La sclérodermie (sclérose systémique, ScS) est une maladie auto-immune du tissu conjonctif caractérisée  par  l’épaississement  de  la  peau,  l’apparition  spontanée de lésions cicatricielles, des maladies   des   vaisseaux   sanguins,   divers   degrés   d’inflammation,   en   association   avec   un   système   immunitaire hyperactif. La pathogénèse exacte de cette maladie est inconnue et aucun traitement approprié   n’est   disponible.   La fibrose est un élément distinctif de la maladie de ScS et est considérée   résulter   d’une   incapacité   à   mettre   fin   de   façon   appropriée   à   la   réponse   normale   de   réparation   des   plaies.   L’analyse   histologique   du   stade   initial   de   la   ScS   révèle   une   infiltration   périvasculaire de cellules mononucléaires dans le derme, associée à une synthèse accrue de collagène dans les fibroblastes environnants. Ainsi, la compréhension des moyens de contrôler le stade inflammatoire de la ScS pourrait être bénéfique pour contrôler la progression de la maladie peu après son apparition. La mPGES-1 est une enzyme inductible qui agit en aval de la cyclo- oxygénase (COX) pour catalyser spécifiquement la conversion de la prostaglandine (PG) H2 en PGE2. La mPGES-1  joue  un  rôle  clé  dans  l’inflammation,  la  douleur  et  l’arthrite;;  toutefois,  le   rôle de la mPGES-1 dans les mécanismes de fibrose, spécifiquement en rapport avec la ScS humaine, est inconnu. Mon laboratoire a précédemment montré que les souris à mPGES-1 nulle sont résistantes à la fibrose   cutanée   induite   par   la   bléomycine,   à   l’inflammation,   à   l’épaississement  cutané,  à  la  production  de  collagène  et  à  la  formation  de  myofibroblastes.  Sur  la   base  de  ces  résultats,  j’ai  formulé  l’hypothèse  que  l’inhibition pharmacologique de la mPGES-1 régulera à la baisse la production de médiateurs pro-inflammatoires et pro-fibreux au cours de la maladie   de   ScS.   Afin   d’explorer   le   rôle   de   la   mPGES-1   dans   l’inflammation   et   la   fibrose   associées  à  la  maladie  de  ScS,  j’ai  d’abord  examiné  l’expression  de  la  mPGES-1 dans la peau normale comparativement à des biopsies de peau extraites de patients atteints de ScS. Mes résultats ont montré que la mPGES-1 est nettement élevée dans la peau de patients atteints de ScS en comparaison avec la peau humaine normale. De plus, les niveaux de PGE2 dérivés de la mPGES-1 étaient également significativement plus élevés dans les fibroblastes cutanés isolés de patients  atteints  de  ScS  comparativement  aux  fibroblastes  isolés  de  témoins  sains.  J’ai  également   étudié  l’effet  de  l’inhibition pharmacologique de la mPGES-1  sur  l’expression  de  marqueurs  pro- fibreux.   Mes   études   ont   montré   que   l’expression   de   médiateurs   pro-fibreux clés (α-SMA, endothéline-1, collagène de type 1 et facteur de croissance du tissu conjonctif (FCTC)) est élevée dans les fibroblastes cutanés ScS en comparaison avec les fibroblastes cutanés normaux. Un traitement avec un inhibiteur de la mPGES-1 a eu pour effet de réduire significativement l’expression  de  l’α-SMA,  de  l’endothéline-1, du collagène de type 1 mais pas du FCTC dans les fibroblastes  ScS,  sans  effet  significatif  sur  les  fibroblastes  normaux.  J’ai  en  outre  examiné  l’effet   de  l’inhibition  de  la  mPGES-1 sur des cytokines pro-inflammatoires clés impliquées dans la pathologie de la ScS, incluant IL-6, IL-8 et MCP-1.  L’inhibition  pharmacologique  de  la  mPGES- 1 a eu pour effet de réduire significativement les niveaux de production de cytokines pro- inflammatoires IL6, IL8 et MCP-1 dans les fibroblastes avec lésion ScS comparativement à des fibroblastes non traités. De plus, les patients atteints de ScS ont présenté des niveaux plus élevés de p-AKT, de p-FAK et de p-SMAD3 en comparaison avec les fibroblastes cutanés normaux. L’inhibiteur  de  la  mPGES-1 a pu réguler à la baisse cette expression accrue de p-AKT et de p- FAK, mais pas de p-SMAD3,  dans  les  fibroblastes  ScS.  Ces  résultats  ont  suggéré  que  l’inhibition   de la mPGES-1 pourrait être une méthode viable pour réduire le développement de sclérose cutanée et constituent une cible thérapeutique potentielle pour contrôler les mécanismes fibreux et inflammatoires associés à la pathophysiologie de la maladie de ScS. L’un   des   autres   processus   critiques   reliés   à   l’évolution de la réponse fibreuse associée à la maladie de ScS est la différenciation des fibroblastes en des cellules activées spécialisées iii iv appelées myofibroblastes, responsables de déclencher une signalisation adhésive excessive et le dépôt excessif de matrice extracellulaire,   conduisant   à   la   destruction   de   l’architecture   de   l’organe.   Ainsi,   l’identification   des   facteurs   endogènes   qui   initient/   favorisent   la   différenciation   fibroblaste-myofibroblaste peut mener à des stratégies thérapeutiques prometteuses pour contrôler  l’excès  de  signalisation  adhésive  et  de  fibrose  associé  à  la  maladie  de  ScS.  Des  études   antérieures  dans  le  domaine  de  la  biologie  du  cancer  ont  suggéré  que  l’éphrine  B2,  une  protéine   transmembranaire appartenant à la famille des éphrines, est impliquée dans la signalisation adhésive   et   le   remodelage   extracellulaire.   Cependant,   son   rôle   dans   la   fibrose   n’a   jamais   été   exploré.   Dans   la   deuxième   partie   de   mon   étude,   j’ai   donc   étudié   le   rôle   de   l’éphrine   B2   dans   la   fibrose.   