O câncer de mama é uma neoplasia altamente maligna e continua a ser a segunda principal causa de mortalidade entre as mulheres. Os efeitos antitumorais de metaloproteinases e desintegrinas de veneno de serpentes têm sido investigados em vários tipos de células tumorais. Neste estudo, foram avaliados os efeitos antitumorais e anti-angiogênicos induzidos pela Bothropoidina, uma metaloproteinase desintegrina-like isolada da peçonha de Bothrops pauloensis em células de câncer de mama humano MDA-MB-231 e células endoteliais. Após 24 horas de tratamento com 100pg/mL de Bothropoidina foi constatado um efeito citotóxico moderado de 30% em MDA-MB-231 contra 10% de citotoxicidade em MCF10A (uma linha de células da mama não tumorigênica), uma diferença significativa que sugere uma possível preferência desta proteína por alvos em células tumorais. Observou-se apoptose e apoptose tardia após tratamento com Bothropoidina (10pg/mL e 40pg/mL) em células MDA-MB-231. Além disso, esta toxina não só inibiu a adesão de células MDA-MB-231 de uma forma dose dependente, como a migração celular em aproximadamente 45%. Bothropoidina reduziu a viabilidade e adesão de células endoteliais em Matrigel e inibiu a angiogênese in vitro estimulada por bFGF em Matrigel, mostrando um número de vasos formados significativamente menor em relação ao controle. Os resultados demonstraram que Bothropoidina tem um potente efeito antitumoral e antiangiogênico in vitro, representando uma ferramenta biotecnológica para elucidar o efeito antitumoral de metaloproteinases desintegrinas-like em células cancerígenas. / Breast cancer is a highly malignant carcinoma and remains the second leading cause of mortality among women. The antitumor effects of metalloproteinases and disintegrins from snake venom on various types of cancer cells have been investigated. In this study, we evaluated the antitumor and antiangiogenic effects on MDA-MB-231 human breast cancer cells and endothelial cells induced by Bothropoidin, a disintegrin-like metalloproteinase isolated from Bothrops pauloensis snake venom. At 24h after treatment at 100pg/mL, Bothropoidin exerted a moderate cytotoxic effect of 30% on MDA-MB-231 versus 10% cytotoxicity against MCF10A (a non-tumorigenic breast cell line), a significant difference that suggests a possible preference by this protein for targets in cancer cells. Early and late apoptosis of MDA-MB-231 was observed after Bothropoidin treatment (10gg/mL and 40gg/mL). Furthermore, this toxin inhibited not only the adhesion of MDA-MB-231 cells in a dose-dependent manner but also cell migration by approximately 45%. In addition, Bothropoidin decreased endothelial cells viability and adhesion in Matrigel and inhibited in vitro angiogenesis in Matrigel stimulated by bFGF, showing significantly fewer formed vessels. The results demonstrated that Bothropoidin has potent in vitro antitumor and antiangiogenic effect and represents a biotechnological tool for elucidating the antitumor effect of disintegrins-like metalloproteinases in cancer cells. / Dissertação (Mestrado)

O veneno do T.nattereri induz uma resposta de memória com a diferenciação de células B B220neg, indicativo de células produtoras de anticorpos de vida longa (ASC). Para avaliar o efeito do veneno na diferenciação de ASCs, camundongos BALB/c foram imunizados e sacrificados nos dias 21, 28, 48, 74 e 120 para avaliação de anticorpos plasmáticos e células B no peritônio, baço e medula óssea. O veneno induziu intensa esplenomegalia, formação de centros germinativos e persistentes níveis de anticorpos específicos IgG1, IgG2a e IgE anafilática. Células B1a e ASC apareceram rapidamente e a população de ASC CD138pos foi dividida em três subtipos (B220highCD43high, B220lowCD43low, e B220negCD43high) que persistiram em diferentes níveis em todos compartimentos. Finalmente, por métodos de neutralização nós sugerimos um papel importante da IL-5 e IL-17 A no desenvolvimento de ASC B220neg e na população B1a e mais ainda, a produção de TNF-a, IL-1b, IL-6, KC bem como a retenção do veneno nas células dendríticas foliculares parece promover os mecanismos para manutenção das ASCs. / T. nattereri fish venom induces a memory immune response with the differentiation of B cells B220neg, an indicative of long-lived antibody-secreting cells - ASC. To assess the effect of the venom on differentiation of ASCs, BALB/c mice were immunized with venom and sacrificed at days 21, 28, 48, 74 and 120 to evaluate plasmatic antibodies and B cell subtypes in peritoneum, spleen and bone marrow. The venom promoted splenomegaly, germinal centers formation and persistent levels of specific antibodies IgG1, IgG2a and anaphylactic IgE. B1a cells and ASC emerged rapidly and CD138pos ASCs can be divided into three subsets (B220high CD43high, B220low CD43low, and B220neg CD43high) that persist at different levels in all compartments. Finally, by neutralization methods we suggested an important role for IL-5 and IL-17A on development of B220neg ASCs and B1a population and moreover the production of TNF-a, IL-1b, IL-6, KC as well as the venom retained in follicular dendritic cells seem to provide mechanisms to explain the maintenance of ASCs.

