Die Organtransplantation stellt ein therapeutisches Verfahren für Patienten mit irreversibel geschädigten Organen dar. Doch weist dieses Behandlungskonzept weiterhin einen wesentlichen Nachteil auf: noch immer wird der langfristige Erfolg der Therapie zu oft durch die so genannte Transplantatabstoßung gefährdet. Hierbei handelt sich um eine vom Organtransplantat ausgelöste Immunantwort, die zu dessen Zerstörung führt. Die derzeit einzige Möglichkeit eine Abstoßung zu verhindern, ist die Unterdrückung des Immunsystems mit so genannten Immunsuppressiva. Auch wenn diese erstmals in den 60er Jahren des 20. Jahrhunderts erfolgreich eingesetzten Medikamente ständig verbessert werden, bleiben die von ihnen ausgelösten Nebenwirkungen weiterhin ein ernstzunehmendes Problem. Sie unterdrücken die gesamte körpereigene Abwehr, was zum Schutz der Organtransplantate vor Abstoßung gewünscht ist, doch fördern sie hierdurch die Entstehung von Tumoren und Infektionen. Bei der Transplantatabstoßung handelt es sich um eine von CD4+ T-Lymphozyten ausgelöste Immunantwort. Diese Lymphozyten werden von allogenen Peptiden, die von Spender-MHC-Molekülen stammen, über den indirekten Weg der Alloantigenerkennung aktiviert. An der Transplantatabstoßung ist zwar eine Vielzahl von Alloantigenen beteiligt, doch ist es möglich, Peptidantigene zu identifizieren, die einen nachweisbaren Effekt auf die Transplantatabstoßung ausüben. So wurde in der eigenen Arbeitsgruppe die für die Abstoßung allogener Organtransplantate beteiligten MHC (RT1u)-Peptidantigene charakterisiert. Insbesondere die Bedeutung des aus 19 Aminosäuren bestehenden allogenen Peptids P1 für die Alloimmunantwort wurde intensiv untersucht. So weisen P1-spezifische T-Lymphozyten ein ausgeprägtes Th1-Cytokin-Muster auf und beschleunigen die Abstoßung von Wistar-Furth-Organtransplantaten in Lewis-Ratten. Das Ziel dieser Arbeit war die Charakterisierung des T-Zellrezeptor Vb-Repertoires P1-spezifischer T-Lymphozyten mit der Methode des PCR-ELISA. In einem ersten Schritt wurden Thymozyten und T-Lymphozyten unterschiedlicher Lymphknotenstationen untersucht. Thymozyten exprimierten alle 22 TCR Vb-Elemente und einzig TCR Vb14 war überrepräsentiert. Die T-Lymphozyten der cervikalen, mesenterialen, iliakalen und poplitealen Lymphknoten zeigten ebenfalls eine charakteristische Überexpression bestimmter TCR Vb-Elemente. So exprimierten cervikale T-Lymphozyten bevorzugt die TCR Vb-Elemente 2, 6, 8.3 und 16, mesenteriale T-Lymphozyten die TCR Vb-Elemente 2, 4, und 8.1, illiakale T-Lymphozyten die TCR Vb-Elemente 2 und 6 und popliteale T-Lymphozyten die TCR Vb-Elemente 2, 4 und 9. Die Immunisierung mit dem nicht-immunogenen Kontrollpeptid (Autoantigen) Ac führte zu einer leichten Veränderung des T-Zellrezeptor-Repertoires, bei der die TCR Vb-Elemente 14 und 16 überexprimiert waren. Das Adjuvant TiterMax beeinflusste kaum das TCR Vb-Repertoire. Die Immunisierung mit dem allogenen Peptid P1 führte zu einer eindeutigen Beeinflussung des T-Zellrezeptor-Repertoires. Popliteale T-Lymphozyten, die 7 Tage nach Immunisierung analysiert wurden, zeigten ein Repertoire, bei dem die TCR Vb-Elemente 15, 16, 17 und 20 überexprimiert waren. Dieses Repertoire war am Tag 3 nach Immunisierung noch nicht so ausgebildet. Wurden die antigenspezifischen T-Lymphozyten nach ihrer Isolierung mit P1 in vitro restimuliert, so waren in diesem T-Zellrezeptor-Repertoire die TCR Vb-Elemente 8.3, 15, 16 und 20 überexprimiert. Zum Vergleich: in naiven T-Lymphozyten waren die Vb-Elemente 2, 4 und 9 überexprimiert. Damit war es zu einer deutlichen Verschiebung im T-Zellrezeptor-Repertoire antigenspezifischer T-Lymphozyten gekommen, die auf das Peptidantigen P1 zurückzuführen