Mes   études   montrent   que   l’expression   de   l’éphrine   B2   est   significativement   augmentée   dans la peau humaine ScS comparativement à la peau normale. Plus important encore, le traitement in vitro de   fibroblastes   de   la   peau   humaine   normale   avec   de   l’éphrine   B2   recombinante est capable de transformer des fibroblastes en cellules myofibroblastiques manifestant toutes les caractéristiques myofibroblastiques typiques, incluant la formation accrue de  fibres  de  tension,  des  adhérences  focales,  l’activation  accrue  de  la  FAK,  un  accroissement  de   l’expression  et  de  la  migration  de  fibroblastes  et  de  leur  adhérence  à  la  fibronectine  à  la  fois  chez   les   fibroblastes   cutanés   normaux   et   ScS.   En   outre,   j’ai   traité   des   souris   avec   de   l’éphrine   B2   recombinante et montré que ces souris ont développé une fibrose cutanée significative associée à une épaisseur dermique et à une synthèse de collagène augmentées, une teneur en hydroxyproline (teneur en collagène) accrue et un nombre accru de myofibroblastes exprimant de   l’α-SMA, une activation augmentée de la FAK et de marqueurs pro-fibreux incluant le collagène de type 1 et le FCTC. Dans  l’ensemble,  mes  études  ont  identifié  deux  médiateurs  endogènes  cruciaux  impliqués  dans  la   propagation  de  l’inflammation  et  de  la  fibrose  associées  à  la  maladie  de  ScS.  L’inhibition  de  la   mPGES-1   pourrait   représenter   une   bonne   stratégie   alternative   pour   contrer   l’inflammation   et   la   fibrose au moins durant les stades précoces de la maladie de ScS. De plus, une signalisation excessive   de   l’éphrine B2 favorise la signalisation adhésive et fibreuse en déclenchant la différenciation   de   fibroblastes   en   myofibroblastes   par   l’activation   de   la   voie   de   signalisation   de   la  FAK.  Ainsi,  l’inhibition  d’éphrine  B2  bloquera  la  formation  de  fibroblastes-myofibroblastes et régulera à la baisse la fibrose associée à la maladie de ScS. En somme, la mPGES-1  et  l’éphrine   B2 semblent toutes deux des cibles attrayantes pour le traitement de la ScS et des troubles fibreux qui y sont reliés. / Scleroderma (Systemic sclerosis, SSc) is an autoimmune disease of the connective tissue featuring skin thickening, spontaneous scarring, and blood vessel disease, varying degrees of inflammation, associated with an overactive immune system. The exact pathogenesis of this disease is unknown and there is no appropriate treatment available. Fibrosis is a hallmark of SSc disease and is considered to arise due to an inability to appropriately terminate the normal wound repair response. Histological analysis of the initial stage of SSc reveals perivascular infiltrates of mononuclear cells in the dermis, which is associated with increased collagen synthesis in the surrounding fibroblasts. Thus understanding how to control the inflammatory stage of SSc may be of benefit in controlling the progression of early onset disease. mPGES-1 is an inducible enzyme that acts downstream of cyclooxygenase (COX) to specifically catalyze the conversion of prostaglandin (PG) H2 to PGE2. mPGES-1 plays a key role in inflammation, pain and arthritis; however, the role of mPGES-1 in fibrotic mechanisms especially with respect to human SSc is unknown. My laboratory has previously shown that mPGES-1-null mice are resistant to bleomycin-induced skin fibrosis, inflammation, cutaneous thickening, collagen production and myofibroblast formation. Based on these results I hypothesized that pharmacological inhibition of mPGES-1 will downregulate the production of pro-inflammatory and pro-fibrotic mediators during SSc disease. To explore the role of mPGES-1 in inflammation and fibrosis associated with SSc disease, I first investigated the expression of mPGES-1 in normal skin compared to skin biopsies extracted from SSc patients. My results showed that mPGES-1 is markedly elevated in SSc skin compared to normal human skin. In addition, the levels of mPGES-1- derived PGE2 were also significantly higher in skin fibroblasts isolated from SSc patients compared to fibroblasts isolated from healthy controls. I further investigated the effect of pharmacological inhibition of mPGES-1 on the expression of pro-fibrotic markers. My studies showed the expression of key pro-fibrotic mediators (α-SMA, endothelin-1, collagen type 1 and connective tissue growth factor) are elevated in SSc skin fibroblasts compared to normal skin fibroblasts. Treatment with mPGES-1 inhibitor resulted in significant reduction in the expression of α-SMA, endothelin-1, collagen type 1 but not CTGF in SSc and normal fibroblasts. Further, I investigated the effect of mPGES-1 inhibition on key pro-inflammatory cytokines implicated in SSc pathology including IL-6, IL-8 and MCP-1. Pharmacological inhibition of mPGES-1 resulted in significant reduction in the production levels of pro-inflammatory cytokines, IL6, IL8 and MCP-1 in SSc-lesioned fibroblasts compared to untreated fibroblasts. In addition, SSc patients exhibited higher levels of p-AKT, p-FAK and p-SMAD3 compared to normal skin fibroblasts. mPGES-1 inhibitor was able to down regulate this increased expression of p-AKT, p-FAK but not p-SMAD3 in SSc fibroblasts. These results suggested that inhibition of mPGES-1 may be a viable method to alleviate the development of cutaneous sclerosis and is a potential therapeutic target to control fibrotic and inflammatory mechanisms associated with the