La mélanogenèse engage 3 enzymes. La tyrosinase catalyse l’oxydation des 2 premiers substrats pour former la DOPAquinone. Celle-ci, en présence de cystéine peut s’y conjuguer pour produire des pheomélanines (pigments clairs). En son absence, la DOPAquinone est convertie en DOPAchrome, précurseur de la voie de synthèse des eumélanines (pigments foncés). Leur formation implique deux autres enzymes : TRP-1 et TRP-2. Un dérèglement de ce processus entraîne des dermatoses de type hyperpigmentations chez les phototypes IV à VI. Traiter ces problématiques efficacement sans effets secondaires nécessite d'identifier de nouvelles molécules actives capables de réguler la synthèse des eumélanines.Pour atteindre l'objectif, nous avons évalué l’effet inhibiteur de 100 venins d’animaux sur l’activité DOPA oxydase de la tyrosinase de champignon. Des analyses HPLC et spectrophotométriques alternées, ont permis de localiser la sous-fraction active conte-nant la molécule d'intérêt. La détermination du poids moléculaire par spectrométrie de masse conjuguée à l’analyse d'AA après hydrolyse acide a permis d'identifier l’Argiotoxine-636 (ARGTX-636). Elle est un inhibiteur mixte de la diphénolase. Des études préliminaires de SAR ont également été réalisées. En parallèle, une méthode analytique par HPLC/MS a été mise au point pour confirmer l'activité inhibitrice de la DHICA oxydase de l'enzyme par l'ARGTX-636.Pour la première fois est reportée l’isolation et la caractérisation d’un nouveau composé extrait d’un venin d’araignée capable de réguler la mélanogenèse. Cette approche peut avoir un impact majeur dans la recherche de nouvelles molécules actives dans le domaine de la dermo-cosmétique. / Melanogenesis involves three enzymes. Tyrosinase catalyses the oxidation of the two first substrates and leads to the formation of DOPAquinone, which may be conjugated with cysteine to produce pheomelanins (red-yellow pigments). In absence of cys-teine, DOPAquinone is spontaneously converted in DOPAchrome, the precursor of eumelanin products (brown-black pig-ments). The formation of eumelanin molecules partially relies on the activity of two other enzymes called Tyrosinase related protein 2 and Tyrosinase related protein 1. A dysregulation of this process engenders hyperpigmentation tasks into skin IV to VI phototypes. To treat these issues effectively without side effects requires the identification of new active molecules able to regulate the eumelanin synthesis.To achieve the objective, we evaluated the inhibitory effect of 100 animals venoms, on the DOPA oxidase activity of mushroom tyrosinase. HPLC and spectrophotometric analysis alternating, we allowed us to localize the active sub-fraction containing the interest molecule. The molecular weight determination by mass spectrometry combined with the AA analysis after acid hy-drolysis identified argiotoxin-636 (ARGTX-636). It is a mixed-type inhibitor on diphenolase activity. SAR preliminary stu-dies have also been conducted. In parallel, an analytical method by HPLC / MS was developed to confirm the DHICA oxy-dase activity inhibitory effect by ARGTX-636.For the first time is reported the isolation and characterization of a novel compound extracted from a spider venom able to regulate melanogenesis process. This approach can have a major impact in the search for new active compounds in the dermo-cosmetics field.

Na África subsaariana, as serpentes do gênero Bitis são de extrema importância, pois suas vítimas apresentam sintomas como dano local, hemorragia e uma severa hipotensão. Este trabalho identificou moléculas capazes de inibir a atividade da enzima conversora de angiotensina I (ECA) presentes no veneno de quatro serpentes do gênero Bitis. Para isto, as porções de baixa massa molecular desses 4 venenos foram fracionadas em RP-HPLC e as frações com boa inibição sobre a atividade da ECA foram analisadas por espectrometria de massas. Foram identificados 34 oligopeptídeos ricos em prolina (PRO), sendo 8 sintetizados e suas constantes de inibição (Ki) determinadas. Em testes com substratos naturais da ECA, angiotensina I e bradicinina, foi constatada a maior inibição da hidrólise da angiotensina I por quatro PROs. Todos os PROs in vivo reduziram a pressão arterial, e seis deles aumentaram a frequência cardíaca em ratos Wistar. Com isto, conclui-se que existem toxinas no veneno de serpentes do gênero Bitis responsáveis pela hipotensão. / In the sub-saharian Africa, snakes from the Bitis genus are of extreme medical importance, since its victims show symptoms as local tissue damage, hemorrhage and a severe hypotension. This work identified molecules that inhibit the angiotensin I converting enzyme (ACE) in the venom of 4 snakes from the Bitis genus. The low molecular portions of the venom of these snakes were fractionated in RP-HPLC and the fractions that efficiently inhibited the ACE activity were analyzed by mass spectrometry. 34 proline-rich oligopeptides were identified, 8 of them synthesized and had their inhibition constants (Ki) determined. In tests using natural substrates of ACE, angiotensin I and bradykinin, the angiotensin I hydrolysis were better inhibited by four PROs. In vivo tests results showed that all PROs decreased the mean arterial pressure and six of them increased the heart rate. Therefore, we can conclude that there are toxins present in the venom of Bitis capable of cause hypotension.