ist. Mit der Methode des PCR-ELISA wurde das T-Zellrezeptor-Repertoire antigenspezifischer T-Lymphozyten bestimmt. Hiermit sind wesentliche Voraussetzungen geschaffen worden, um T-Zellklone zu etablieren und ihre Bedeutung für die Transplantatabstoßung genauer zu untersuchen. / Organ transplantation is an important medical therapy for patients with irreversibly damaged organs. However, this therapeutic intervention has a great disadvantage because the long-term success of organ allografts is still too often endangered by the so called allograft rejection, an immune response directed to the allograft and leading to its destruction. The major approach for the prevention and management of allograft rejection is to suppress the immune system with immunosuppressive agents. These agents were introduced into transplantation medicine in the 1960s and since that time they have been continually improved. However, their side effects remain a seriousness problem because the suppression of the immune defence inhibits the allograft rejection on the one hand but causes infections and increases tumour incidence on the other hand. The allograft rejection is mediated by CD4+ T lymphocytes and the responsible antigens recognized by them are allogenic peptides processed from donor MHC molecules. Although a large number of allgenenic peptides are involved in allograft rejection, it is possible to identify certain peptide antigens involved in allograft rejection. In our group allogeneic peptides from MHC class I molecules from Wistar Furth rats were investigated. The significance of the allogeneic peptide P1, consisting of 19 amino acids, in inducing allograft rejection was analysed in detail. P1-specific T lymphocytes demonstrated a Th1-cytokine dominated profile and accelerated the rejection of allografts in Lewis rats donated by Wistar Furth rats. The aim of this study was to characterise the T cell receptor (TCR) Vb repertoire of P1-specific T lymphocytes with the PCR-ELISA technique. First, thymocytes and T lymphocytes from different lymph nodes were analysed. Thymocytes expressed all 22 TCR Vb elements and only TCR Vb14 was overrepresented. The T lymphocytes of cervical, mesenteric, iliacal und popliteal lymph nodes also demonstrated a certain repertoire of overrepresented TCR Vb elements. In cervical T lymphocytes the expression levels of the TCR Vb elements 2, 6, 8.3 and 16 were increased; in mesenteric T lymphocytes the TCR Vb elements 2, 4 and 8.1; in illiacal T lymphocytes the TCR Vb elements 2 and 6; and in popliteal T lymphocytes the TCR Vb elements 2, 4 and 9. The immunisation with the autoantigen Ac led to a small variation of the TCR Vb repertoire and the TCR Vb elements 14 and 16 were overrepresented. The adjuvant TiterMax did not influence the TCR Vb repertoire. In contrast, the immunisation with the allogeneic peptide P1 clearly influenced the T cell receptor repertoire. Popliteal T lymphocytes analysed 7 days after immunisation demonstrated a TCR Vb repertoire where the TCR Vb elements 15, 16, 17 und 20 were overrepresented. On day 3 after immunisation this repertoire was not yet clearly developed. In P1-specific T lymphocytes restimulated in vitro the expression levels of the TCR Vb elements 8.3, 15, 16 and 20 were increased. In comparison, in naïve T lymphocytes the TCR Vb elements 2, 4 and 9 were overrepresented. These results underline that the immunisation with peptide P1 induces a characteristic TCR Vb repertoire. The TCR Vb repertoire of P1-specific T lymphocytes was analysed with the PCR-ELISA technique. The determination of the T cell receptor repertoire is a prerequisite to establish T cell clones and to analyse their involvement in allograft rejection.