pathophysiology of SSc disease. One of the other critical processes associated with the evolution of fibrotic response associated with SSc disease is the differentiation of fibroblasts into specialized activated cells called myofibroblasts responsible for triggering excessive adhesive signaling and deposition of excessive extracellular matrix (ECM) leading to the destruction of organ architecture. Thus identifying endogenous factors which initiate/promote fibroblast-myofibroblast differentiation can lead to promising therapeutic strategies to control excessive adhesive signaling and fibrosis associated with SSc disease. Previous studies in cancer biology have suggested that ephrin B2, a transmembrane protein belonging to the family of ephrins, is involved in adhesive signaling and extracellular remodeling. However its role in fibrosis has never been explored. Therefore, in second part of my study, I investigated the role of ephrin B2 in fibrosis. My studies show ephrin v vi B2 expression is significantly enhanced in human SSc skin versus normal skin. Most importantly, in vitro treatment of normal human skin fibroblasts with recombinant ephrin B2 is able to transform fibroblasts into myofibroblastic cells exhibiting all typical myofibroblastic- characteristics including increased stress fibre formation, focal adhesions, increased activation of FAK, increased expression of and enhanced fibroblast migration and adhesion to fibronectin in both normal and SSc skin fibroblasts. Further, I treated mice with recombinant ephrin B2 and showed that these mice developed significant skin fibrosis associated with enhanced dermal thickness and collagen synthesis, increased hydroxyproline content (collagen content) and increased number of α-SMA-expressing myofibroblasts, enhanced activation of FAK and pro- fibrotic markers including type-I collagen and CTGF. Overall, my studies have identified two crucial endogenous mediators involved in propagating inflammation and fibrosis associated with SSc disease. mPGES-1 inhibition may present a good alternative strategy to counteract inflammation and fibrosis at least during early stages of SSc disease. Further, excessive ephrin B2 signaling promotes adhesive and fibrotic signaling by triggering fibroblast to myofibroblast differentiation via activation of the FAK signaling pathway. Thus, inhibition of ephrin B2 will block fibroblast-myofibroblast formation and downregulate fibrosis associated with SSc disease. Overall, both mPGES-1 and ephrin B2 seems to be attractive targets for treatment of SSc and related fibrotic disorders.

Diss. (sammanfattning) Linköping : Linköpings universitet, 2005. / Härtill 4 uppsatser.

No description available.

Modélisations moléculaires réalisés avec le logiciel HyperChem 8. / L’effet de différentes alkylations sur l’activité biologique et la stabilité enzymatique d’un peptide linéaire L, énantiomère du modulateur allostérique des récepteurs prostaglandine F2α ont été étudiés. Dans une étude antérieure, le peptide D PDC-31 avait montré un potentiel d’inhibition des contractions du myomètre et permettait de retarder l’accouchement dans des modèles animaux et humains. Il est possible que le peptide L possède une activité semblable, mais les protéases, abondantes dans le tissu myométrial, le dégradent probablement avant qu’il ne puisse atteindre le site actif. La synthèse peptidique sur support solide suivie d’une amination réductive a permis d’obtenir différents peptides portant différentes chaines alkyle et PEG N-terminales. La protection de l’amine terminale par un groupement ortho-nitrobenzène sulfonyle suivie par une réaction de Mitsunobu a permis l’obtention d’un analogue portant une chaine farnesyle. Malgré le fait que ni l’analogue PEGylé, ni l’analogue farnesylé n’aient montrés la moindre activité, certains analogues alkylés se sont avérés actifs dans l’essai tissulaire de contractions myométriales. Le peptide L portant une chaine dodecyle s’est avéré posséder une activité statistiquement significative et reproductible. Qui plus est, l’analogue D du peptide possédant une chaine de 12 carbones s’est avéré posséder une activité inférieure à l’analogue L portant la même chaine, ce qui représente une perte d’activité significative par rapport au peptide D nonmodifié (PDC-31). / The application of hydrophobic grafts to prolong the biological activity of rapidly metabolized peptides has been explored by modification of the L-peptide of the prostaglandin F2α receptor modulator PDC-31. The all-D peptide PDC-31 has previously been shown to inhibit myometrial contractions and delay labour in various animal models as well as in humans. The L-peptide may have activity; however, proteases, which are abundant in myometrial tissue, may likely degrade the peptide before it is capable of showing activity. Solid-phase peptide synthesis followed by Nterminal modification by reductive aminations with different aldehydes provided linear aliphatic alkyl and PEG-grafted peptide analogs. Alternatively, ortho-nitrobenzensulfonylation of the peptide followed by Mitsunobu alkylation with farnesol and deprotection gave a farnesylated analog. Although the PEG and fanesylated analogs exhibited no activity, certain N-alkyl analogs exhibited inhibitory activity on myometrial contractions, with the most active analog possessing a dodecyl chain. Moreover, the N-dodecyl analog of PDC-31, exhibited lower activity than its L-counterpart in the myometrial contraction assay, and with reduced potency relative to its unmodified structure.

