Objetivos: Determinar os efeitos celulares e o mecanismo de ação da mitomicina C tópica na prevenção e tratamento da opacidade corneana em coelhos submetidos à ceratectomia fotorrefrativa (PRK). Métodos: Foram submetidos à cirurgia de PRK 224 coelhos para correção de -9 dioptrias esféricas, associada à aplicação de mitomicina C tópica ou solução salina balanceada. O nível de opacidade corneana foi avaliado por meio de análise à lâmpada de fenda. Os animais foram sacrificados quatro horas, 24 horas, quatro semanas e seis meses após a cirurgia. A análise imunohistoquímica foi realizada com as técnicas de TUNEL e foram utilizados os anticorpos Ki67 e alpha-SMA para a análise da apoptose celular, replicação celular e formação de miofibroblastos, respectivamente. Resultados: Todos os grupos submetidos à aplicação de mitomicina C apresentaram um maior número de células positivamente marcadas pelo ensaio com TUNEL (indicando maior taxa de apoptose celular) e um menor número de células positivamente marcadas pelo anticorpo Ki67 (indicando menor taxa de replicação celular). Uma menor quantidade de miofibroblastos (células positivamente marcadas pelo anticorpo alpha-SMA) foi identificada após a aplicação profilática da mitomicina C, comparada com sua aplicação com finalidade terapêutica. Além disso, identificou-se uma zona de acelularidade no estroma anterior de córneas tratadas com mitomicina C, persistente por um período mínimo de seis meses. Conclusões: A aplicação da mitomicina C diminuiu signficativamente a formação de opacidade corneana em coelhos. Apesar da mitomicina C ter induzido uma maior apoptose de ceratócitos e miofibroblastos, seu principal mecanismo de ação, responsável pela prevenção da opacidade corneana, decorreu do bloqueio da replicação dos ceratócitos ou outras linhagens celulares progenitoras dos miofibroblastos. A aplicação da mitomicina C na concentração de 0,002% mostrou-se tão eficiente quanto sua aplicação na concentração de 0,02%. Não obstante, uma persistente diminuição da densidade de ceratócitos no estroma anterior pode representar um sinal de alerta para possíveis complicações a longo prazo / Purpose: To determine cellular effects and the mechanism through which topical mitomycin C prevents and treats corneal haze after photorefractive keratectomy (PRK) in rabbits. Methods: Minus nine diopters PRK with mitomycin C or balanced salt solution was performed in two hundred and twenty four New Zealand rabbits. Haze level was graded at the slit lamp. Rabbits were sacrificed at 4 hours, 24 hours, 4 weeks or 6 months after surgery and immunohistochemistry was performed with TUNEL assay, Ki67 and alpha-SMA to analyze keratocyte cells apoptosis, keratocyte cells replication and myofibroblast cells formation, respectively. Results: TUNEL-positive cells increased in all mitomycin C groups (representing more keratocyte cells undergoing apoptosis) while Ki67-positive cells decreased significanlty (representing a decreased keratocyte cells replication) following mitomycin C application. A greater decrease in myofibroblasts was noted with prophylactic mitomycin C treatment than therapeutic mitomycin C treatment. There was, however, an anterior stromal acellular zone in eyes treated with mitomycin C that persisted out to the maximum follow-up of 6 months. Conclusion: Mitomycin C application significantly reduced corneal haze formation in rabbits. Its treatment induces apoptosis of keratocytes and myofibroblasts, but the predominate effect in inhibiting or treating haze appears to be at the level of blocked replication of keratocytes or other progenitor cells of myofibroblasts. Treatment with 0.002% mitomycin C appears to be just as effective as higher concentrations (0.02%) in the rabbit model. However, a persistent decrease in keratocyte cells density in the anterior stroma could be a warning sign for future complications