Os objetivos desse estudo foram: 1) Padronizar a pesquisa do anti-tTg, comparando-o com o anticorpo antiendomísio (AAE) e 2) Avaliar as interações da tTg com a fibronectina. 49 celíacos e 124 controles com AAE negativo foram avaliados. O AAE foi pesquisado por imunofluorescência indireta e a reatividade contra a tTg e a fibronectina por ELISA in house e com kits comerciais. O antitTg foi positivo em 46,9% e 100% dos celíacos com o ELISA in house e com kits comerciais, respectivamente. A adição de fibronectina não melhorou a sensibilidade do ELISA. Em conclusão: a detecção do antitTg por ELISA apresenta percentual elevado de falso-positivos, não podendo substituir a pesquisa do AAE / The aims of the current study were: to standardize the detection of anti-tTg antibodies, comparing them with antiendomysial antibodies (EMA) and to assess the interaction of tTg with fibronectin. 49 celiac patients and 124 controls were enrolled. EMA was detected by indirect immunofluorescence reaction and tTg and fibronectin reactivity by in house ELISA and with commercially available kits. Seropositivity to anti-tTG was found in 46.9% and 100% of patients by the in house technique and by commercial kits, respectively. Fibronectin addition did not improve the ELISA sensibility. In conclusion, ELISA for anti-tTG detection has a high rate of false positive results and does not replace EMA

No indivíduo adulto, a angiogênese ocorre particularmente em situações patológicas como nos tumores em crescimento, no desenvolvimento de metástases e no processo de cicatrização, sendo o fator de crescimento do endotélio vascular (VEGF) o principal fator envolvido na angiogênese tumoral. Por essa razão, o presente estudo teve como objetivo avaliar os níveis circulantes de VEGF no soro de cães com sarcoma de tecidos moles e sua relação com as células sanguineas, comparados com um grupo de animais sem câncer (controle), a expressão do VEGF e a densidade de microvasos nos espécimes tumorais dos cães com sarcoma de tecidos moles. O grupo controle foi composto de 30 cães machos e o grupo de animais com sarcoma de tecidos moles foi de 25 cães (18 machos e 7 fêmeas castradas) os quais foram avaliados prospectivamente. A coleta sangüínea foi realizada apenas uma vez no grupo controle e em três tempos nos animais com sarcoma (pré-operatório, duas semanas e três meses de pós-operatório) da mesma maneira. Após a colheita o sangue foi processado, para extração do soro e determinação dos níveis de VEGF a partir de um método quantitativo ELISA (enzime-linked immunosorbent assay). A expressão do VEGF e a densidade de microvasos foi investigada por meio da prova de imunoistoquiímica utilizando-se anticorpos anti-VEGF e anti-fator VIII respectivemente. Não houve diferença entre o nível sérico de VEGF dos animais controles e portadores de sarcoma de tecidos moles no tempo pré-operatório. O nível de VEGF sérico no pré-operatório mostrou-se discretamente aumentado em relação a duas semanas e três meses de pós-operatório. Houve correlação positiva entre VEGF sérico e contagem neutrófilos e correlação negativa entre o VEGF e quantidade de hemoglobina nos animais com sarcoma. Houve uma tendência dos animais com hemangiopericitoma apresentarem níveis maiores de VEGF sérico em relação aos portadores de tumor maligno da bainha neural periférica. Houve expressão do VEGF em 65% dos casos, sendo o hemangiopericitoma aquele que expressou maior quantidade de VEGF intratumoral. Não houve diferença na densidade de microvasos entre os tumores negativos e positivos ao VEGF. Os resultados encontrados sugerem contribuição das células do sangue circulante para os níveis de VEGF do soro de cães portadores de sarcomas de tecidos moles, células tumorais e outros tipos celulares parecem ser responsáveis pela angiogênese tumoral, mas não contribuem para elevação da concentração sérica desse fator. / In adult individual, angiogenesis occurs particularly in pathological situations as tumors growth, development of metastasis and in the wound healing process. The vascular endothelial growth factor (VEGF) is the main agent involved in tumor angiogenesis. Therefore, the aim of the