La naissance avant terme constitue un important problème de santé périnatale partout dans le monde. Chaque année près de 15 millions de bébés naissent prématurément. Notre laboratoire s’intéresse depuis une décennie à la conception et le développement de nouvelles classes thérapeutiques (appelés agents tocolytiques) capables d’inhiber les contractions utérines et prolonger le temps de gestation. Due à son implication directe dans le déclanchement des contractions utérines, la prostaglandine F2α (FP) est devenue notre cible de choix. Le PDC 113.824 et aza-glycinyl-proline sont apparus comme des inhibiteurs allostériques puissants du récepteur de la prostaglandine F2α, capables de prolonger la durée de gestation chez les souris. Le travail réalisé au cours de ce mémoire a pour objectif d’étudier le tour β nécessaire pour la reconnaissance sur le récepteur de la prostaglandine F2α. Dans la conception de mime peptidique sélectif efficace et puissant, le repliement β est un élément structural essentiel pour le maintien ou l’amélioration de l’activité du mime peptidique. Les études conformationelles du PDC113.824 et l’aza-glycinyl-proline montrent que les squelettes centraux de ces mimes peptidiques pourraient adopter essentiellement un tour β de type I ou II`, ce qui suggère l’implication des deux types de tours dans l’activité biologique. La synthèse de mimes peptidiques adoptant le tour β de type I et II` est devenue une stratégie logique dans la recherche de nouveaux agents tocolytiques. Au cours de ces études quatre analogues du PDC113.824 ont été synthétisés, un azabicyclo[5.3.0]alkanone et un dipeptide macrocyclique (mimes du tours β de type I) pour étudier l’implication du tour β type I. Par la suite la synthèse de glycinyl-proline et le D-alaninyl-proline (des mimes du tour β du type II`) a été réalisée afin de valider l’implication du tour β type II` dans la reconnaissance sur le récepteur. Nous avons utilisé une stratégie de synthèse efficace pour obtenir les deux analogues (azabicyclo [5.3.0]alkanone et dipeptides macrocycles) à partir d’un intermédiaire commun, en procédant par une cyclisation transannulaire diastéréosélective du tétra-peptide. / Preterm birth is a major perinatal health problem. Worldwide each year, more than one baby on ten is born premature, accounting for approximately 15 million babies per year. Pursuing molecules that can delay labour, so called tocolytics, we have targeted the prostaglandin F2α receptor (FP), because of its intimate involvement with the stimulation of uterine contractions leading to birth and preterm birth. Our laboratory has previously demonstrated that PDC-113.824 and azapeptide can modulate FP function by an allosteric mechanism featuring biased signalling, and can delay labour in a mouse model. The objective of the present work is to study the active turn geometry responsible for the modulatory effects of our lead tocolytic agents on the prostaglandin F2α receptor. Crystal structure and computational analyses of indolizidin-2-one amino acid and aza-glycinyl-proline components have shown them to adopt geometry that mimic ideal type I and II’ β-turns suggesting the potential involvement of such turns in the biological activity. To study the relevance of a type I β-turn on activity, we have prepared analogs possessing macrocyclic and pyrroloazepinone dipeptide mimics, because of their reported propensities to mimic type I β-turns. The syntheses of analogs possessing glycinyl-proline and D-alaninyl-proline were conducted to validate the involvement of a type II` β-turn in receptor recognition. In addition, we reported an efficient strategy for preparing the macrocyclic and pyrroloazepinone dipeptide mimics from a common intermediate using a diastereoselective transannular cyclization on a tetrapeptide precursor.

Resultados obtidos pelo nosso grupo demonstraram, in vitro, que a PGE2 é capaz de modular a sobrevivência de linfócitos TCD4+ protegendo essas células da morte. Dentro do modelo de EAE, nossa hipótese é que a PGE2 liberada pelas APCs, durante a fase de indução, module a sobrevivência de linfócitos autorreativos específicos induzindo a doença. Realizamos o tratamento de camundongos submetidos à EAE com indometacina durante 5 dias e notamos que houve redução da EAE associada à redução de linfócitos produtores de IFN-γ, IL-17 e GM-CSF, e macrófagos infiltrantes e microglias ativadas, no SNC. O tratamento alterou a freqüência de células em proliferação e a frequência de células produtoras de IFN-γ e IL-17 na periferia e a concentração dessas citocinas. Esses resultados sugerem que a indometacina reduz o desenvolvimento da EAE e sua resposta antígeno-específica demonstrando a sua importância na modulação das respostas de linfócitos T na autoimunidade. / Results obtained by our group demonstrated in vitro that PGE2 is able to modulate CD4+ T cells survival protecting these cells from death. Within the EAE model, we hypothesized that PGE2 released by APCs during the induction phase, modulate survival of autoreactive specific lymphocytes by induction the disease. We carried out the treatment of EAE in mice subjected to indomethacin for 5 days and noticed that there is reduction of EAE associated with decreased IFN-γ, IL-17 and GM-CSF producing T cells, and infiltrating macrophages and activated microglia in the CNS. The results suggest that indomethacin reduces EAE and its antigen-specif response demonstrating their importance in the modulation of T lymphocyte responses in autoimmunity.