In der vorliegenden Arbeit wurde ein onkolytisches Vaccinia-Virus unter Ausnutzung seiner Eigenschaft als Vektorsystem mit dem Designer-Zytokin Hyper-IL-6 ausgestattet (GLV 1h90). Bei Hyper IL 6 handelt es sich um ein Fusionsprotein bestehend aus humanem Interleukin-6 und der Liganden-Bindungsdomäne des löslichen Interleukin-6-Rezeptors, welche kovalent über einen flexiblen Linker miteinander verbunden sind. Dieses chimäre Designer-Zytokin erlaubt die Untersuchung von IL-6-Effekten, welche über das IL-6-Trans-Signaling vermittelt werden. Daraus ergibt sich einerseits eine beträchtliche Erweiterung des Wirkspektrums und darüber hinaus weist Hyper-IL-6 sowohl in vitro als auch in vivo eine 100-1000fach verstärkte biologische Aktivität auf. Aufgrund der Tatsache, dass Hyper-IL-6, neben seiner Tumor-inhibierenden Wirkung, eine Vielzahl weiterer Effekte zugeschrieben wird, wurde in dieser Arbeit durch die Kombination des Designer-Zytokins mit einem onkolytischen Vaccinia-Virus nicht nur additive Effekte auf die Tumorregression, sondern darüber hinaus auch mögliche systemisch-vermittelte Hyper-IL-6-Effekte untersucht. Nach intravenöser Injektion von GLV-1h90 in DU-145-Tumor-tragende Mäuse konnte neben der intratumoralen Replikation des Virus und der Expression des Markerproteins Ruc-GFP zusätzlich die Expression des integrierten Designer-Zytokins Hyper-IL-6 im Tumor nachgewiesen werden. Von entscheidender Bedeutung war der zusätzliche Nachweis des Designer-Zytokins in Serum-Proben von GLV-1h90-injizierten Mäusen. Nach einer aktiven Hyper-IL-6-Sekretion von infizierten Tumorzellen, bildet der Transport in die Blutbahn die Voraussetzung für systemisch-vermittelte Hyper-IL-6-Effekte. In dieser Arbeit wurde untersucht, ob sich durch die Überexpression von Hyper-IL-6 im Tumor, zusätzlich zu den onkolytischen Eigenschaften des Vaccinia-Virus, additive anti-Tumor-Effekte ergeben. Eine systemische Injektion von GLV 1h90 bzw. GLV 1h68 in DU-145-Tumor-tragende Mäuse führte zu einer signifikanten Reduktion des Tumorvolumens im Vergleich zu PBS-injizierten Mäusen. Neben Effekten, welche mit Entzündungsprozessen assoziiert sind, wie eine Rotfärbung der Haut, eine signifikanten Vergrößerung der Leber sowie eine massive Stimulation der Akute-Phase-Antwort in der Leber, konnte in GLV-1h90-injizierten Mäusen ein verbesserter Gesundheitszustand auf der Basis einer signifikanten Gewichtszunahme, verbunden mit einer beschleunigten Wundheilung Virus-induzierter Schwanzläsionen, beobachtet werden. Darüber hinaus konnte für Hyper-IL-6 eine Stimulierung der Megakaryopoese im Knochenmark nachgewiesen werden, welche zu einer signifikanten Erhöhung der Thrombozyten-Zahl im Blutkreislauf von GLV-1h90-injizierten Mäusen führte. Es ist von entscheidender Bedeutung anzumerken, dass alle beobachteten systemischen Hyper-IL-6-Effekte eine zeitliche Limitierung aufwiesen, welche sich höchstwahrscheinlich auf die Virus-bedingte Zerstörung Hyper IL 6-produzierender Tumorzellen zurückführen lässt. Dies impliziert zudem, dass eventuelle Komplikationen, welche durch die Überexpression des Designer-Zytokins hervorgerufen werden können, ebenfalls selbstlimitierend sind. Es konnte bereits mehrfach gezeigt werden, dass eine Kombinationstherapie aus onkolytischen Viren und Chemotherapie über synergistische Effekte zu einer signifikant verbesserten Tumorregression führt. Allerdings kommt es in Folge einer Chemotherapie oft zu einer Vielzahl von gefährlichen Nebenwirkungen, da alle schnell proliferierenden Zellen des Körpers betroffen sind. Thrombozytopenie ist eine der am häufigsten vorkommenden Nebenwirkung und beschreibt eine massive Reduktion der Thrombozyten-Zahl im Blut. Im Hinblick auf eine mögliche klinische Anwendung von GLV 1h90 wurde deshalb untersucht, ob in einer Kombinationstherapie mit Mitomycin C, neben einer Verstärkung der therapeutischen Effekte des Virus, basierend auf den beobachteten Hyper-IL-6-Effekten, zusätzlich der Gesundheitszustand der behandelten Mäuse verbessert werden kann. Die Experimente belegen, dass eine Kombination onkolytischer Vaccinia-Virus-Konstrukte mit Mitomycin C zu einer signifikant verbesserten Tumorregression im Vergleich zu den jeweiligen Monotherapien führt. Von bedeutender Relevanz war die Beobachtung, dass in einer Kombinationstherapie von Mitomycin C und GLV-1h90, im Gegensatz zu GLV-1h68, eine signifikante zeitliche Verkürzung der auftretenden Thrombozytopenie erreicht wird. Zusammenfassend konnte in dieser Arbeit gezeigt werden, dass eine systemische Injektion von GLV-1h90 zu einer funktionellen Expression des Designer-Zytokins Hyper-IL-6 führte, welches in der Lage ist eine erfolgreiche Kombinationstherapie aus einem onkolytischen Vaccinia-Virus und dem Chemotherapeutikum Mitomycin C durch eine Reduktion der Nebenwirkungen zusätzlich zu optimieren. / In this thesis, an oncolytic vaccinia virus was armed with the designer cytokine Hyper-IL-6 by recombinant integration (GLV-1h90), exploiting its features as a vector system. Hyper IL-6 is composed of human interleukin-6 (IL-6) and the cytokine-binding domain of its soluble receptor sIL-6R which are bond covalently by a flexible