present study was to evaluate the circulating levels of VEGF in the serum of dogs suffering from soft tissue sarcoma and its relation with the blood cells, and also the expression of VEGF and the micro vascular density in tumors specimens. A group of health animals was used as control. The control group and treatment group were composed of 30 male dogs 25 dogs (18 males and 7 castrated females) respectively. The blood collection was carried once in the control group and three times in the animals with sarcoma (timely, pre-surgery, two weeks and three months post-surgery) following the same protocol. The blood was processed and a quantitative method ELISA (enzime-linked immunosorbent assay) was used to determinate of the levels of VEGF. The expression of the VEGF and the microvascular density were investigated by means of the immunohistochemical test using anti-VEGF and anti-factor VIII antibodies respectively. There was no difference between serum level of VEGF of the controls animals and serum level of VEGF in the animal with soft tissue sarcoma in pre-surgical time. Serum level of VEGF in pre-surgical time revealed discrete increased in relation the two weeks and three months of post-surgery. There was a positive correlation between serum VEGF and neutrophils counting and negative correlation between the VEGF and amount of haemoglobin in the animals with sarcoma. The animals with hemangiopericytoma showed a trend to higher levels of VEGF in relation to the malignant peripheral nerve sheath tumor. The expression of the VEGF was detected in 65% of the cases; the hemangiopericytoma is the one that expressed greater intratumoral amount of VEGF. There was no difference in the microvascular density between the negative and positive tumors to the VEGF. The results suggest contribution of the circulating blood cells for the levels of serum VEGF of the of the dogs with of soft tissue sarcomas, tumors cells and other cells types seem to be responsible for tumor angiogenesis, but they don\'t contribute for rise serum level of VEGF.

INTRODUÇÃO: Apesar de ser a droga de primeira escolha na prevenção da hemorragia pós-parto, o uso da ocitocina em cesarianas permanece empírico. O objetivo deste estudo foi dosar a ocitocina sérica após a administração profilática de diferentes regimes de ocitocina em pacientes submetidas à cesariana eletiva. MÉTODOS: 30 pacientes que se apresentaram para cesariana eletiva foram randomizadas para receber ocitocina intravenosa, após o clampeamento do cordão umbilical, nos seguintes grupos: G1 (n=9): 10 UI de ocitocina infundidas em 30 minutos (0,33 UI/min), G2 (n=11): 10 UI de ocitocina infundidas em 3 minutos e 45 segundos (2,67 UI/min) e G3 (n=10): 80 UI de ocitocina infundidas em 30 minutos (2,67 UI/min). Este estudo foi encoberto para as pacientes e para os cirurgiões. A avaliação do tono uterino foi realizada pela equipe cirúrgica e a dosagem da concentração sérica de ocitocina foi feita pela técnica de enzimoimunoensaio (ELISA), antes da anestesia (T0) e nos tempos 5 (T5), 30 (T30) e 60 (T60) minutos após o início da infusão da ocitocina. RESULTADOS: Os níveis de ocitocina sérica (média ± erro padrão, ng/mL) foram semelhantes entre os grupos em T0 (0,062 ± 0,021; 0,039 ± 0,019 e 0,067 ± 0,041; respectivamente, p = 0,76) e em T60 (0,648 ± 0,257; 0,356 ± 0,257 e 0,683 ± 0,257; respectivamente, p = 0,58). G3 apresentou níveis maiores de ocitocina que G1 em T5 (3,651 ± 0,741 versus 0,709 ± 0,268; p = 0,01). Em T30, G3 apresentou níveis de ocitocina sérica maiores que G1 (6,190 ± 1,195 versus 1,174 ± 0,375; p < 0,01) e, também, que G2 (6,190 ± 1,195 versus 0,411 ± 0,206; p < 0,01). Os parâmetros hemodinâmicos foram semelhantes entre os grupos. O tono uterino foi considerado satisfatório em todos os intervalos estudados e não houve a necessidade de utilização de uterotônico complementar. CONCLUSÃO: Foram demonstradas dosagens séricas de ocitocina por ELISA em gestantes submetidas à cesariana eletiva. A administração de 80 UI de ocitocina em 30 minutos resulta em níveis séricos de ocitocina maiores que os outros dois métodos de administração aos 5 e 30 minutos, porém, estas concentrações