O objetivo do experimento I foi avaliar a luteólise causada por três doses de PGF2&#945; (12,5; 25 e 50 mg de Dinoprost tometamina) nos dias 5 e 7 do ciclo estral. Foram utilizadas 339 vacas não lactantes da raça Nelore. Os animais foram divididos em dois grupos de acordo com a apresentação do estro, recebendo a dose de PGF2&#945; nos dias 5 e 7 do ciclo estral. Cada grupo foi subdividido em três, que receberam os seguintes tratamentos de PGF2&#945; (12,5 mg; 25 mg; 50 mg). Através da concentração de P4 foram estimadas as taxas de regressão luteal 1 e 0,5 (1 - animais com concentração de P4 abaixo de 1 ng/mL; 0,5 animais com concentração de P4 abaixo de 0,5 ng/mL). Não houve interação entre a dose de PGF2&#945; e o dia do ciclo estral. A aplicação de PGF2&#945; no dia 7 do ciclo estral apresentou maior taxa de regressão luteal 1 e 0,5 quando comparada com o dia 5 do ciclo estral (1 - 76,9 vs 37,0 % - P = 0,0001; 0,5 - 57,5 vs 21,7 %, P = 0,0001, respectivamente). A taxa de regressão luteal 1 aumentou de acordo com a dose de PGF2&#945; administrada (12,5 mg 39,0 %; 25 mg 56,9 %; 50 mg 76,5 % P <0,0001). Quando a PGF2&#945; foi administrada no dia 5 do ciclo estral a concentração média de P4 segui o padrão de luteólise parcial e foi dependente da dose de PGF2&#945;. Quando a PGF2&#945; foi aplicada no dia 7 do ciclo estral a concentração média de P4 caiu drasticamente de 0 para 24 h e não voltou a se elevar em 48 h. A concentração de P4 em 48 h após a aplicação de PGF2&#945; foi menor na dose de 50 mg (0,51 ± 0,07 ng/mL). A taxa de luteólise foi menor no dia 5 do ciclo estral comparado com o dia 7 do ciclo estral. À medida que a dose de PGF2&#945; foi aumentada, a porcentagem de regressão luteal se elevou. O objetivo do experimento II foi avaliar se a substituição das aplicações do GnRH por BE, ECP e eCG no protocolos de 5 dias de P4 causa dupla ovulação e se a utilização de 1 ou 2 mg de BE no início do protocolo influencia na taxa de ovulação dupla. Foram utilizadas 85 multíparas da raça Nelore. No dia 0 do protocolo, as vacas receberam um implante de P4 e a dose de BE segundo o tratamento pertencente (tratamento A 1 mg de BE, tratamento B 2 mg de BE). Cinco dias após, o dispositivo foi retirado e aplicou-se PGF2&#945;, ECP eCG. Houve 5,9 % de dupla ovulação. O tratamento A causou menor porcentagem folículos maiores que 8 mm no dia 7 protocolo que o tratamento B (28,7 vs 50,6 P = 0,0460). Não houve diferença significativa na taxa de ovulação dupla, no diâmetro do folículo dominante no dia 5 e no dia 7 do protocolo e na taxa de crescimento folicular entre os tratamentos A e B. O protocolo de 5 dias de P4 com BE, ECP e eCG causou uma baixa taxa de dupla ovulação. O objetivo do experimento III foi comparar o aparecimento e a frequência de estro e a taxas de ovulação e gestação entre os protocolos de 5 dias de P4 + GnRH / GnRH e de 7 dias de P4 + BE / ECP + eCG em nulíparas, primíparas e multíparas. Foram utilizadas 411 fêmeas da raça Nelore (nulíparas - n = 198; primíparas - n = 80; multíparas - n = 133). No protocolo de 7 dias de P4, os animais receberam no dia 0 um implante de P4 e BE. No dia 7, o dispositivo foi retirado e aplicou-se PGF2&#945;, ECP e eCG. No protocolo de 5 dias de P4, os animais receberam no dia 0 o implante de P4 e GnRH. No dia 5, o dispositivo foi retirado e aplicaram-se 2 doses de PGF2&#945; com intervalo de 6 h entre as doses de PGF2. Os animais que não apresentaram estro até a hora da IA receberam 100 &#956;g de GnRH no momento da IA. A taxa de prenhez utilizando o protocolo de 5 ou 7 dias de P4 variou de acordo com a categoria da fêmea (nulíparas - 41,0 vs 51,0 % - P = 0.1608; primíparas 25,6 vs 31,7 % - P = 0,5513; multíparas - 58,4 vs 32,8 % - P = 0,0041, respectivamente). A taxa de apresentação de estro no protocolo de 7 dias de P4 foi maior para todas as categorias de fêmeas quando comparado com o protocolo de 5 dias deP4. (nulíparas 95,8 vs 66,0 % - P <0,0001; primíparas 48,7 vs 0 %; multíparas - 76,9 vs 13,4 % - P <0,0001, respectivamente). A resposta ao protocolo de 5 dias com GnRH foi pior nas multíparas. / The objective of the experiment I was to evaluate the luteolysis caused by three doses of PGF2&#945; (12.5, 25 and 50 mg of Dinoprost tometamina) when applied on the 5th and 7th days of the estrous cycle. Three hundred thirty-nine (339) nonlactating Nelore cows were used. The animals were divided into two groups according to the onset of estrus, and received the PGF2&#945; dose on the 5th or 7th day of the estrous cycle. Each group was divided into three subgroups, which were submitted to the treatments of PGF2&#945; with dose of 12.5 mg, 25 mg or 50 mg. Through the P4 concentration, the rates of 1 and 0.5 luteal regression were estimated (1 - animals with P4 concentration below 1 ng/mL and 0.5 - animals with P4 concentration below 0.5 ng/mL). There was no interaction between the PGF2&#945; dose and the day of the estrous cycle. The PGF2&#945; application on the 7th day of the estrous cycle had higher rates of the 1 and 0.5 luteal regression, when compared to the PGF2&#945; application on the 5th day of the estrous cycle (1 - 76.9 vs 37.0 % - P = 0.0001; 0,5 - 57.5 vs 21.7 %, P = 0.0001, respectively). The rate of 1 luteal regression increased with the PGF2&#945; dose (12.5 mg - 39.0 %; 25 mg - 56.9 %; 50 mg - 76.5 %, P < 0.0001). The average concentrations of P4, when the PGF2&#945; was administered on the 5th day of the estrous cycle, follow the partial luteolysis standard that dependent on the PGF2&#945; dose. When the PGF2&#945; is applied on 7th day of the estrous cycle, the average concentration of P4 drops dramatically from 0 to 24 h and it do not rise