peptide linker. Hyper-IL-6 is a multifunctional cytokine which exhibits not only anti-tumor activity, but also a variety of other effects. For this reason, the combination of the designer cytokine and an oncolytic vaccinia virus was used to study possible improvements regarding tumor regression and more importantly additional systemically mediated Hyper IL-6 effects. In addition to intratumoral replication and visualization of the marker gene ruc-gfp, intratumoral expression of the inserted designer cytokine Hyper-IL-6 could be detected after systemic administration of GLV-1h90 into DU-145-tumor-bearing mice. Of special interest was the presence of hyper-IL-6 in blood serum samples of GLV-1h90-injected mice. Following active hyper-IL-6 secretion of infected tumor cells, the transport into the blood circulation is essential for its ability to induce signal transduction pathways outside the tumor. IL-6 is a pro-inflammatory cytokine which is postulated to exhibit both, tumor promoting as well as tumor inhibiting effects. However, growth or proliferation inhibition of tumors could only be observed after addition of soluble IL-6 receptor and is consequently associated with the IL 6-trans-signaling pathway. Therefore, the thesis deals with the question of whether overexpression of hyper-IL-6 can further enhance the pre-existing oncolytic effects of vaccinia virus. Systemic administration of either GLV-1h90 or GLV-1h68 led to significant tumor regression compared to PBS-treated mice. Comparison of the two viral constructs demonstrated a slightly increased oncolytic activity of GLV-1h90. However, further studies have to clarify to which extend this improvement is resulting from an intratumoral overexpression of hyper IL 6. Following the detection of hyper-IL-6 in the blood circulation as a consequence of GLV 1h90-mediated overexpression in the tumor, functionality of the designer cytokine was analyzed regarding systemically mediated effects. Besides effects which can be associated with inflammatory processes, such as red skin, significant enlargement of the liver as well as enormous stimulation of the acute-phase-response, GLV-1h90-injected mice showed improved healthiness. Health status was assessed by significant gain in body weight associated with accelerated epithelial barrier repair of virus-induced tail lesions. Moreover, it could be demonstrated that Hyper-IL-6 stimulates megakaryopoiesis in the bone marrow, which in turn leads to significantly elevated levels of blood platelets in GLV-1h90-injected mice. It is particularly important to note that all observed systemic Hyper-IL-6 effects occurred only temporarily, which could be explained by virus-mediated oncolysis, reducing the amount of viable Hyper-IL-6 producing tumor cells. The results also implicate that potential complications associated with the overexpression of the designer cytokine can be self-limiting due to the destruction of the virus replication site. Recently, we and others demonstrated that the combination of oncolytic virotherapy and chemotherapy could lead to synergistic interactions that ultimately result in enhanced tumor regression. On the other hand, chemotherapy is often associated with serious side effects, since all fast proliferating cells are affected. Among the most frequently observed adverse effects is thrombocytopenia, which is characterized by a massive reduction of blood platelets. With regard to a possible clinical application of GLV 1h90, combination therapy of the hyper IL 6 encoding vaccinia-virus strain and the chemotherapeutic agent mitomycin C was investigated. Besides therapeutic effects of the virus, the issue was addressed, whether the health status of mice can be improved based on the observed hyper-IL-6 effects. Experimental results clearly demonstrated that combination therapy of mitomycin C and oncolytic vaccinia viruses led to a significantly improved DU-145 tumor regression compared to the respective monotherapies. Of particular importance was the finding that as compared to GLV-1h68, a combination of GLV-1h90 and mitomycin C reduced the time interval during which treated mice suffered from thrombocytopenia significantly. Taken together, this thesis revealed that systemic injection of GLV-1h90 leads to functional expression of the designer cytokine hyper-IL-6, which is able to further optimize the already effective combination therapy of the oncolytic virus GLV-1h90 and the chemotherapeutic agent mitomycin C by reducing of serious adverse effects.