Die Prävalenz einer Insektengiftallergie beträgt 2,8 %. Am häufigsten sind Honigbienen (Apis melliferi) und Faltenwespen (Vespula vulgaris, Vespula germanica) Auslöser einer Insektengiftallergie in Deutschland. Sie ist eine allergische Typ-I-Reaktion und durch eine Immunglobulin-E-vermittelte (IgE) Immunreaktion charakterisiert. IgE führt zur Sensibilisierung der an einer Immunreaktion beteiligten Mastzellen und basophilen Granulozyten. Durch den Zweitkontakt erfolgt die Aktivierung jener mit Degranulation von Histamin, Serinproteasen, Prostaglandinen, Leukotrienen und Zytokinen. Dies spiegelt sich in einer allergischen Reaktion mit Vasodilatation, Tachykardie, Hypotonie, Bronchokonstriktion, Pruritus, Schmerzen, Erythem oder Flush, Nausea, Vomitus und Diarrhoe wieder. Mittels Hauttests wie Prick- und Intrakutantest kann eine IgE-vermittelte Sensibilisierung auf das Insektengiftallergen nachgewiesen werden. Es werden die Serumparameter Gesamt-IgE und spezifisches IgE auf native und rekombinante Allergene des Insektengiftes gemessen. Der Basophile Aktivierungstest (BAT) kann ergänzt werden. Hierbei wird die immunologische Quantifizierung der Rezeptorenaktivität auf basophilen Granulozyten mittels Detektion der membranständigen Aktivierungsmarker CD63 und CD203c vor und nach Antigenexposition gemessen. CD63 befindet sich intragranulär gespeichert in ruhenden basophilen Granulozyten, Mastzellen, Makrophagen und Monozyten. In vitro konnte eine verstärkte Expression von CD63 v.a. allergen-induziert und FcεRI-vermittelt beobachtet werden. CD203c ist ein hochspezifischer Marker für die basophile Differenzierungslinie. Nach Allergenstimulation wird eine rasche Expression von CD203c beobachtet, welche FcεRI-vermittelt ist. Am Ausmaß der Expression von CD63 und CD203c kann die allergene Eigenschaft abgeleitet und bei Kreuzreaktivität i.R. der in-vitro-Diagnostik das auslösende Allergen identifiziert werden. Die spezifische Immuntherapie (SIT) ist die einzige kausale Therapie einer Insektengiftallergie. Pathophysiologisch wird eine immunmodulatorische Wirkung angenommen. Die Stichprovokation schätzt die Therapieeffektivität einer SIT auf Grund von Mangel an validen laborchemischen Kriterien ein. Bei Asymptomatik oder lokaler allergischer Reaktion hat das Ergebnis einen hohen prädiktiven Wert für die Verträglichkeit weiterer Stiche. Bei ausbleibender systemischer Reaktion nach 3 bis 5 Jahren SIT kann jene beendet werden, wenn SIT oder Stichprovokation ohne Nebenwirkungen vertragen wurden. Ein Feldstich ist der Stichprovokation ebenbürtig, wenn das allergieauslösende Insekt sicher identifiziert wurde. Nach SIT kommt es bei bis zu 15% der Patienten zum Verlust des Schutzes nach 5-10 Jahren (Epidemiology of Insect Venom Sensitivity | JAMA | The JAMA Network, 2017). Die Leitlinie empfiehlt daher ein Notfallset dauerhaft mitzuführen. Vor einer Stichprovokation muss eine 109 Risiko-Nutzen-Abwägung bei relevanten Nebenerkrankungen oder vorbekannter Mastozytose erfolgen. Da die Aktivität des Insektes sowie die Giftzusammensetzung in Abhängigkeit der Jahreszeit variieren, geht die Stichprovokation mit einer eingeschränkten Beurteilbarkeit einher. Auf Grund von erhöhter Angst und Arbeitsausfall für bis zu 3 Tage wird eine Provokation häufig nicht durchgeführt. Daher besteht die Notwendigkeit nach Messmethoden, welche die Therapieeffektivität einer SIT sicher beurteilen. Ziel der vorliegenden Arbeit ist den BAT bezüglich seines Nutzens als Monitoringinstrument während einer SIT zu prüfen und die Therapieeffektivität jener beurteilen zu können. Es wurden 50 Probanden während einer SIT begleitet (6 Kinder mit 4x Bienengift- und 2x Wespengiftallergie, 44 Erwachsene mit 4x Bienengift- und 40x Wespengiftallergie). Die Probanden erklärten sich zu Blutentnahmen vor der SIT und alle 6 Monate bis 3 Jahre während der SIT einverstanden. Hieraus erfolgte die Bestimmung der Serumparameter mittels des ImmunoCAP® 250 der Firma Thermo Fisher Scientific/ Phadia. Mit Hilfe des Flow CAST® Kit (FK-CCR) von der Firma Bühlmann Laboratories AG wurde die Expression der Oberflächenmarker basophiler Granulozyten CD63 und CD203c im BAT gemessen. Anhand des a2-Wertes wurde der relative Anteil an aktivierten basophilen Granulozyten [%] auf die Stimulation mit der Allergenkonzentration von 56,8 ng/ml (c2) bestimmt. Der kalkulierte c50-Wert definiert die mindest notwendige Konzentration an Allergen, um eine Aktivierung von 50% aller in der Testprobe vorliegenden basophilen Granulozyten zu induzieren. Klinische Daten wurden in halbjährlichen Visiten telefonisch, vor Ort, mittels Fragebogen und durch Einsicht in Patientenakten erhoben. Probanden wurden nach Stichprovokation oder Feldstich während SIT und Studie zu Verträglichkeit und Klinik befragt. 