again in 48 h. The P4 concentration is lower in the 50 mg (0.51 ± 0.07 ng/mL), 48 h after the PGF2&#945; application. The luteolysis rate was low on the 5th day of the estrous cycle. The luteal regression percentage increased with increase of the PGF2&#945; dose. The objective of the experiment II was to evaluate whether the replacement of the GnRH applications by BE, ECP and eCG in the 5-day protocols of P4 cause double ovulation and if the use of 1 or 2 mg of BE at the beginning of the protocol influences the double ovulation rate. Eighty-five (85) multiparous Nelore cows were used. On day 0 of the protocol, the cows received a P4 implant and a dose of BE that depending on the treatment to which the cows belong (Treatment A - 1 mg of BE, treatment B - 2 mg of BE). Five days later, the device was removed and the PGF2&#945;, ECP and eCG were applied. In the experiment II, there was 6.5 % of double ovulation. There was no significant difference in the double ovulation rate, dominant follicle diameter on the 5th and 7th day of the protocols, and follicular growth rate between the treatments A and B. The 5-day protocol of P4 with BE, eCG and ECP caused a low rate of the double ovulation, and there was no difference between 1 or 2 mg of BE to synchronize the follicular development wave. The objective of the experiment III was to compare the onset and frequency of estrus and the ovulation and pregnancy rates among the protocols of 5 days of P4 with GnRH and 7 days of P4 with BE, ECP and eCG in nulliparous, primiparous and multiparous. Four hundred eleven (411) Nellore females were used (nulliparous - n = 198; primiparous - n = 80; multiparous - n = 133). In 7-day protocol of P4, the animals received an implant of P4 and BE on day 0. On day 7, the device was removed and the PGF2&#945;, ECP and eCG were applied in the cows. In 5-day protocol of P4, the animals received implants of GnRH and P4 on the day 0. On day 5, the device was removed and two doses of PGF2&#945; were applied with interval of 6 h. The animals that did not show estrus until the AI time received 100 mg of GnRH at this moment. The pregnancy rate varied according to the female category in both protocols (nulliparous - 41.0 vs 51.0 % - P = 0.1608; primiparous - 25.6 vs 31.7 % - P = 0.5513; multiparous - 58.4 vs 32.8 % - P = 0.0041, respectively). The rate estrus onset in the 5-day protocol of P4 with GnRH was lower for all female categories, when compared to the 7-day protocol (nulliparous - 95.8 vs 66.0 % - P <0.0001; primiparous 0 vs 48.7 %; multiparous - 76.9 vs 13.4 % - P <0.0001, respectively). The response in the 5-day protocol with GnRH was worse in multiparous cows.

Interação entre angiotensina-(1-7) [Ang-(1-7)] e bradicinina (BK) foi determinada no mesentério de ratos Wistar anestesiados utilizando-se microscopia intravital. Aplicação tópica de BK e Ang-(1-7) induziram vasodilatação que foi abolida por HOE-140 e A-779, respectivamente. Ang-(1-7) (100 pmol) potencializou a vasodilatação de BK (1 pmol) mas não a vasodilatação promovida por acetilcolina, nitroprussiato de sódio, histamina e ácido araquidônico. O efeito potencializador de Ang-(1-7) sobre BK foi abolido por A-779, HOE-140, indometacina, L-NAME e TEA, entretanto, losartan não bloqueou este efeito. Enalaprilato aumentou a resposta vasodilatadora de BK e Ang-(1-7) e não alterou o efeito potencializador do segundo sobre o primeiro. Conclui-se que: 1)efeito potencializador de Ang-(1-7) sobre BK depende da interação de ambos com seus respectivos receptores; 2)é dependente de óxido nítrico, produtos da cicloxigenase e hiperpolarização de membrana via canais de potássio; 3) o mecanismo de potencialização parece não depender da atividade catalítica da ECA. / The interaction between angiotensin-(1-7) [Ang-(1-7)] and bradykinin (BK) was determined in the mesentery of anesthetized Wistar rats using intravital microscopy. The response-induced by topical application of BK (1, 10 and 30 pmol), Ang-(1-7) (1, 10, 100 and 1000 pmol) and Ang-(1-7) (100 pmol) + BK (1 pmol) was determined in mesenteric arterioles (15-20 mm diameter). The BK (1 pmol)- and Ang-(1-7) (100 pmol)- induced vasodilation was abolished by BK B2 receptor antagonist HOE-140 (100 pmol applied during 60 seconds) and the Ang-(1-7) antagonist [d-Ala7]-Ang-(1-7)] (A-779) (100 pmol applied during 15 seconds), respectively. Indomethacin (5 mg/kg; IM, 30 min before), a cyclooxygenase inhibitor; L-NAME (10 nmol; topical application, 3 min before), a NO synthase inhibitor, decreased the Ang-(1-7)-induced vasodilation. However, TEA (90 pmol; topical application), a non specific K+ channels blocker, did not alter the response to BK or Ang-(1-7). BK (1 pmol)-induced vasodilation, however, was potentiated by Ang-(1-7) 100 pmol. Sodium nitroprusside (38 pmol), acetylcholine (1,6 nmol), histamine (5,4 nmol) and arachdonic acid (10 nmol) responses were not modified by Ang-(1-7) 100 pmol. The Ang-(1-7)-potentiating effect on BK-induced vasodilation was abolished by A-779, HOE-140, indomethacin, L-NAME and TEA. Losartan (15 mg/kg.IV, 40 min before), an AT1 angiotensin receptor antagonist was without effect. On the other hand, enalaprilat treatment (10 mg/kg; IV, 30 min before), to inhibit angiotensin-converting enzyme (ACE), enhanced the BK and Ang-(1-7)-induced vasodilation but did not modify the effect of Ang-(1-7) on BK vasodilation. In conclusion, the potentiation of BK-induced vasodilation by Ang-(1-7) is a receptor-mediated phenomenon dependent on cyclooxygenase-related products, NO release and K+ channel-mediated membrane hyperpolarization. The potentiating mechanism, apparently, is not related to ACE catalytic activity.