não diferem aos 60 minutos / BACKGROUND: The use of oxytocin to prevent postpartum hemorrhage after elective cesarean delivery still remains empirical. The purpose of this study was to determine oxytocin serum levels following differents regimens of prophylactic oxytocin administration in pregnant women undergoing elective cesarean delivery. METHODS: 30 healthy pregnant patients were randomized to receive intravenous oxytocin, after clamping of the umbilical cord, into the following groups: G1 (n=9), 10 IU of oxytocin infused over 30 minutes (0.33 IU/min); G2 (n=11), 10 IU of oxytocin infused over 3 minutes and 45 seconds (2.67 IU/min) and G3 (n=10), 80 IU of oxytocin infused over 30 minutes (2.67 IU/min). Both patient and surgeon were blinded to the study group allocation. Uterine tone was assessed by palpation by the surgeon. Serum oxytocin concentration was determined by enzyme immunoassay (EIA) before anesthesia (T0) and at 5 (T5), 30 (T30) and 60 (T60) minutes following the start of oxytocin infusion. RESULTS: Serum oxytocin levels (mean ± standard error, ng/mL) were similar in the groups at T0 (0.062 ± 0.021, 0.039 ± 0.019 and 0.067 ± 0.041, respectively, P = 0.76), and T60 (0.648 ± 0.257, 0.356 ± 0.257 and 0.683 ± 0.257, respectively, P = 0.58). G3 presented higher serum oxytocin than G1 at T5 (3.651 ± 0.741 versus 0.709 ± 0.268, P = 0.01). At T30, serum oxytocin levels of G3 were higher than G1 (6.190 ± 1.195 versus 1.174 ± 0.375, P < 0.01) and also than G2 (6.190 ± 1.195 versus 0.411 ± 0.206, P < 0.01). Hemodynamic data were similar in all groups. Uterine tone was considered satisfactory in all intervals studied and no additional uterotonic agent was needed. CONCLUSION: We demonstrate serum oxytocin determinations by EIA in healthy pregnant women presented for elective cesarean delivery. Administering 80 IU in 30 min results in higher serum oxytocin levels at 5 and 30 min than the other two methods of oxytocin administration, but the concentrations did not differ at 60 min

O crescimento cada vez maior da industrialização e a existência de um mercado globalizado vêm exigindo dos produtores rurais à utilização de modernas tecnologias ligadas à produção animal. O baixo custo de produção e o aumento da qualidade do produto final gerado têm sido a grande meta dos pecuaristas e centros de pesquisa voltados à agropecuária. Devido à necessidade de aumento na produção, propriedades agropecuárias têm recorrido ao uso de todo e qualquer recurso disponível que possa promover melhorias na atividade produtiva.O uso indiscriminado dos antimicrobianos com a intenção de aumentar a produção animal pode resultar em concentrações residuais nos produtos como carne, ovos e leite acima das doses aceitáveis para o consumo humano. Inicialmente, os antibióticos eram usados como medida terapêutica, mas com o avanço do conhecimento e desenvolvimento de novos compostos, passaram a ser utilizados também como medida preventiva e como promotores do crescimento.O que se deve questionar não é a presença destes resíduos, mas sim o tipo e as concentrações dos mesmos. De fato, as modernas tecnologias analíticas têm permitido a detecção de partes por bilhão de substâncias químicas, componentes ou metabólitos de medicamentos de uso veterinário em alimentos e, nestas quantidades, dificilmente elas representariam perigo à saúde dos consumidores. Portanto, há necessidade de se estabelecer metodologias analíticas mais eficientes que possam auxiliar os órgãos de fiscalização da agricultura no controle do uso desses antimicrobianos na produção animal. Assim como auxiliar no controle de qualidade, principalmente em indústrias exportadoras que almejam expansão de mercado, onde essa prática é quase uma imposição no contexto do comércio internacional de produtos pecuários \"in natura\" e processados. Devido a essas necessidades, o objetivo do presente foi padronizar e desenvolver reagentes imunoenzimáticos para a pesquisa da ciprofloxacina em produtos de origem animal. Os resultados obtidos no desenvolvimento desses imunoreagentes foram positivos quando correlacionado (r2 = 0,9588) com o kit referência RIDASCREEN® Enro/Cipro da