Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / A vacina anti-diftérica de uso corrente no Brasil (DTP), embora de alta eficácia na prevenção da difteria, está associada com episódios de toxicidade e reatogenicidade no recipiente vacinal, resultantes de proteínas residuais derivadas do processo de produção ou detoxificação. Estratégias para o desenvolvimento de vacinas menos reatogênicas e ao mesmo tempo mais eficazes e economicamente viáveis contra a difteria têm sido alvo de intensa investigação. A alternativa proposta por nosso grupo é a utilização da vacina contra a tuberculose (Mycobacterium bovis BCG sub-cepa Moreau), como vetor do gene que codifica o fragmento B da toxina diftérica (dtb) de 58,3 kDa. Neste trabalho o dtb foi clonado no vetor micobacteriano bifuncional (pUS977) de expressão citoplasmática e os clones recombinantes (pUS977dtbPW8), após a transformação do BCG, foram testados com relação a expressão do DTB em BCG e quanto a antigenicidade frente a anticorpos policlonais anti-toxóide diftérico por Immunobloting. A integridade do gene dtb e a identidade das sequências de DNA da construção plasmidial pUS977dtbPW8 foram confirmadas por sequenciamento de DNA e análise de similaridade. A imunogenicidade do BCGr pUS977dtbPW8 expressando o DTB foi investigada em camundongos BALB/c, os resultados obtidos revelaram uma soroconversão específica (IgG). A infectividade e atividade microbicida do BCGr pUS977dtbPW8 no ambiente intracelular foi avaliada através da infecção de linhagens de células de monócitos humano (THP-1), os dados obtidos indicaram que houve sobrevivência intracelular em até 12 dias. Nesse contexto, esplenócitos dos camundongos imunizados com 30 e 60 dias foram extraídos, mostrando que o BCGr pUS977dtbPW8 persistiu até 60 dias na ausência de pressão seletiva e a viabilidade celular não sofreu alteração significativa durante o período testado. Por outro lado, o BCGr pUS977dtbPW8, quando submetido a seis sub-cultivos consecutivos in vitro não apresentou diferença significativa na capacidade de expressar o DTB, demonstrando portanto a persistência da estabilidade funcional da linhagem recombinante. A estabilidade estrutural da construção pUS977dtbPW8 também foi avaliada por PCR confirmando a presença do gene dtb em colônias do BCGr pUS977dtbPW8 . Adicionalmente, foi possível avaliar preliminarmente in vitro a capacidade soroneutralizante dos soros de camundongos imunizados com BCGr pUS977dtbPW8 após 30 e 60 dias em células VERO. A ação citotóxica da toxina diftérica entre as diluições de 1/4 e 1/16 foram neutralizadas com o pool de soros imunes com 60 dias. Finalmente, em nosso estudo foi possível avaliar o potencial da vacina BCG como vetor de expressão de um antígeno de Corynebacterium diphtheriae in vitro e in vivo. / The diphtheria vaccine currently used in Brazil (DTP), despite its history of high efficacy in the prevention of diphtheria, is associated with episodes of toxicity and vaccine reactogenicity in the vaccinee, resulting from the presence in the vaccine of residual proteins derived from the production process or detoxification. Strategies for the development of new vaccines more effective and economically viable against diphtheria have been the subject of intense investigation. The alternative proposed by our group is the use of the vaccine against tuberculosis (Mycobacterium bovis BCG Moreau sub strain) as a vector for the gene that encodes the 58.3 kDa fragment B of the diphtheria toxin (DTB). In our project the dtb gene was cloned into the bifunctional vector pUS977 for cytoplasmic expression and recombinant BCG (rBCG) clones, selected after transformation of BCG, were tested for expression of the DTB polypeptide and antigenicity against polyclonal antibodies anti- diphtheria toxoid by immunoblotting. The integrity and identity of the DNA sequence encoding the dtb gene carried by the plasmid construct pUS977dtbPW8 was confirmed by DNA Sequencing and Analysis of Similarity. The immunogenicity of the rBCG expressing the DTB was investigated in BALB/c mice and the results revealed a specific seroconversion (IgG). Also, infectivity and microbicidal activity were analyzed in the intracellular environment by infecting human monocytes (THP-1 cell line) with rBCG. The data obtained indicated intracellular survival within 12 days. In this context, splenocytes collected from mice at days 30 and 60 after immunization were removed and assayed for live bacteria. The results showed that rBCG persisted viable up to 60 days in the absence of selective pressure and cell viable counts did not change significantly during testing. Additionally, the rBCG subjected to six consecutive sub-cultures in vitro showed no significant difference in the ability to express the DTB, thus demonstrating the functional stability of the recombinant vaccine. The structural stability of the construct pUS977dtbPW8 was also confirmed by PCR detection of the dtb gene in rBCG colonies. Also, it was possible to have a preliminary evaluation of the neutralizing capacity of sera from mice immunized with BCGr 30 and 60 days after immunization. The cytotoxic action of diphtheria toxin, between dilutions 1/ 4 and 1/16, was neutralized by mice sera in an in vitro assay using VERO cells. Finally, in our study it was possible to evaluate the potential of BCG as a vector for expression of an antigen of Corynebacterium diphtheriae in vitro and in vivo.