Três experimentos foram desenvolvidos para avaliar protocolos hormonais de sincronização de estro e ovulação em fêmeas Nelore, extratificadas nas três principais categorias de fêmeas (vacas secas, novilhas e vacas paridas) comumente encontradas nas propriedades. Os experimentos possuíam objetivos de comparar a performance reprodutiva das categorias supra citadas, sobre a taxa de detecção do estro, taxa de prenhez na IA (exp. I e II) e/ou IATF (exp III) e no repasse, de fêmeas submetidas a protocolos de sincronização do estro e ovulação. EXP I: Foram utilizadas vacas Nelore não lactantes (n=286), blocadas em um esquema fatorial 2x3, e receberam no D0 o CIDR e 2mg de benzoato de estradiol (BE). O CIDR permaneceu por 5 dias (D5) ou 7 dias (D7), no dia da remoção do CIDR, as vacas receberam PGF2&alpha; (12,5mg, 25mg ou 50mg), formando os tratamentos: 5d12,5mg, 5d25mg, 5d50mg, 7d12,5mg, 7d25mg e 7d50mg. O estro foi detectado em 83.2% (238/286) das vacas, onde D7 (88,0%) foi superior (P<0,05) ao D5 (78,7%). Não houve diferença no tempo médio para detecção do estro que foi de 70,1 ±17,2 h. A taxa de concepção não diferiu entre os tratamentos. A taxa de prenhez à IA foi maior (P<0,05) para o D7 (54,2%) quando comparado ao D5 (39,6%). A taxa de prenhez no repasse e na prenhez total não diferiram entre os tratamentos. EXP II: Utilizou-se 407 novilhas Nelore pré-puberes, blocadas em um esquema fatorial 4x2, as novilhas receberam no D0 o CIDR e 2 mg de BE, o CIDR permaneceu por 5, 7, 9 ou 11 dias. No dia da remoção do CIDR, as novilhas receberam 25mg de PGF2&alpha; e 300 UI eCG. Além disso metade das novilhas receberam 1 mg de BE 48 horas após a retirada do CIDR. A detecção do estro foi maior (P<0,05) para as novilhas que receberam BE (87,3%) quando comparado aos s/BE (54,6%). O tempo médio para detecção do estro foi de 68,1 ±17,3 h. A taxa de concepção não foi influênciada pelos tratamentos, porém a taxa de prenhez da inseminação da sincronização foi maior para o grupo BE (18,8%) quando comparado ao s/BE (12,4%). A taxa de prenhez final não diferiu entre os tratamentos e foi de 62,3%. EXP III: Foram utilizadas vacas Nelore lactantes (n=191), divididas em dois tratamentos: 1xPGF-5d (as vacas permaneceram com o CIDR por 7 dias, sendo que 25mg de PGF2&alpha; foi aplicada no D5); 2xPGF-0/7d (as vacas permaneceram com o CIDR por 7 dias, sendo realizadas duas aplicações de 12,5mg de PGF2&alpha;, no D0 e D7). O CIDR foi retirado no D7, e aplicou-se 0,3 ml de ECP e 300 UI de eCG. Após 50 horas todas as vacas foram submetidas à IATF. No momento da inserção do CIDR 24% das vacas estavam ciclícas. O estro foi detectado em 54,45% (104/191) das vacas, sendo similar entre os tratamentos. O tempo médio para detecção do estro foi de 36,4 ± 8,7 h. Não houve diferença na concepção à IATF, que foi de 59,5 e 59,6% para os tratamentos 1xPGF-5d e 2xPGF- 0/7d, respectivamente. A taxa de prenhez ao final da estação de monta não diferiu e foi de 82,9% para 1xPGF-5d e 86,6% para 2xPGF-0/7d. Em todos os experimentos foi realizada a detecção do estro, com o auxilio de rufiões por até 5 dias após a remoção do CIDR, sendo novamente detectado 10 dias após por um periodo de até 10 dias. Os animais dos exp. I e II foram inseminadas de acordo com o protocolo AM/PM. O diagnóstico de gestação foi realizado de acordo com a caracteristica de cada experimento, mas sempre com o objetivo de identificar e quantificar a origem da prenhez (IA ou repasse). Em vacas Nelore não lactantes, a utilização de protocolos de 7 dias, possui uma performance reprodutiva melhor do que 5 dias de permanência do CIDR. Em novilhas pré-puberes a utilização de BE 48 horas após a remoção do CIDR, aumentou a taxa de detecção de estro, impactanto diretamente no incremento da taxa de prenhez à IA. A utilização do protocolo 7d BE+CIDR, apresenta bons resultados reprodutivos, sendo que podemos reduzir o número de manejos sem o comprometimento da performance reprodutiva, realizando a aplicação de 12,5mg na inserção e na retirada do CIDR. / Three experiments were designed to evaluate estrus synchronization protocols in Nellore females of three different class (dry cows, heifers and lactating cows) commonly found in properties. The experiments had aim to compare a reproductive performance of the categories mentioned above, at the estrus detection, pregnancy rate at AI (exp. I and II) or TAI (exp. III), pregnancy rate at rebreeding, of females submited to estrus synchronization program and ovulation. EXP I: Nonlactating Nellore cows (n=286), were randomized in a 2x3 fatorial arrangement. All animals received the CIDR and 2 mg of EB at D0, the CIDR remained for 5 days (D5) or 7 days (D7). At day of CIDR removal, cows received PGF2&alpha; treatments (12.5mg, 25mg and 50mg), the treatments followed: 5d12.5mg, 5d25mg, 5d50mg, 7d12.5mg, 7d25mg and 7d50mg. Estrus was