Das Ziel dieser Studie war der Vergleich zweier intraoperativer Applikationsmethoden von Mitomycin C (MMC) im Rahmen der Trabekulektomie. Zu diesem Zweck wurde eine retrospektive Analyse vorgenommen. 100 Patienten hatten intraoperativ eine MMC-Applikation mittels Schwämmchen und 100 Patienten mittels Streifen erhalten. Vor dem Beginn der Studie wurden Labormessungen durchgeführt, um die standartisierte Applikationsdosis von MMC zu bestimmen, die sich jeweils in einem Schwämmchen beziehungsweise in 4 Streifen befand. Die Laborergebnissen zeigten eine wirkliche MMC-Dosis von 17,59±3,15µg für die Schwämmchen und von 12,38 ±1,75µg für die Streifen. Überprüft wurde sowohl die absolute (= complete success, ohne lokale Medikation) als auch die relative Erfolgsrate (= qualified success, mit lokaler Medikation). Beide Methoden zeigten eine zufriedenstellende IOD-senkende Wirkung. Für die Erfolgskriterien ≤21+20%, <16, <12 mmHg gab es keinen statistisch signifikanten Unterschied zwischen den zwei Gruppen weder für die absolute noch für die relative Erfolgsrate 1 Jahr nach der Operation. Für das Erfolgskriterium <18 mmHg gab es einen statistisch signifikanten Unterschied bezüglich der relativen Erfolgsrate zugunsten der Streifen-Gruppe. Zwei Erklärungen dafür sind möglich: Zum einen könnte dies mit der höheren Anzahl von Interventionen (Faden-LKs) in der frühen postoperativen Phase zu tun haben. Zum anderen könnte sich die Behandlung eines größeren Areals mit MMC günstiger auf die Vernarbungsreaktion auswirken und damit auch auf die Erfolgsrate. Die Streifen-Gruppe hatte zudem – trotz weniger MMC – eine statistisch signifikant günstigere Sickerkissen-Morphologie 12 Monate nach der Operation. Auch dies ließe sich durch die größere Applikationsfläche von MMC bei den Streifen erklären. Die Schwämmchen-Gruppe hatte statistisch signifikant mehr Hornhaut-Erosiones in der frühen postoperativen Phase. Auf der anderen Seite hatte die Streifen-Gruppe zu dem Zeitpunkt eine bessere relative Erfolgsrate für die Erfolgskriterien <18 und <12 mmHg. Eine mögliche Erklärung wäre, dass die 8x8-mm-Schwämmchen mehr MMC aufnehmen und näher zur Hornhaut appliziert werden. Im Gegensatz zu den Streifen, die über eine breitere Applikationsfläche in der Peripherie weniger MMC abgeben. Möglich wäre aber auch, dass die Erosiones durch die in der Schwämmchen-Gruppe statistisch signifikant vermehrt vorhandenen Voroperationen erklärbar sind. Diese präoperativen Eingriffe können zu Augentrockenheit führen, welche eine Epitheliopathie der Hornhaut begünstigt. Im Bezug auf die Sickerkissen-Needlings und 5-FU-Injektionen hatten die Schwämmchen-Patienten statistisch signifikant mehr Interventionen als die Streifen-Patienten. Dass die Schwämmchen-Gruppe einen statistisch signifikanten höheren Bedarf an 5-FU-Injektionen zeigte, könnte durch die schlechtere Sickerkissen-Morphologie dieser Patienten erklärt werden. Abschließend sei erwähnt, dass die Schwämmchen-Gruppe statistisch signifikant mehr drucksenkende Augentropfen ein Jahr nach der Operation benötigten, damit der erwünschte IOD postoperativ erreicht werden könnte. Zusammenfassend zeigt die Streifen-Gruppe eine tendenziell etwas günstigere Erfolgsrate als die Schwämmchen-Gruppe und benötigte dafür eine geringere Anzahl von postoperativen Interventionen und weniger MMC. Diese Befunde sprechen für die These, dass die Streifen das MMC effektiver applizieren, indem sie eine größere Fläche behandeln. Aufgrund dieser positiven Tendenz hat die Würzburger Klinik den Einsatz der Schwämmchenapplikation eingestellt. / The aim of this study was to compare two methods of intraoperative application of mitomycin C (MMC ) during trabeculectomy . For this purpose, a retrospective analysis was performed. 100 patients received intraoperative MMC application through a sponge and 100 patients through stripes. Before the start of the study laboratory measurements were made to determine the standardized application dose of MMC which was in the sponge or in the 4 stripes. The laboratory results showed a real MMC dose of 17.59 ± 3.15 mcg for the sponge and of 12.38 ± 1.75 mcg for the stripes . The study tested both the absolute ( = complete success , without local medication) and the relative success rate ( = qualified success , with local medication) . Both methods showed a satisfactory IOP lowering effect. For the success criteria ≤ 21 +20 %, < 16, <12 mmHg , there was no statistically significant difference between the two groups for either the absolute or the relative success rate 1 year after surgery. For the success criterion <18 mmHg , there was a statistically significant difference in the relative success rate in favor of the strip Group. Two explanations are possible : At first, this might have to do in the early postoperative period with the higher number of interventions ( suturelysis). Secondly, the treatment of a larger area with MMC could be more beneficial in case of scaring and thus on the success rate . The stripes group - despite less MMC - had a statistically significant favorable bleb morphology 12 months after surgery. Again, this could be explained by the larger application area of MMC through the stripes. The sponge group had statistically significantly more corneal erosions in the early postoperative period . On the other hand, the stripes group had at that point a better success rate for the success criteria had < 18 and <12 mmHg at the time. A possible explanation could be that the 8x8-mm sponge apply more amount of MMC and nearer to the cornea . In contrast to the stripes, that apply less MMC in a wider area in the periphery. The sponge group had statistically significant more prior surgery and this could explain the number of corneal erosions. These preoperative interventions can lead to eye dryness and superficial punctate keratitis. The sponge group had statistically significant more interventions(needlings of the bleb and the subkonjunctival 5-FU injections) than the stripes group. The sponge group showed a statistically significant higher demand for 5-FU injections, which could be explained by the worse bleb morphology. Finally, it should be mentioned that the sponge group needed statistically significant more eye pressure lowering eye drops one year after trabeculectomy in order to reach the desired IOP postoperatively. In summary, the stripes group showed a better success rate than the sponge group and required a smaller number of post-surgical interventions and less MMC. These findings support the theory that the stripes applied MMC effectively over a larger area. Because of this positive trend, the Würzburg university eye hospital uses the application of MMC with stripes.