90,9% der Erwachsenen und 100% der Kinder boten eine allergische Reaktion Stadium II oder III nach Ring & Messmer. Anaphylaktische Reaktionen wurden nicht beschrieben oder beobachtet. Es waren 40 Erwachsene mit einer Wespengift-SIT zu verzeichnen. Für CD203c sind jene im BAT alle Responder, für CD63 waren 4 Probanden Nonresponder. Für CD63 zeigten sich für 60% steigende und für etwa 30% konstante c50-Werte i.V. der SIT. Dies spiegelt sich in fallenden a2-Werten bei etwa 60% aller Probanden dieser Gruppe wieder. Für CD203c konnten für 60-75% der Probanden steigende sowie für 20% konstante c50-Werte kalkuliert werden. Nach einem Jahr SIT boten 20%, nach zwei Jahren 32,5% und nach drei Jahren SIT 47,5% aller Probanden eine geringere Aktivierung basophiler Granulozyten. In der vorliegenden Studie konnte für 4 Testkonzentrationen (c1 = 284 ng/ml, c2 = 56,8 ng/ml, c3 =11,4 ng/ml sowie c4 = 2,27 ng/m) in allen untersuchten Studiengruppen (Kinder mit Bienengift-SIT, Erwachsene mit Bienengift-SIT, Kinder mit Wespengift-SIT und Erwachsene mit Wespengift-SIT) fallende am-Werte über die Zeit der SIT ermittelt werden. 110 Dies korrelierte mit steigenden c50-Werten über die Dauer der SIT. Die Routinetest-konzentration des Flow CAST® Kit (56,8 ng/ml) gibt hierbei die beste Diskriminierung wieder. Alle 40 Erwachsenen mit einer Wespengiftallergie beschrieben eine mildere klinische Symptomatik unter SIT. Für alle 21 Probanden, welche an einer Stichprovokation teilnahmen oder einen Feldstich erlitten, bestätigte sich dies im BAT. Anhand von Serologie oder Kinetikmessungen lässt sich in der vorliegenden Studie keine Aussage zur Immunmodulation einer SIT und ihrer Therapieeffektivität treffen. Der BAT hingegen ist ein mögliches valides Messverfahren, um die Effektivität einer SIT zu prüfen. Hierfür eignen sich bei guter Korrelation zu Klinik und Stichprovokation/ Feldstich der a2-Wert sowie der kalkulierte c50-Wert. Das Ergebnis eines BAT ist reproduzierbar. Es können Arbeitsausfall und ein erhöhtes Angstempfinden vermieden werden. Ein Routineeinsatz des BAT kann anhand der Studie noch nicht abgeleitet werden. Hausmann et. al konnten jedoch 2014 den c50-Wert als valides Monitoringinstrument der Effektivität bei guter Korrelation zur Stichprovokation belegen. Der BAT ist daher in Fällen interessant, in denen eine Stichprovokation nicht möglich ist (Kontraindikationen, Schwangerschaft, Patientenwunsch). Eine Langzeitwirkung der SIT könnte ggf. mit begleitenden BAT-Messungen geprüft werden. Damit ließe sich die Effektivität einer SIT zum Beispiel auch nach 10 oder 15 Jahren prüfen. Dann wäre eine Aussage darüber möglich, ob das Notfallset lebenslang indiziert ist. Weitere Studien sind notwendig, um die vorliegenden Ergebnisse zu stützen. Eine multizentrische Studie mit standardisiertem BAT und begleitender Stichprovokation ist hierfür Voraussetzung.:Abbildungsverzeichnis Tabellenverzeichnis Abkürzungsverzeichnis 1 Einleitung und Zielstellung 2 Hintergrund und Wissensstand 2.1. Insektengiftallergie 2.1.1. Terminologie der Allergie 2.1.2. Prävalenz der Insektengiftallergie 2.1.3. Hymenoptera - Arten, Taxonomie und Gifte 2.1.4. Immunologische Grundlagen der allergischen Reaktion 2.2. Diagnostik allergischer Reaktionen 2.2.1. Anamnese 2.2.2. Klinik 2.2.3. Hauttests 2.2.4. In-vitro-Allergiediagnostik 2.2.5. Zelluläre Testverfahren 2.3. Therapie 2.3.1. Allgemeine Maßnahmen 2.3.2. Notfalltherapie 2.3.3. Spezifische Immuntherapie (SIT) nach aktueller Leitlinie 2.3.4. Stichprovokation 3 Material und Methoden 3.1. Patientenkollektiv 3.2. Blutentnahmen im Rahmen der Studie während der SIT 3.3. Serologische Untersuchungen 3.4. Zelluläre Testverfahren 3.4.1. Basophiler Aktivierungstest (BAT) 3.4.2. Kinetikuntersuchungen 3.5. Stichprovokation und Feldstiche 3.6. Anamnestische Datenerhebung 3.7. Statistische Methoden 4 Ergebnisse 4.1. Studienpopulation 4.2. Stichereignisse 4.3. Aktivierung basophiler Granulozyten im BAT 4.3.1. Aktivierung basophiler Granulozyten im BAT - c50 und a2 4.3.2. Zeitlicher Verlauf der mittleren Aktivierung basophiler Granulozyten während SIT, Dosis-Wirkungs-Kurve 4.4. Verlauf der serologischen Messdaten 4.5. Kinetikuntersuchungen im BAT 4.6. Anamnese und Klinik 4.7. Korrelation BAT-Ergebnisse und Anamnese/ Klinik 4.8. Korrelation BAT-Ergebnisse und Stichprovokationen/ Feldstiche 5 Diskussion 5.1. Prävalenz der Insektengiftallergie und Verteilungsmuster der allergischen Reaktion nach Ring und Messmer 5.2. Diagnostik und Therapie einer Insektengiftallergie, Studienpopulation 5.3. State of the art - Lücken in Diagnostik und Therapie einer Insektengiftallergie 5.4. Alternative Methoden der Beurteilung