detected in 83.2% (238/286) of cows, where D7 (88.0%) was higher (P<0.05) than D5 (78.7%). The onset of estrus did not differ among groups and was 70.1 ± 17.2 hours. Conception rate did not differ among treatments. The pregnancy rate at AI was greater (P<0.05) for D7 (54.2%) compared to D5 (39.6%). The pregnancy rate at rebreeding and overall pregnancy did not differ among treatments. EXP II: Prepubertal Nellore heifers (n=407), randomized in a 4x2 factorial arrangement. Heifers received CIDR and 2 mg EB at D0, CIDR remained for 5, 7, 9 or 11 days. On day of CIDR removal, heifers received 25mg of PGF2&alpha; and 300 IU of eCG. Half of the heifers received 1 mg of EB 48 hours after CIDR removal. Estrus detection was greater in EB (87.3%) when compared to NoEB (54.6%). The onset of estrus did not differ among treatments and was 68.1 ± 17.3 hours after CIDR removal. Conception rate was similar among treatments. The pregnancy rate at AI of synchronization was higher for EB (18.8%) compared with NoEB (12.4%). The final pregnancy rate did not differ among treatments. EXP III: Lactating Nellore cows (n=191) were randomized in two groups, 1xPGF-5d (cows received CIDR for 7 days, and 25 mg of PGF was applied on D5); 2xPGF-0/7d (cows received CIDR for 7 days, and two injections of 12.5mg of PGF, on D0 an D7). CIDR was removed on D7, and was associate with injections of 0.3 ml ECP and 300 IU of eCG. TAI was performed 50 h after CIDR removal. Estrus was detected in 54.45% (104/191), and did not differ between groups. The onset of estrus did not differ, and was 36.4 ± 8.7 hours after CIDR removal. The conception rate at TAI did not differ and was 59.5 and 59.6% for 1xPGF-5d and 2xPGF-0/7d, respectively. The final pregnancy rate at the end of breeding season did not differ and was 82.9% for 1xPGF-5d and 86.6% for 2xPGF- 0/7d. All experiments were performed estrus detection with teasers for 5 days after CIDR removal, and was detected again after 10 days, and performed for 10 days. All animals of exp. I and II were inseminated according AM/PM protocol. The pregnancy diagnosis was performed according to caracteristic of each experiment, but always with the goal of identifying and quantifying the origin of pregnancy (AI or rebreeding). Data were analyzed using the statistical program SAS 9.9, for binominal variables we used GLIMMIX procedure and MIXED procedure for continuous variables. In nonlactating Nellore cows, the use of protocols for 7 days, has a better reproductive performance than 5 days of CIDR. In prepubertal heifers using BE 48 hours after CIDR removal, allows for greater estrus detections and increased number of inseminations, and increasing the pregnancy rate at AI. The use of 7d EB+CIDR program yields good reproductive results, and we can reduce a number of handlings without reduce a reproductive performance.

Neste trabalho nós mostramos que células dendríticas (DCs) de camundongos BALB/c expressam receptor para o PAF (Fator ativador de Plaquetas) e que sua ativação promove um fenótipo tolerogênico, associado à produção de IL10 e PGE2. O bloqueio do PAF-receptor por antagonistas aumentou a capacidade das DCs induzirem proliferação de linfócitos T. O antagonista WEB2170 potencializou a resposta imune in vivo a concentração de anticorpo IgG2a OVA-específico aumentou 30 vezes no grupo tratado; a concentração de IgG1 foi semelhante nos dois grupos. O bloqueio do PAFR em camundongos imunizados com OVA em adjuvante completo de Freund, aumentou a produção de IgG1 e IgG2a OVA-específicos. Em camundongos imunizados com OVA/alum o antagonista não alterou a produção de IgG1. Estes resultados indicam que a ativação do PAFR em DCs modula a sua função apresentadora de antígenos pela produção de IL10 e PGE2. O bloqueio do PAFR pode ser útil na ativação das DCs em protocolos de vacinação com DCs e/ou como co-adjuvante em protocolos de imunização. / In the present work we show that BALB/c mice dendritic cells (DCs) express the PAF (platelet-activating factor) receptor and that its activation promotes a tolerogenic phenotype via IL10 and PGE2 production. Blocking PAFR by selective antagonists markedly enhanced DCs ability to induce T cell proliferation. The antagonist WEB2170 potentiated the in vivo immune response the IgG2a OVA-specific levels were 30 fold increased in the treated group; IgG1 concentration was similar for both groups. The PAFR blockade in mice immunized with OVA in complete Freunds adjuvant enhanced both IgG1 and IgG2a OVA-specific antibody production. In OVA/alum immunized mice, the antagonist did not change IgG1 production. These results suggest that PAFR activation in DCs modulates their antigen-presenting function through IL10 and PGE2 production. Blocking PAFR may be useful to induce DCs activation in DCs-based vaccination protocols and/or as a co-adjuvant in immunization protocols.