A central theme within the concept of enzyme-directed bioreductive drug development is the potential to predict tumour response based on the profiling of enzymes involved in the bioreductive activation process. Mitomycin C (MMC) is the prototypical bioreductive drug that is reduced to active intermediates by several reductases including NAD(P)H:quinone oxidoreductase (NQO1) and NADPH cytochrome P450 reductase (P450R). The purpose of our study was to determine whether NQO1 and P450R protein expression in a panel of low-grade, human superficial bladder tumours correlates with clinical response to MMC. A retrospective clinical study was conducted in which the response to MMC of 92 bladder cancer patients was compared to the immunohistochemical expression of NQO1 and P450R protein in archived paraffin-embedded bladder tumour specimens. A broad spectrum of NQO1 protein levels exists in bladder tumours between individual patients, ranging from intense to no immunohistochemical staining. In contrast, levels of P450R were similar with most tumours having moderate to high levels. All patients were chemotherapy naïve prior to receiving MMC and clinical response was defined as the time to first recurrence. A poor correlation exists between clinical response and NQO1, P450R or the expression patterns of various combinations of the 2 proteins. The results of our study demonstrate that the clinical response of superficial bladder cancers to MMC cannot be predicted on the basis of NQO1 and/or P450R protein expression and suggest that other factors (other reductases or post DNA damage events) have a significant bearing on tumour response.

Made available in DSpace on 2014-06-11T19:31:09Z (GMT). No. of bitstreams: 0 Previous issue date: 2007-02-28Bitstream added on 2014-06-13T20:21:58Z : No. of bitstreams: 1 mamede_fv_dr_jabo.pdf: 467286 bytes, checksum: f692f69fc0d5d4f1a73028aaad9d672a (MD5) / Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) / Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) / Fundação para o Desenvolvimento da UNESP (FUNDUNESP) / Mioplastias extra-oculares podem ensejar aderências entre o músculo operado e os tecidos adjacentes, produzindo, não raro, estrabismos cicatriciais. Com intuito de se minimizar a ocorrência de aderências, investigaram-se os efeitos da mitomicina C (MMC), um antifibrótico, em concentrações ascendentes de 0,008, 0,02 e 0,04%, aplicada no per-operatório de mioplastias do reto dorsal do bulbo do olho de coelhos. Para tal, foram operados 56 animais, os quais foram divididos em sete grupos. Para o pós-operatório, instituíram-se limpeza com solução fisiológica das áreas operadas, profilaxia antimicrobiana e antiinflamatória, na forma de colírio. Procederam-se avaliações clínica, histológica, morfométrica e quanto à imunoistoquímica, em que se estudou o Fator de Crescimento Fibroblástico-básico (FGF-2). Encontraram-se, clinicamente, mais aderências nos olhos controle, comparativamente aos tratados, no entanto, sem significação estatística (p>5%). À histologia, verificou-se que a MMC ensejou retardo da cicatrização junto às áreas das mioplastias, notadamente no grupo que a recebeu, à concentração de 0,02%. A imunoistoquímica revelou marcação do FGF-2 em fibroblastos e macrófagos, indistintamente, entre os grupos. Com base nos resultados, permite-se admitir que a MMC, nas concentrações em que fora empregada, foi capaz de retardar a cicatrização e, por conseguinte, o estrabismo secundário, sem ensejar efeitos colaterais. / Extraocular myoplasties may cause adhesions to adjacent tissues, resulting cicatricial strabismus. With the purpose of reducing to a minimum the occurrence of adhesion, we studied the effects of mitomycin C (MMC), an antifibrotic, in concentrations of 0,008%, 0,02%, and 0,04% applied during intraoperative of myoplasties of the superior rectus muscle of laboratory albine rabbits. Fifty six animals were operated on and were divided in seven groups. During the postoperative the operated areas were cleaned with physiological solution. Eyedrop instillation to prevent inflammation and bacterial infection were used. The method to analyze the results consisted of clinical and histological evaluation and statistical analyzes. We also evaluated at the same time the amount of basic fibroblast growth factor (FGF-2) by immunohistochemical study. Clinically we found more adhesions in the eyes of the control group than in the groups of treated eyes. However there was no significant statistics difference between the two groups (p>5%). Histologically we found that MMC caused a delayed cicatrization in the mioplastic areas, specially in the group who received the 0,02% concentration. The immunohistochemical showed FGF-2 marking in fibroblasts and macrophages, but between the groups there wasn’t no difference. Based on those results we conclude that MMC in the utilized concentrations was capable of delaying the cicatrization and consequently avoid the secondary strabismus without undesirable side effects.