R-Biopharm. Diante disso, é possível concluir que o Brasil possui infra-estrutura adequada para padronizar esses testes rápidos, evitando com isso um grande gasto na importação desses kits / The growth each time higher of industrialization and the existence of a global market forces the rural producers to use modern technologies related to animal production. The low production cost and the increasing of obtained final product quality have been the great aim of cattle farmers and cattle farming research centers. Due to the necessity of increasing the production, rural properties have required the use of any available source able to promote better productive activities. The indiscriminated antibiotics use aiming to increase animal production may result in residual concentrations found in products such as meat, eggs and milk over acceptable doses to the human consumption. Firstly, antibiotics were used as therapeutic prescriptions although the advances on knowledge and development of new compounds were also used as a preventive way and as growth promoters. What must be questionable its not the presence of these residuals but surely what kind of residual and their concentrations. In fact, the modern analytic technologies make possible the detection of parts per billions (ppb) of chemical substances, compounds or medicine metabolites of veterinary use in food and in these quantities, hardly they would be dangerous to the consumer health. However, it\'s necessary to establish more efficient analytic methodologies to help on agriculture supervising organs in the control of these antibiotics use in the animal production. Thus, how to help in the quality control, mainly in industries that export to get the market expansion, where this activity is almost an imposition at the international trade context of farming products \"in natura\" and industrialized ones. Due to these necessities, the goal of our present work was to standard and develop immunoenzymatics reagents to the ciprofloxacin researching from animal products. The results obtained on development of these immunoreagents were positive when correlated (r2= 0,9588) to the kit RIPASCREEN® Enro/Cipro from R-Biopharm reference. At the light of these knowledge, it?s possible to conclude that Brazil has adequate proper-structure to standardize these quick tests, to avoid in this way a high cost to import these kits.

INTRODUÇÃO: Apesar de ser a droga de primeira escolha na prevenção da hemorragia pós-parto, o uso da ocitocina em cesarianas permanece empírico. O objetivo deste estudo foi dosar a ocitocina sérica após a administração profilática de diferentes regimes de ocitocina em pacientes submetidas à cesariana eletiva. MÉTODOS: 30 pacientes que se apresentaram para cesariana eletiva foram randomizadas para receber ocitocina intravenosa, após o clampeamento do cordão umbilical, nos seguintes grupos: G1 (n=9): 10 UI de ocitocina infundidas em 30 minutos (0,33 UI/min), G2 (n=11): 10 UI de ocitocina infundidas em 3 minutos e 45 segundos (2,67 UI/min) e G3 (n=10): 80 UI de ocitocina infundidas em 30 minutos (2,67 UI/min). Este estudo foi encoberto para as pacientes e para os cirurgiões. A avaliação do tono uterino foi realizada pela equipe cirúrgica e a dosagem da concentração sérica de ocitocina foi feita pela técnica de enzimoimunoensaio (ELISA), antes da anestesia (T0) e nos tempos 5 (T5), 30 (T30) e 60 (T60) minutos após o início da infusão da ocitocina. RESULTADOS: Os níveis de ocitocina sérica (média ± erro padrão, ng/mL) foram semelhantes entre os grupos em T0 (0,062 ± 0,021; 0,039 ± 0,019 e 0,067 ± 0,041; respectivamente, p = 0,76) e em T60 (0,648 ± 0,257; 0,356 ± 0,257 e 0,683 ± 0,257; respectivamente, p = 0,58). G3 apresentou níveis maiores de ocitocina que G1 em T5 (3,651 ± 0,741 versus 0,709 ± 0,268; p = 0,01). Em T30, G3 apresentou níveis de ocitocina sérica maiores que G1 (6,190 ± 1,195 versus 1,174 ± 0,375; p < 0,01) e, também, que G2 (6,190 ± 1,195 versus 0,411 ± 0,206; p < 0,01). Os parâmetros hemodinâmicos foram semelhantes entre os grupos. O tono uterino foi considerado satisfatório em todos os intervalos estudados e não houve a necessidade de