estudadas utilizando sangue base ovino ou uma solução de matriz sintética (SMS), de acordo com a concentração do analito no sangue base. Os limites de detecção (3s/coeficiente angular) para determinação de 27Al, 75As, 111Cd, 59Co, 63Cu, 55Mn, 98Mo, 208Pb, 195Pt, 123Sb, 82Se, 120Sn, 130Te, 205Tl e 66Zn em sangue por q-ICP-MS foram 0,223; 0,014; 0,003; 0,011; 0,304; 0,009; 0,009; 0,003; 0,001; 0,005; 0,264; 0,006; 0,010; 0,001; 0,834 µg L-1, respectivamente, e para determinação de 52Cr e 51V em sangue por DRC-ICP-MS utilizando o gás de reação amônia os limites de detecção foram de 0,007 e 0,006 µg L-1. Para a determinação de 27Al, 63Cu, 65Cu, 64Zn e 66Zn em sangue por q-ICP-MS através da calibração com ajuste de matriz com SMS os limites de detecção foram 0,083; 0,090; 0,055; 0,281; 0,306 µg L-1, respectivamente. A validação do método foi realizada por meio da análise de materiais de referência do INSP-Canadá, NYSDOH-EUA e Seronorm-Noruega. / The use of inductively coupled plasma mass spectrometry with quadrupole (q-ICP-MS) and dynamic reaction cell (DRC-ICP-MS) was evaluated for sequential determination of Al, As, Cd, Co, Cu, Cr, Mn, Mo, Pb, Pt, Sb, Se, Sn, Te, Tl, V and Zn in whole blood by q-ICP-MS or DRC-ICP-MS methods. Prior to analysis, sample (200 µL) were diluted 1:50 v/v in a solution containing 0.01% v/v Triton® X-100 and 0.5% v/v nitric acid. For all elements, except Cr and V, the instrument was operated in q-ICP-MS mode. The use of DRC was only mandatory for Cr and V. NH3 was evaluated as the reaction gas. Selection of best flow rate of reaction gas and optimization of the quadrupole dynamic bandpass rejection parameter (RPq) were carried out, using base whole blood or synthetic matrix solution (SMS), in according with element base blood concentration. Method detection limits (3s/slope) for 27Al, 75As, 111Cd, 59Co, 63Cu, 55Mn, 98Mo, 208Pb, 195Pt, 123Sb, 82Se, 120Sn, 130Te, 205Tl and 66Zn determination in whole blood by q-ICP-MS against matrix matching calibration (base blood) were 0.223; 0.014; 0.003; 0.011; 0.304; 0.009; 0.009; 0.003; 0.001; 0.005; 0.264; 0.006; 0.010; 0.001; 0.834 µg L-1, respectively, and for 52Cr and 51V determination in whole blood by DRC-ICP-MS the detection limits were 0.007 e 0.006 µg L-1. For 27Al, 63Cu, 65Cu, 64Zn and 66Zn determination by q-ICP-MS through matrix matching calibration with SMS the detection limits were 0.083; 0.090; 0.055; 0.281; 0.306 µg L-1, respectively. Method validation was accomplished by the analysis of reference materials from INSP-Canada, NYSDOH-USA, Seronorm-Norway.