der Effektivität einer SIT 5.5. Aktivierung basophiler Granulozyten im BAT 5.5.1 Aktivierung basophiler Granulozyten BAT - a2 und c50 5.5.2. Kinetik der Immunmodulation unter SIT 5.5.3. Interleukin-3 und Expressionskinetik der Oberflächenmarker CD63/ CD203c 5.5.4. Responder versus Nonresponder 5.5.5. Korrelation BAT-Ergebnisse und Anamnese/ Klinik 5.5.6. Korrelation BAT-Ergebnisse und Stichprovokation 5.6. Verlauf der serologischen Messdaten 5.7. Fehleranalysen 5.8. Ausblick in die zukünftige Forschung 6 Zusammenfassung 7 Literaturverzeichnis 8 Danksagung Anhang A Curriculum vitae Anhang B. Veröffentlichungen Anhang C. Anlage 1 - Eröffnung Promotionsverfahren Anhang D. Anlage 2 - Einhaltung gesetzlicher Vorgaben Anhang E. Anlage 3 - Eidesstattliche Erklärung Anhang F. Anlage 4 - Fragebogen Anhang G. Anlage 5 - Experimentelle Daten

Im Fokus dieser Dissertation steht die Zerstörung des Lebensraumes von Schlangen durch anthropogene Einflüsse im Zusammenhang mit dem Auftreten von Schlangenbissen bei Menschen. Zum Schutz der Schlangen und dem Problem der Schlangenbisse wurden vier wesentliche Anforderungen erarbeitet. Mit Hilfe eines Artenverteilungsmodells wurden die ökologischen Nischen von 29 Giftschlangenarten aus Bangladesch und von zehn Kobraarten (Naja) aus Asien ermittelt. Auf lokaler Ebene wurde festgestellt, dass Überschwemmungen, Waldtypen, Ökosysteme und klimatische Parameter das Verbreitungsgebiet der Schlangenarten in Bangladesch beeinflussen. Auf regionaler Ebene sind anthropogene Faktoren wie die Zerstörung von Schlangenlebensräumen, Handel, Ausbeutung und Tötung von Schlangen wahrscheinlich die Hauptursachen für den Rückgang der Populationen der asiatischen Naja. Auf beiden Ebenen führt der Klimawandel zu einer Verkleinerung der ökologischen Nischen von Schlangenarten in Bangladesch und Asien. In Bangladesch könnten die untersuchten Arten innerhalb der nächsten 50 Jahre mehr als 90 % ihren derzeitigen Lebensraum verlieren (Asiatische Naja durchschnittlich 56 % (12 bis 100 %)). Wenn günstige Nischen nicht erhalten werden können, könnten viele Schlangenarten in einigen Ländern Asiens, darunter Bangladesch, in wenigen Jahrzehnten aussterben. Darüber hinaus wurde in der vorliegenden Studie geschätzt, dass die Zahl der jährlichen Schlangenbisse in Bangladesch mehr als doppelt so hoch sein könnte wie bisher angenommen. Da der Klimawandel zu einer geografischen Verschiebung günstiger Nischen führen kann, kann sich diese Veränderung auch auf die Häufigkeit von Schlangenbissen auswirken. Bei der Analyse von Giftvariationen und der Wirksamkeit von Antivenomen wurde festgestellt, dass isolierte Populationen deutliche intraspezifischere Giftvariationen zeigen. / Snakes are threatened by habitat degradation and anthropogenic impacts, while accidental encounters between humans and snakes may result in snakebite incidents. Thus, the conservation of snakes snakebite problem, a global public health emergency, are interconnected. I found four primary needs for a combined solution to satisfy both above causes. A species distribution model was employed to identify the ecological niches of 29 venomous snake species from Bangladesh and of ten cobra species (Naja) from Asia. At the local scale, flood events, forest types, ecosystems, and climatic parameters were found to shape the range of snake species in Bangladesh. At the regional scale, anthropogenic factors, like snake habitat destruction, trade, exploitation, and snake killings are likely to be the main causes for the decrease in populations of Asian Naja. At both levels, climate change causes shrinking and fragmenting the ecological niches of snake species in Bangladesh and Asia. In Bangladesh, the studied species may lose more than 90% of their current climatic niche within the next 50 years. In the same timeframe, Asiatic Naja may lose an average of 56% (12- 100%) of the area of suitable climatic niches. If favourable niches cannot be preserved, many snake species might go extinct from several countries in Asia in a few decades, including Bangladesh. In addition, the present study estimated that there might be more than double the amount of annual snakebite incidents in Bangladesh than previously estimated. As climate change may cause geographical shifting of favourable niches, this change may also have consequences for the frequency of snakebites, so snakebite should be considered a climate driven dynamic public health problem. While analysing venom variations and the efficacy of antivenom, I found that venom should be sampled from geographically or ecologically isolated populations of each venomous snake, because isolated populations have shown distinct intraspecific venom variations.