Objetivos: Determinar os efeitos celulares e o mecanismo de ação da mitomicina C tópica na prevenção e tratamento da opacidade corneana em coelhos submetidos à ceratectomia fotorrefrativa (PRK). Métodos: Foram submetidos à cirurgia de PRK 224 coelhos para correção de -9 dioptrias esféricas, associada à aplicação de mitomicina C tópica ou solução salina balanceada. O nível de opacidade corneana foi avaliado por meio de análise à lâmpada de fenda. Os animais foram sacrificados quatro horas, 24 horas, quatro semanas e seis meses após a cirurgia. A análise imunohistoquímica foi realizada com as técnicas de TUNEL e foram utilizados os anticorpos Ki67 e alpha-SMA para a análise da apoptose celular, replicação celular e formação de miofibroblastos, respectivamente. Resultados: Todos os grupos submetidos à aplicação de mitomicina C apresentaram um maior número de células positivamente marcadas pelo ensaio com TUNEL (indicando maior taxa de apoptose celular) e um menor número de células positivamente marcadas pelo anticorpo Ki67 (indicando menor taxa de replicação celular). Uma menor quantidade de miofibroblastos (células positivamente marcadas pelo anticorpo alpha-SMA) foi identificada após a aplicação profilática da mitomicina C, comparada com sua aplicação com finalidade terapêutica. Além disso, identificou-se uma zona de acelularidade no estroma anterior de córneas tratadas com mitomicina C, persistente por um período mínimo de seis meses. Conclusões: A aplicação da mitomicina C diminuiu signficativamente a formação de opacidade corneana em coelhos. Apesar da mitomicina C ter induzido uma maior apoptose de ceratócitos e miofibroblastos, seu principal mecanismo de ação, responsável pela prevenção da opacidade corneana, decorreu do bloqueio da replicação dos ceratócitos ou outras linhagens celulares progenitoras dos miofibroblastos. A aplicação da mitomicina C na concentração de 0,002% mostrou-se tão eficiente quanto sua aplicação na concentração de 0,02%. Não obstante, uma persistente diminuição da densidade de ceratócitos no estroma anterior pode representar um sinal de alerta para possíveis complicações a longo prazo / Purpose: To determine cellular effects and the mechanism through which topical mitomycin C prevents and treats corneal haze after photorefractive keratectomy (PRK) in rabbits. Methods: Minus nine diopters PRK with mitomycin C or balanced salt solution was performed in two hundred and twenty four New Zealand rabbits. Haze level was graded at the slit lamp. Rabbits were sacrificed at 4 hours, 24 hours, 4 weeks or 6 months after surgery and immunohistochemistry was performed with TUNEL assay, Ki67 and alpha-SMA to analyze keratocyte cells apoptosis, keratocyte cells replication and myofibroblast cells formation, respectively. Results: TUNEL-positive cells increased in all mitomycin C groups (representing more keratocyte cells undergoing apoptosis) while Ki67-positive cells decreased significanlty (representing a decreased keratocyte cells replication) following mitomycin C application. A greater decrease in myofibroblasts was noted with prophylactic mitomycin C treatment than therapeutic mitomycin C treatment. There was, however, an anterior stromal acellular zone in eyes treated with mitomycin C that persisted out to the maximum follow-up of 6 months. Conclusion: Mitomycin C application significantly reduced corneal haze formation in rabbits. Its treatment induces apoptosis of keratocytes and myofibroblasts, but the predominate effect in inhibiting or treating haze appears to be at the level of blocked replication of keratocytes or other progenitor cells of myofibroblasts. Treatment with 0.002% mitomycin C appears to be just as effective as higher concentrations (0.02%) in the rabbit model. However, a persistent decrease in keratocyte cells density in the anterior stroma could be a warning sign for future complications

The one-way mixed lymphocyte culture (MLC) is used to assess histocompatibility between donor and recipient. First introduced in 1966, this method involves the co-culture of lymphocytes from the peripheral blood of the donor and the recipient for a period of 6 to 7 days: antigen disparities, primarily in the HLA-DR region, stimulate proliferation of the responding cells, which is detected by addition of 3H-labelled thymidine and subsequent measurement of radioactivity. The lymphocytes of either the donor, used to predict graft-versus-host disease (GVHD) or recipient, used to predict host-versus-graft disease (HVGD)/graft rejection, are inactivated by exposure to radiation or mitomycin C, so that the observed proliferation is that of the other set of lymphocytes, hence the name “one-way” MLC. The amount of measured radioactivity is directly proportional to the amount of DNA synthesized, which is a reflection of the number of disparities at the major histocompatibility