utilização de uterotônico complementar. CONCLUSÃO: Foram demonstradas dosagens séricas de ocitocina por ELISA em gestantes submetidas à cesariana eletiva. A administração de 80 UI de ocitocina em 30 minutos resulta em níveis séricos de ocitocina maiores que os outros dois métodos de administração aos 5 e 30 minutos, porém, estas concentrações não diferem aos 60 minutos / BACKGROUND: The use of oxytocin to prevent postpartum hemorrhage after elective cesarean delivery still remains empirical. The purpose of this study was to determine oxytocin serum levels following differents regimens of prophylactic oxytocin administration in pregnant women undergoing elective cesarean delivery. METHODS: 30 healthy pregnant patients were randomized to receive intravenous oxytocin, after clamping of the umbilical cord, into the following groups: G1 (n=9), 10 IU of oxytocin infused over 30 minutes (0.33 IU/min); G2 (n=11), 10 IU of oxytocin infused over 3 minutes and 45 seconds (2.67 IU/min) and G3 (n=10), 80 IU of oxytocin infused over 30 minutes (2.67 IU/min). Both patient and surgeon were blinded to the study group allocation. Uterine tone was assessed by palpation by the surgeon. Serum oxytocin concentration was determined by enzyme immunoassay (EIA) before anesthesia (T0) and at 5 (T5), 30 (T30) and 60 (T60) minutes following the start of oxytocin infusion. RESULTS: Serum oxytocin levels (mean ± standard error, ng/mL) were similar in the groups at T0 (0.062 ± 0.021, 0.039 ± 0.019 and 0.067 ± 0.041, respectively, P = 0.76), and T60 (0.648 ± 0.257, 0.356 ± 0.257 and 0.683 ± 0.257, respectively, P = 0.58). G3 presented higher serum oxytocin than G1 at T5 (3.651 ± 0.741 versus 0.709 ± 0.268, P = 0.01). At T30, serum oxytocin levels of G3 were higher than G1 (6.190 ± 1.195 versus 1.174 ± 0.375, P < 0.01) and also than G2 (6.190 ± 1.195 versus 0.411 ± 0.206, P < 0.01). Hemodynamic data were similar in all groups. Uterine tone was considered satisfactory in all intervals studied and no additional uterotonic agent was needed. CONCLUSION: We demonstrate serum oxytocin determinations by EIA in healthy pregnant women presented for elective cesarean delivery. Administering 80 IU in 30 min results in higher serum oxytocin levels at 5 and 30 min than the other two methods of oxytocin administration, but the concentrations did not differ at 60 min

Aberrant expression of short non-coding micro RNAs (miRNA) in many human diseases, along with remarkable stability in physiological media, has made them attractive clinical biomarkers. In particular, miRNA-122 is substantially elevated in plasma of patients with established drug-induced liver injury and can also be used to identify early liver injury when current markers, such as alanine aminotransferase (ALT), still show normal levels. The development of a rapid test for miRNA-122 e.g. in drug poisoning would allow earlier and more sensitive clinical diagnosis of liver injury. Nucleic acids are traditionally analysed by polymerase chain reaction (PCR), which has a high degree of sensitivity but suffers from high cost and is prone to sample contamination. The aim of this project is to develop a PCR free method to directly detect miRNA- 122 in biological samples using SMART technology. The SMART technology takes advantage of dynamic chemistry for sequence specific recognition of nucleic acids using aldehyde-modified nucleobases (SMART nucleobases), and target-complementary peptide nucleic acid (PNA) probe containing an “abasic” position (so called modified PNA probe). In this study, this unique detection method was used in a fluorescent detection with the use of light up probes, which are probes with an environmental dye as nucleobase; a FRET system was also designed to allow the discrimination between perfect match target and mismatched one. The SMART technology was also transferred onto magnetic beads to develop an ELISA like assay allowing sensitive and rapid detection of single stranded DNA mimic of the miRNA-122. With its potential PCR free approach, this easily adapted platform promises to transform and expand routine clinical diagnostic testing and screening for circulating miRNAs.