Rapid development of bacterial resistance and multiresitance to conventional antibiotics has resulted in an intensive search for alternative antimicrobial agents. Antimicrobial peptides (AMPs) belong to promising anti-infective candidates since they do not development bacterial resistance. They kill microbes by disturbing or permeabilizing the cytoplasmic membrane, or may target putative key intracellular compartments. Their advantages include fast action and selectivity between prokaryotic and eukaryotic cells. We have isolated several novel AMPs from the venom of wild bees: halictines (HAL-1 and HAL-2) from Halictus sexcinctus, lasiocepsin (Las) from Lasioglossum laticeps and macropin (MAC-1) from Macropis fulvipes. They are active against Gram-positive and Gram- negative bacteria and against yeast Candida albicans. While halictines and macropin have moderate hemolytic activity, Las shows no hemolytic activity. A novel AMP was isolated also from the mucus of Xiphydria camelus. This AMP belongs to the category of insect defensins. It contains 55 amino acid residues, three disulphide bridges and its C-terminus is amidated. CD and NMR studies of HAL-1, HAL-2 and MAC-1 revealed propensity to form amphipathic α-helical structure in the presence of sodium dodecyl sulfate or trifluoroethanol. For the...

O transplante de ilhotas pancreáticas humanas representa uma estratégia promissora para a cura do diabetes mellitus tipo 1 (DM1), mas a aplicação a todos os pacientes diabéticos ainda é impraticável devido à limitada disponibilidade de ilhotas ou células β e à necessidade de utilização de drogas imunossupressoras pelo paciente transplantado. O tratamento com imunossupressores após o transplante de ilhotas pode ser abolido quando se realiza o microencapsulamento das ilhotas pancreáticas. Neste trabalho investigou-se um novo biomaterial, Biodritina® (alginato/sulfato de condroitina) adequado ao microencapsulamento que gelifica na presença de íons de cálcio ou bário. A biocompatibilidade das microcápsulas tem sido avaliada segundo o grau de pureza do alginato utilizado na sua confecção. Amostras de alginato comercial purificado foram analisadas, comprovando-se a presença de impurezas (polifenóis, endotoxinas, proteínas) em níveis elevados, que impedem sua aplicação clínica. Optou-se, portanto pela utilização do alginato comercial ultrapurificado nos experimentos descritos neste trabalho. Das formulações de biomateriais avaliadas, as microcápsulas de bário-Biodritina apresentaram o melhor desempenho em testes de estabilidade físico-química. Estas microcápsulas mantiveram sua morfologia e estabilidade estrutural após permanecerem 30 dias na cavidade peritoneal de camundongos, conforme demonstrado por microscopia eletrônica de varredura (MEV). Análises histológicas mostraram que microcápsulas de bário-Biodritina explantadas, não possuíam adesão celular em sua superfície. Estudos de permeabilidade demonstraram que o tamanho médio dos poros das microcápsulas de bário-Biodritina permite passagem de proteínas de até 70 kDa, enquanto os poros daquelas de cálcio-Biodritina comportam proteínas de até 100 kDa. Experimentos de coResumo | x cultivo de macrófagos peritoneais com ilhotas de rato microencapsuladas demonstraram uma capacidade imunoprotetora maior das microcápsulas de bário-Biodritina em relação às de cálcio- Biodritina, sendo que as primeiras não ativaram os macrófagos. A manutenção da viabilidade e função de ilhotas humanas microencapsuladas com bário-Biodritina foi confirmada através de ensaio funcional in vitro, no qual ilhotas microencapsuladas apresentaram níveis de secreção de insulina idênticos aos de ilhotas nuas. A prova de conceito do biomaterial foi realizada através do implante de ilhotas humanas microencapsuladas em bário-Biodritina em camundongos com DM1 induzido por estreptozotocina. A hiperglicemia desses animais foi corrigida pelo implante por um período superior a 60 dias, durante os quais o teste oral de tolerância à glicose mostrou-se normal, demonstrando completa funcionalidade e eficiência das ilhotas microencapsuladas com bário-Biodritina. Partindo de observações de que animais inoculados com a peçonha do escorpião Tityus serrulatus apresentam nesidioblastose, foi realizado o fracionamento do veneno por HPLC de fase reversa e 24 frações obtidas foram submetidas a ensaios de proliferação celular através da incorporação de 3H-timidina em células de insulinoma de rato RINm5F. Uma dessas frações foi capaz de induzir a proliferação das células RINm5F e quando aplicada a ilhotas humanas isoladas, elevou o índice de secreção de insulina e induziu um aumento da expressão dos mRNAs de insulina e PCNA. Portanto, demonstrou-se que o biomaterial bário-Biodritina possui as características necessárias para microencapsular células/ilhotas com eficiência e que a \"fração ativa\" do veneno do escorpião T. serrulatus induz proliferação de células RINm5F e melhora a secreção de insulina de ilhotas humanas. / Islet transplantation has been proposed as a promising therapeutic strategy for the cure of type 1 diabetes mellitus (DM), however, its application to all diabetic patients is still not possible due to the limited source of islets or β cells and to the need of an immunosuppressive treatment of the recipient to avoid graft rejection. The use of immunosupressors may be abolished when pancreatic islets are microencapsulated prior to transplantation. Here, we investigated the use of a new biomaterial suitable for cell microencapsulation, namely, Biodritin®, composed of alginate and chondroitin sulphate, which is capable of gelation in the presence of barium or calcium ions. Microcapsules biocompatibility has been evaluated according to the purity of the alginate used in its production. Samples of purified commercial alginate were analyzed, but the high levels of contaminants (proteins, endotoxins and polyphenols) detected prevented its use in clinical applications. On the other hand, also commercially available ultrapure alginate fulfills the requirements for this application, therefore, this biomaterial was chosen for our experiments. Among the different biomaterial formulations evaluated, barium-Biodritin microcapsules displayed the best performance in the physico-chemical