complex (MHC).Previous studies have established that inactivation of the lymphocytes by radiation and mitomycin C, has a negative effect on the structure/expression of HLA-DR molecules on the cell surface, which provides the primary stimulus for the MLC reaction. The laboratory research presented in this dissertation was designed i) to compare the viabilities and HLA-DR levels on stimulator cells exposed to cyclosporin A, mitomycin C and ionizing irradiation , in order to determine whether cyclosporin A can be used as an alternative to mitomycin C or radiation as inactivator of the stimulator cells in the one-way MLC; ii) to improve sensitivity and accelerate the MLC reaction by addition of IL-2; iii) establish a flow cytometric mixed lymphocyte assay using the fluorochrome 5,6 carboxyfluorescein diacetate succinimidyl ester (CFSE). Cyclosporin A showed striking similarities to mitomycin C and ionizing radiation in its effect on viability and reduction/structural changes in HLA-DR molecules of the stimulator cells. Exposure of the stimulator cells to 20ìM cyclosporin A, demonstrated a significant loss of both cell viability and HLA-DR molecule cell surface expression. Thus, in evaluating these three methods of inactivation of the stimulator cells in the one-way MLC, it was concluded that a one-way MLC may not in fact be an accurate and qualitative reflection of the histocompatibility between donor and recipient. Instead the two-way MLC , in which neither the donor’s nor recipient’s cells are inactivated, may be a more reliable alternative. The only limitation associated with a two-way MLC is the inability to distinguish between a host-versus-graft-rejection and a graft-versus-host reaction in the observed allogeneic response Addition of 5 and 10 IU/ml IL-2 to the MLC showed the opposite effect to that intended, inhibiting proliferation in the MLC. Previous studies have shown that an excess of IL-2 results in the production of suppressor T cells. The amount of IL-2 produced during the MLC depends on the number of disparities in the MLC between donor and recipient, which will be different for each MLC reaction. Since the number of allogeneic T cells involved in the MLC reaction is not known, the amount of IL-2 produced during the allogeneic immune response in the MLC can not be predicted and addition of exogenous IL-2 may result in production of suppressor T cells and an inhibition of proliferation. The two-way MLC was modified by staining one of the participating set of lymphocytes (donors or recipients) with CFSE and tracking proliferation in this population, using flow cytometry. The two-way CFSE-based MLC analyzed in this study were counterstained with CD25 (IL-2R). An increase in CD25 expression on the cell surface is an indicator of cell activation and proliferation. Proliferation, as indicated by a progressive loss of CFSE fluorescence correlated well with the corresponding increase in CD25 expression and accumulated daughter cells. In addition, by loading only one of the participating donors in the two-way MLC, the responder/stimulator interaction, observed in the one-way MLC, is re-established. Thus the modified, CFSE-based two-way MLC can be used to predict GVHD. To conclude, the use of CFSE labeling and flow-cytometry to measure proliferation in a two-way MLC, together with CD25 counterstaining provides an alternative, reliable and probably superior method to 3H thymidine uptake. / Dissertation (MSc)--University of Pretoria, 2009. / Immunology / unrestricted

No / The indolequinone EO9 demonstrated good preclinical activity but failed to show clinical efficacy against a range of tumours following intravenous drug administration. A significant factor in EO9's failure in the clinic has been attributed to its rapid pharmacokinetic elimination resulting in poor drug delivery to tumours. Intravesical administration of EO9 would circumvent the problem of drug delivery to tumours and the principal objective of this study is to determine whether or not bladder tumours have elevated levels of the enzyme NQO1 (NAD(P)H:quinone oxidoreductase) which plays a key role in activating EO9 under aerobic conditions. Elevated NQO1 levels in human bladder tumour tissue exist in a subset of patients as measured by both immunohistochemical and enzymatic assays. In a panel of human tumour cell lines, EO9 is selectively toxic towards NQO1 rich cell lines under aerobic conditions and potency can be enhanced by reducing extracellular pH. These studies suggest that a subset of bladder cancer patients exist whose tumours possess the appropriate biochemical machinery required to activate EO9. Administration of EO9 in an acidic vehicle could be employed to reduce possible systemic toxicity as any drug absorbed into the blood stream would become relatively inactive due to an increase in pH.

No description available.