Corynebacterium diphtheriae, Pseudomonas aeruginosa, Ricinus communis, Shigella dysentariae, and Vibrio cholerae produce AB toxins which share the same basic structural characteristics: a catalytic A subunit attached to a cell-binding B subunit. All AB toxins have cytosolic targets despite an initial extracellular location. AB toxins use different methods to reach the cytosol and have different effects on the target cell. Broad-spectrum inhibitors against these toxins are therefore hard to develop because they use different surface receptors, entry mechanisms, enzyme activities, and cytosolic targets. We have found that grape seed extract provides resistance to five different AB toxins: diphtheria toxin (DT), P. aeruginosa exotoxin A (ETA), ricin, Shiga toxin, and cholera toxin (CT). To identify individual compounds in grape seed extract that are capable of inhibiting the activities of these AB toxins, we screened twenty common phenolic compounds of grape seed extract for anti-toxin properties. Three compounds inhibited DT, four inhibited ETA, one inhibited ricin, and twelve inhibited CT. Additional studies were performed to determine the mechanism of inhibition against CT. Two compounds inhibited CT binding to the cell surface and even stripped bound CT off the plasma membrane of a target cell. Two other compounds inhibited the enzymatic activity of CT. We have thus identified individual toxin inhibitors from grape seed extract and some of their mechanisms of inhibition against CT. This work will help to formulate a defined mixture of phenolic compounds that could potentially be used as a therapeutic against a broad range of AB toxins.

Abstract Wnt4, a member of the Wnt family of secreted factors, is essential for kidney organogenesis since the kidney fails to develop in its absence. Besides the kidney, Wnt4 signaling is involved in the control of development of several other organs such as the gonads, adrenal glands and pituitary gland. In the context of the embryonic kidney, Wnt4 signaling induces mesenchymal to epithelial transition of the progenitor cells in the metanephric mesenchyme, an early step in nephrogenesis. Wnt4 signaling may also be relevant in the development of a childhood kidney tumor, the Wilms’ tumor, that involves the function of Wilms’ tumor suppressor protein 1 (WT1). Wilms’ tumor is thought to arise from the early metanephric mesenchymal cells of the embryonic kidney, but the detailed mechanisms are not known. The main aim of this project was to study the mechanisms that regulate expression of the Wnt4 gene by using immortalized embryonic kidney mesenchyme-derived mK4 cells as a model. The Wnt4 gene expression was also analyzed in vivo in the frog embryonic pronephros. Through the use of reporter assays and a two-hybrid screen, Sox11, a member of the SoxC family of transcription factors, was identified as a synergistic protein that interacts with WT1. Immunoprecipitation studies provided further evidence that Sox11 and WT1 may physically interact with each other in the developing embryonic kidney. Indeed, Sox11 and WT1 may regulate the Wnt4 gene expression in vivo since the morpholino-based knock-down of either WT1 or Sox11 led to notable downregulation of the Wnt4 gene expression in the frog embryonic pronephros. The other general aim of this thesis was to develop novel tissue targeting and therapy tools to the cell lineages regulated by the Wnt4 signals, including the podocytes. For this purpose, we utilized mice carrying a floxed expression cassette for the avidin-LDL receptor fusion protein, Lodavin, in the constitutively active Rosa-26 locus. Three Cre driver mice, including the Wnt4-Cre knock-in line, were used to activate Lodavin expression in the respective cells of the embryonic kidney. Moreover, we generated a podocyte injury model by expressing the human receptor for diphtheria toxin specifically in the podocytes. This was achieved by crossing mice containing a floxed expression cassette for this receptor in the Rosa-26 locus with those expressing the Cre recombinase under the nephrin promoter. Administration of diphtheria toxin led initially to podocyte damage only, followed by a