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Mycoplasmas conform most closely with the theoretical concept of 'minimum cells', existing as the smallest, free-living organisms capable of self-replication. They survive as parasites of plants, insects, animals or humans, with the most common human colonising species being Mycoplasma hominis. M. hominis has been characterised as a human pathogen responsible for a variety of infections, which pose a significant threat particularly to immunocompromised patients and neonates. However little has been elucidated about the cell physiology and molecular structure of this organism. Of interest to this study were the investigation of the heat shock response of M. hominis and the diagnostic assays used for its detection. The heat shock response is a ubiquitous physiological feature of all organisms and displays unprecedented conservation. This phenomenon is particularly evident in the 70 kDa family of heat shock proteins (hsp70) which exhibits a high degree of homology between different species. The hsp70 gene from M. hominis was cloned and preliminary partial sequencing indicated the similarity with other hsp70 homologs. The regulation of hsp70 expression at the transcriptional and translational levels was investigated. The level of hsp70 mRNA was found to increase correspondingly in response to heat shock, more visibly than the level of hsp70 protein. However imrnunochemical studies of the M. hominis hsp70 translation product demonstrated further the homology with other species. To facilitate rapid diagnosis of M. hominis infections, a PCR ELISA diagnostic assay was developed and optimised. The amplification of a conserved region of the M. hominis 16S rRNA gene was linked to subsequent hybridisation to an appropriate capture probe in a microtiter plate format. The sensitivity of the assay was comparable to other molecular assays although the PCR ELISA produces more rapid results and is less labour intensive. / Thesis (M.Sc.)-University of Natal, Pietermaritzburg, 1998.

Biological/biochemistry analyses traditionally require bulky instruments and a great amount of volume of biological/chemical agents, and many procedures have to be performed in certain locations such as medical centers or research institutions. These limitations usually include time delay in testing. The delays may be critical for some aspects such as disease prevention or patient treatment. One solution to this issue is the realization of point-of-care (POC) testings for patients, a domain in public health, meaning that health cares are provided near the sites of patients using well-designed and portable medical devices. Transportation of samples between local and central institutions can therefore be reduced, facilitating early and fast diagnosis. A closely related topic in engineering, lab-on-a-chip (LOC), has been discussed and practiced in recent years. LOC emphasizes integrating several functions of laboratory processes in a small portable device and performing analysis using only a very small amount of sample volume, to achieve low-cost and rapid analysis. From an engineer's point of view, LOC is the strategy to practice the idea of POC testing. This dissertation aimed at exploring the POC potentials of porous membrane-base LOC devices, which can be used to simplify traditional and standard laboratory procedures. In this study, three LOC prototypes are shown and discussed. First the protein sensor incorporating with silica nanofiber membrane, which has shown 32 times more improvement of sensitivity than a conventional technique and a much shorter detection time; secondly the bacteria filter chip that uses a sandwiched aluminum oxide membrane to stabilize the bacteria and monitor the efficacy of antibiotics, which has reduced the test time from 1 day of the traditional methods to 1 hour; the third is the sensor combining microfluidics and silica nanofiber membrane to realize Surface Enhanced Raman Spectroscopy on bio-molecules, which has enhancement factor 10^9 and detection limit down to nanomolar, but simple manufacturing procedures and reduced fabrication cost. These results show the porous-base membrane LOC devices may have potentials in improving and replacing traditional detection methods and eventually be used in POC applications.