tests. Scanning electronic microscopy revealed that barium-Biodritin microcapsules maintained their morphology and structural stability after being implanted for 30 days in the peritoneal cavity of mice. No cellular adhesion was detected on the surface of explanted barium-Biodritin microcapsules by histological analysis. Permeability studies determined the medium pore size of barium-Biodritin microcapsules, which allows proteins of up to 70 kDa to pass through the biomaterial, while calcium-Biodritin pores accomodate proteins of up to 100 kDa. Co-culture of peritoneal macrophages with microencapsulated rat islets, revealed a superior immunoprotective capacity of barium-Biodritin microcapsules, which were capable of protecting the islets with no macrophage activation. Microencapsulated and naked human islets presented identical insulin secretion levels upon stimulation with glucose in vitro, confirming that barium-Biodritin microencapsulation maintains the function and viability of human islets. Proof-of-concept experiments in which barium-Biodritin microencapsulated human islets were implanted into chemically-induced diabetic mice, showed that these animals maintained normal blood glucose levels for more than 60 days, during which oral glucose tolerance tests were normal, demonstrating the complete functionality and efficiency of barium-Biodritin microencapsulated human islets. From the observation that animals inoculated with the venom of the scorpion Tityus serrulatus presented nesidioblastosis, we decided to fractionate the venom to isolate the active principle. The venom was fractionated by reversed phase HPLC and 24 fractions were obtained and submitted to cellular proliferation assays, in which rat insulinoma RINm5F cells evaluated for 3H-timidina incorporation. One of these fractions was capable of inducing cell proliferation and was also applied to isolated human islets. Treated islets presented a higher insulin secretion index and an increase in insulin and PCNA mRNA expression. In conclusion, we demonstrated that the barium-Biodritin biomaterial possesses all characteristics required for efficient cell/islet microencapsulation and that the active fraction of Tityus serrulatus venom induces the proliferation of RINm5F cells and improves insulin secretion in human islets.

O transplante de ilhotas pancreáticas humanas representa uma estratégia promissora para a cura do diabetes mellitus tipo 1 (DM1), mas a aplicação a todos os pacientes diabéticos ainda é impraticável devido à limitada disponibilidade de ilhotas ou células β e à necessidade de utilização de drogas imunossupressoras pelo paciente transplantado. O tratamento com imunossupressores após o transplante de ilhotas pode ser abolido quando se realiza o microencapsulamento das ilhotas pancreáticas. Neste trabalho investigou-se um novo biomaterial, Biodritina® (alginato/sulfato de condroitina) adequado ao microencapsulamento que gelifica na presença de íons de cálcio ou bário. A biocompatibilidade das microcápsulas tem sido avaliada segundo o grau de pureza do alginato utilizado na sua confecção. Amostras de alginato comercial purificado foram analisadas, comprovando-se a presença de impurezas (polifenóis, endotoxinas, proteínas) em níveis elevados, que impedem sua aplicação clínica. Optou-se, portanto pela utilização do alginato comercial ultrapurificado nos experimentos descritos neste trabalho. Das formulações de biomateriais avaliadas, as microcápsulas de bário-Biodritina apresentaram o melhor desempenho em testes de estabilidade físico-química. Estas microcápsulas mantiveram sua morfologia e estabilidade estrutural após permanecerem 30 dias na cavidade peritoneal de camundongos, conforme demonstrado por microscopia eletrônica de varredura (MEV). Análises histológicas mostraram que microcápsulas de bário-Biodritina explantadas, não possuíam adesão celular em sua superfície. Estudos de permeabilidade demonstraram que o tamanho médio dos poros das microcápsulas de bário-Biodritina permite passagem de proteínas de até 70 kDa, enquanto os poros daquelas de cálcio-Biodritina comportam proteínas de até 100 kDa. Experimentos de coResumo | x cultivo de macrófagos peritoneais com ilhotas de rato microencapsuladas demonstraram uma capacidade imunoprotetora maior das microcápsulas de bário-Biodritina em relação às de cálcio- Biodritina, sendo que as primeiras não ativaram os macrófagos. A manutenção da viabilidade e função de ilhotas humanas microencapsuladas com bário-Biodritina foi confirmada através de ensaio funcional in vitro, no qual ilhotas microencapsuladas apresentaram níveis de secreção de insulina idênticos aos de ilhotas nuas. A prova de conceito do biomaterial foi realizada através do implante de ilhotas humanas microencapsuladas em bário-Biodritina em camundongos com DM1 induzido por estreptozotocina. A hiperglicemia desses animais foi corrigida pelo implante por um período superior a 60 dias, durante os quais o teste oral de tolerância à glicose mostrou-se normal, demonstrando completa funcionalidade e eficiência das ilhotas microencapsuladas com bário-Biodritina. Partindo de observações de que animais inoculados com a peçonha do escorpião Tityus serrulatus apresentam nesidioblastose, foi realizado o fracionamento do veneno por HPLC de fase reversa e 24 frações obtidas foram submetidas a ensaios de proliferação celular através da incorporação de 3H-timidina em células de insulinoma de rato RINm5F. Uma dessas frações foi capaz de induzir a proliferação das células RINm5F e quando aplicada a ilhotas humanas isoladas, elevou o índice de secreção de insulina e induziu um aumento da expressão dos mRNAs de insulina e PCNA. Portanto, demonstrou-se que o biomaterial bário-Biodritina possui as características necessárias para microencapsular células/ilhotas com eficiência e que a \"fração ativa\" do veneno do escorpião T. serrulatus induz proliferação de células RINm5F e melhora a secreção de insulina de ilhotas humanas. / Islet transplantation has been proposed as a promising therapeutic strategy for the cure of type 1 diabetes mellitus (DM), however, its application to all diabetic patients is still not possible due to the limited source of islets or β cells and to the need of an immunosuppressive treatment