progression to glomerular sclerosis. As a summary, Sox11 and WT1 serve as synergistic transcription factors that may regulate expression of the Wnt4 gene in vivo. The transgenic mouse models generated and used provide the basis to generate acute and chronic kidney disease models and the potential to purify the respective cells for developing cell-based therapy avenues for the kidney. Moreover, the Lodavin-based approaches may enable targeted delivery of biotinylated small compounds, proteins, viruses or even cells and novel means for in vivo imaging and functional studies. / Tiivistelmä Wnt-4 kuuluu signaloivien proteiinien Wnt-perheeseen ja sen toiminta on välttämätöntä munuaisen kehityksessä. Ilman Wnt-4 proteiinia munuainen ei kehity. Munuaisen lisäksi Wnt4-signalointi on mukana useiden muiden elinten, kuten sukurauhasten, lisämunuaisen ja aivolisäkkeen säätelyssä. Alkion munuaisessa Wnt4-signalointi saa aikaan mesenkymaalisen kantasolukon epitelisoitumisen, edustaen näin ollen nefronin kehityksen varhaisia vaiheita. Wnt4-signaloinnilla on myös merkittävä asema lapsuusiän munuaiskasvaimen, niin kutsutun Wilmsin kasvaimen kehittymisessä. Tämän tyyppisessä kasvaimessa keskeisenä on Wilmsin tuumoriproteiinin WT1:n toiminta, mutta myös Wnt4:n toiminnalla voi olla merkitystä. Wilmsin kasvaimen arvellaan saavan alkunsa varhaisista sikiöaikaisista jälkimunuaisen soluista, mutta yksityiskohtaisia mekanismeja ei vielä tunneta. Tämän projektin tarkoituksena oli tutkia Wnt4-geenin ilmentymistä sääteleviä mekanismeja käyttäen mallina mK4-soluja eli alkion munuaisesta saatuja, immortalisoituja soluja. Wnt4-geenin ilmentymistä analysoitiin myös in vivo sammakon alkion alkumunuaisessa. Tuplahybridi-analysoinnin avulla tunnistettiin transkriptiotekijäperhe SoxC:n jäsen Sox11 samantoimiseksi proteiiniksi transkriptiotekijä WT1:n kanssa Wnt4-geenin ilmentymisen säätelyssä. Immunopresipitaatiotutkimukset tukivat ajatusta, että Sox11 ja WT1 voisivat olla fyysisessä vuorovaikutuksessa säädellessään nefroninmuodostuksen alullepanossa ratkaisevan Wnt4-geenin ilmentymistä. Sox11 ja WT1 voivat mahdollisesti säädellä Wnt4-geenin ilmentymistä myös in vivo, sillä morfoliineihin perustuvissa kokeissa sekä WT1:n että Sox11:n hiljennys laski Wnt4-geenin ilmentymistasoa sammakon alkumunuaisessa. Tämän väitöstutkimuksen toinen yleinen tavoite oli kehittää uusia kudoskohdennus- ja terapiakeinoja Wnt4-signaloinnin säätelemille solulinjoille, kuten podosyyteille. Tätä tarkoitusta varten kloonattiin siirtogeeninen hiiri, jossa floksattu avidiini-LDL -reseptorifuusioproteiini, Lodavin, kohdennettiin jatkuvasti aktiiviseen Rosa-26 -lokukseen. Kolmea eri Cre-hiirilinjaa käytettiin aktivoimaan Lodavinin ilmentyminen kussakin tietyssä alkion munuaisen solupopulaatiossa. Yksi näistä Cre-linjoista oli Wnt4-Cre. Jotta kyettäisiin vahingoittamaan samoja soluja, jotka ilmentävät Lodavinia, hyödynnettiin difteriamyrkyn ihmisen reseptoria (iDTR). IDTR:n ilmentäminen tietyissä hiiren soluissa tekee ne alttiiksi tappavalle difteriamyrkylle. IDTR-perusteisen munuaisvauriomallin kehittämiseksi käytettiin floksattua iDTR-hiirimallia, ja geenin ilmentyminen aktivoitiin Wnt4-indusoiduissa munuaissolulinjoissa, erityisesti podosyyteissä Nephrin Cre -välitteisesti. NephrinCre;R26RiDTR hiiriä altistettiin difteriamyrkylle ja niiden munuaiskerästen muutoksia seurattiin. Tutkimukset antavat viitteitä siitä, että R26R-floksatut iDTR-hiiret toimivat hyvänä mallina kehitettäessä sekä akuutteja että kroonisia munuaistautimalleja. Yhteenvetona voidaan todeta, että Sox-11 ja WT-1 ovat samantoimisia transkriptiotekijöitä, jotka voivat säädellä Wnt4-geenin ilmentymistä in vivo. Tutkimuksessa kehitetyt ja käytetyt siirtogeeniset hiirimallit tarjoavat perustan kehittää sekä akuutteja että kroonisia munuaistautimalleja. Samalla ne mahdollistavat kulloistenkin solujen eristämisen uusien soluperusteisten hoitomenetelmien kehittelemiseksi. Lisäksi Lodavin-perusteiset lähestymistavat voivat mahdollistaa biotinyloitujen pienten yhdisteiden, proteiinien, virusten tai jopa solujen kuljetuksen kohdennetusti sekä avata uusia mahdollisuuksia in vivo -kuvantamiselle ja toiminnallisille tutkimuksille.