INTRODUÇÃO: É importante estudar a efetividade da trabeculectomia e estratégias para manter sua ação ao longo do tempo. Para melhorarmos o prognóstico das revisões cirúrgicas é imprescindível entendermos melhor a estrutura da bolha filtrante e suas possíveis causas de falência. Formas alternativas para se restabelecer a drenagem e o funcionamento da trabeculectomia não funcionante foram desenvolvidas, entre elas a Revisão Interna de Simmons. OBJETIVOS: Avaliar os efeitos da Revisão Interna de Simmons sobre a PIO; sobre o aspecto biomicroscópico da bolha no pós-operatório e sua correlação com achados biométricos obtidos pela biomicroscopia ultra-sônica. CASUÍSTICA E MÉTODOS: Foram incluídos e estudados de forma prospectiva por 6 meses, 29 pacientes portadores de glaucoma primário, submetidos a trabeculectomia prévia,há pelo menos três meses, que apresentavam PIO acima do esperado para o controle do glaucoma, apesar de medicação hipotensora utilizada. Foram fatores de inclusão a conjuntiva livre e o óstio interno da trabeculectomia patente à gonioscopia. O critério de sucesso foi: abaixamento da PIO, igual ou maior que 30% da PIO em relação à PIO pré-operatória e/ou pressão final menor ou igual a 15mmHg, com ou sem medicação antiglaucomatosa associada. RESULTADOS: Houve uma queda estatisticamente significante da PIO e do número de medicações utilizadas após a cirurgia. A PIO média antes da operação era de 23,72 ± 4,10 mmHg e ao término do estudo, de 15,04 ± 4,00 mmHg (p=0,000016). A média das pressões da curva tonométrica antes da cirurgia era de 22,36 ± 3,91mmHg, caindo para 15,01 ± 3,95mmHg no PO180d (p=0,0000001). Antes da cirurgia, a média do número de medicações usadas era de 2,1 ± 0,77 e ao término do estudo, 1,22 ± 1,01 (p=0,001117). Houve um aumento do tamanho da bolha filtrante, avaliado pela biomicroscopia. As bolhas apresentaram altura média de 0,28 ± 0,65 e terminaram o estudo com altura de 0,78 ± 0,85 (p=0,000016). Não houve diferença na altura da bolha, medida pela biomicroscopia ultra-sônica após a cirurgia. A pressão intra-ocular apresentou relação inversamente proporcional à dimensão da bolha filtrante, com significância estatística. As complicações mais encontradas foram: descolamento da membrana de Descemet, no local da incisão principal em 5 doentes (17,2%), abertura inadvertida da conjuntiva em 4 (13,8%), e descolamento de coróide, em 3 (10,3%). CONCLUSÕES: A Revisão Interna de Simmons proporcionou diminuição de 30% da PIO em 52% dos pacientes e redução do número de medicações antiglaucomatosas associadas, quando utilizadas. As bolhas se mostraram-se estatisticamente maiores após o procedimento. Houve uma correlação perfeita entre a classificação da altura da bolha entre a medida biomicroscópica baseada na escala de Indiana e a biométrica obtida pela biomicroscopia ultra-sônica. Não houve aplanamento da curva tonométrica apesar desta ter diminuído 32%. Não foi observada mudança significativa no vício de refração ou na acuidade visual após a operação. A biomicroscopia ultra-sônica demonstrou uma associação inversa entre um maior número de complicações pós-operatórias e o tamanho menor do óstio interno da trabeculectomia. / INTRODUCTION: It is important to study the trabeculectomy effectiveness and strategies to keep its action within long time. To improve the prognostic presented on glaucoma surgical reviews it is essential to understand better the bleb structure and its possible cause of failing. Alternative ways of restoring the drainage and the good performance of trabeculectomy have been studied, among them the Internal Revision of Simmons. PURPOSE: Evaluate the surgery effects on the IOP; on the biomicroscopic aspect of the bleb and its co-relation with biometric founding gotten through UBM. METHODS: For six months, 29 patients with open angle glaucoma who had been submitted to trabeculectomy before, for at least three months, with IOP above the expected to the control of glaucoma, despite the medication used, were included and studied in a prospective way. These patients should present the conjunctive tissue not too scarry and internal ostio of trabeculectomy present on the gonioscopia. The success criterion was: lowering the IOP, equal or above 30% on pre-operatory IOP and/or final pressure below 15 mmHg, with or without eye drops. RESULTS: There was a statistically fall of IOP and amount of medication used after the surgery. The average of IOP before surgery was 23.27 ± 4.10 mmHg and at the end of study was 15.04 ± 4.00 mmHg (p=0.000016). The average of the IOP curve pressure before surgery was 22.36 ± 3.91mmHg, dropped to 15.01 ± 4.0 mmHg at PO180d (p=0.0000001). Before the surgery the average of medication used was 2.1 ± 0.77 and at the end of the study it was 1.22 ± 1.01 (p=0.001117). There was an increase of the filtering bleb size clinically measured. The blebs had an average height of 0.28 ± 0.65 and at the end of the study their height was 0.78 ± 0.85 (p=0.000016). The UBM didn?t show any difference in the bleb size. The intra-ocular pressure presented an inversely proportional relation to the dimension of the filtering bleb, with statistic significance. The most frequently problems found were: detachment of Descemet membrane where the main incision was performed, in 5 (17.28%) patients; hole in the conjuntiva in 4 (13.88%), detachment of the choroid in 3 (10.38%). CONCLUSIONS: The surgery was effective, for provided 30% decrease of initial IOP in 52% without or with eye drops statistically in small number than in presurgery. The blebs have appeared, statistically, bigger after the procedure. There was a perfect correlation between the bleb height classification, within the clinical measurement, based on the Indian scale, and the biometric one, gotten by UBM. There was no flattening of the IOP curve pressure despite this one had been 32% lower than the initial one. No significant change has occurred in the vicious of refraction or in the visual accuracy after the surgery. The UBM demonstrated an association between a larger number of complications post-surgery and the internal size of the trabeculectomy ostio.