of the recipient to avoid graft rejection. The use of immunosupressors may be abolished when pancreatic islets are microencapsulated prior to transplantation. Here, we investigated the use of a new biomaterial suitable for cell microencapsulation, namely, Biodritin®, composed of alginate and chondroitin sulphate, which is capable of gelation in the presence of barium or calcium ions. Microcapsules biocompatibility has been evaluated according to the purity of the alginate used in its production. Samples of purified commercial alginate were analyzed, but the high levels of contaminants (proteins, endotoxins and polyphenols) detected prevented its use in clinical applications. On the other hand, also commercially available ultrapure alginate fulfills the requirements for this application, therefore, this biomaterial was chosen for our experiments. Among the different biomaterial formulations evaluated, barium-Biodritin microcapsules displayed the best performance in the physico-chemical tests. Scanning electronic microscopy revealed that barium-Biodritin microcapsules maintained their morphology and structural stability after being implanted for 30 days in the peritoneal cavity of mice. No cellular adhesion was detected on the surface of explanted barium-Biodritin microcapsules by histological analysis. Permeability studies determined the medium pore size of barium-Biodritin microcapsules, which allows proteins of up to 70 kDa to pass through the biomaterial, while calcium-Biodritin pores accomodate proteins of up to 100 kDa. Co-culture of peritoneal macrophages with microencapsulated rat islets, revealed a superior immunoprotective capacity of barium-Biodritin microcapsules, which were capable of protecting the islets with no macrophage activation. Microencapsulated and naked human islets presented identical insulin secretion levels upon stimulation with glucose in vitro, confirming that barium-Biodritin microencapsulation maintains the function and viability of human islets. Proof-of-concept experiments in which barium-Biodritin microencapsulated human islets were implanted into chemically-induced diabetic mice, showed that these animals maintained normal blood glucose levels for more than 60 days, during which oral glucose tolerance tests were normal, demonstrating the complete functionality and efficiency of barium-Biodritin microencapsulated human islets. From the observation that animals inoculated with the venom of the scorpion Tityus serrulatus presented nesidioblastosis, we decided to fractionate the venom to isolate the active principle. The venom was fractionated by reversed phase HPLC and 24 fractions were obtained and submitted to cellular proliferation assays, in which rat insulinoma RINm5F cells evaluated for 3H-timidina incorporation. One of these fractions was capable of inducing cell proliferation and was also applied to isolated human islets. Treated islets presented a higher insulin secretion index and an increase in insulin and PCNA mRNA expression. In conclusion, we demonstrated that the barium-Biodritin biomaterial possesses all characteristics required for efficient cell/islet microencapsulation and that the active fraction of Tityus serrulatus venom induces the proliferation of RINm5F cells and improves insulin secretion in human islets.

In the past couple of decades, the zebrafish has been widely used to study hemostatic disorders. In this study, we generated a CRISPR/Cas9 mediated zebrafish mutant that contains a 55-nucleotide insertion in exon 29 of the von Willebrand factor (vwf) gene. The mutants had impaired ristocetin-mediated agglutination of whole blood, prolonged PTT and more bleeding in the lateral incision compared to wild-type fish. The bleeding phenotype observed here is similar to the phenotype observed in vwf knockout mice and patients with von Willebrand disease (VWD). The mutant model developed here can thus be used for exploring the role of Vwf in angiogenesis and for developing gene therapy. The deficiency of VWF causes VWD and the etiology remains unknown in 30% of Type 1 VWD cases. Previous studies have identified that the ABO blood group and ST3GAL4 (glycosyltransferases) are involved in the regulation of VWF levels. Since VWF is heavily glycosylated, we hypothesized that other glycosyltransferases may also be involved in regulating VWF. We performed a knockdown screen of 234 glycosyltransferase genes and identified 14 genes that altered Vwf levels. The sequencing of these genes in Type 1 VWD patients could help identify novel mutations to decipher the molecular basis for the unknown etiologies in Type 1 VWD. Moreover, therapeutic interventions could be designed in the future by modulation of these gene products to control bleeding or thrombosis.Zebrafish has three f9 genes, f9a, f9b, and f9l and the ortholog to human F9 is unknown. RNA analysis showed an age-dependent increase in expression of all three genes from larval stages to adults, comparable to those observed in mice and humans while mass spectrometry and immunohistochemistry confirmed the presence of all three proteins in the fish. Based on coagulation assays performed after individual gene knockdown and immunodepletion, we identified that zebrafish f9a has functional activity similar to human F9 and Fixl is functionally similar to Fx. Thus, the zebrafish could be used to identify factors controlling f9 gene expression with age and for modeling Hemophilia B in the quest to develop gene therapy protocols. In zebrafish, dilute plasma with exogenously added human fibrinogen was used for kinetic coagulation assays. Here, we developed a microkinetic assay using 25% zebrafish or 30% human plasma followed by the addition of coagulation activators and CaCl2. Our results showed both zebrafish and human plasmas yielded kinetic PT, kinetic PTT, and kinetic Russel's viper venom time curves similar to previously established human kinetic curves. Moreover, clotting times derived from these kinetic curves were identical to human PT, PTT, and Russel's viper venom time. Thus, the microkinetic assay developed here could measure blood coagulation activity in small animal models like zebrafish and human blood samples obtained from a finger prick in adults or heel prick in infants.