This thesis describes investigations into Placental Protein 14 (PP14), a immunomodulator involved in human reproduction. The studies included the development of a purification procedure and an investigation of the activity of the protein. In addition the cDNA coding for the protein was cloned and expressed as a recombinant fusion protein and the molecular structure of the protein was predicted and analysed using computer-assisted modelling. Finally the clinical significance of the protein was studied in a range of patient groups. The purification scheme consisted of ion exchange, hydrophobic interaction and gel filtration chromatography, and the pure protein obtained was analysed by SDS-PAGE and Western blotting. The results demonstrate that the purification procedure is a suitable method to obtain PP14 in large quantity and with high purity. PP14 purified by this method retained its activity and was shown to suppress, in a dose-dependent manner, the uptake of 3H-Thymidine by peripheral blood mononuclear cells stimulated with interleukin-2. Purified PP14 was also shown to suppress the uptake of 3H-Thymidine by the cell line U937, also in a dose-dependent manner. This suppression could be removed by the incubation of the PP14 sample with an immunoabsorbent gel linked to monoclonal antibodies against PP14, demonstrating that PP14 was the molecule responsible for the observed activity. Based on the suppression by PP14 of U937 cell growth a bioassay for PP14 was developed, this assay was used to express the specific activity of PPM in Units/ml. To obtain recombinant PP14, mRNA was purified from a tissue sample and reverse transcription used to prepare cDNA. Specific primers were used to amplify the portion of cDNA coding for PP14 which was then ligated into the plasmids pUC 18 and pGEX-KG. Recombinant PP14 was then expressed as a fusion protein with glutathione-S-transferase. The expression conditions were optimised and the fusion protein was purified using affinity chromatography. The structure of PPM was investigated using computer assisted modelling. PPM is a member of the lipocalin family of proteins which share the feature of binding small hydrophobic molecules. The X-ray coordinates of two lipocalins known to share sequence homology with PP14 were used as a basis to model a predicted structure for PP14. An analysis of the structural motifs of the protein was carried out, and it was established that PP14 shares many of the characteristic features of this family of proteins including the presence of a binding pocket. The model was then used to predict potential ligands for PP14.PP14 was measured by radioimmunoassay in uterine flushings from fertile women, women with unexplained infertility and women suffering from recurrent miscarriages, and in plasma samples from fertile and infertile women. The results from the uterine flushings from fertile women showed that PP14 levels rose during the second half of the menstrual cycle reaching ug/ml levels by the end of the cycle. These physiological concentrations are in the same range as the concentrations at which the immunomodulatory activity of PP14 was observed in vitro. The levels of PP14 measured in uterine flushings were lower in infertile women than in fertile women, indicating that a deficiency in PP14 may be associated with infertility. The levels measured in plasma samples from these two groups of women did not pick up this difference. These results suggests that the measurement of proteins such as PP14 in uterine flushings instead of plasma samples may be a more sensitive indicator of local uterine function. In women suffering from recurrent miscarriage a significant lack of secretion of PP14 was observed around the time of implantation. This may be conected with the failure of implantation in these patients. A correlation was observed between the PP14 levels measured in uterine flushings from recurrent miscarriage patients and the level of endometrial development.

Conselho Nacional de Desenvolvimento Científico e Tecnológico / The major goal of bovine viral diarrhea virus (BVDV) vaccines is to confer fetal protection and thus prevent reproductive losses and the production of persistently infected (PI) calves. This dissertation reports the antibody response and fetal protection in pregnant cows conferred by an experimental vaccine containing two attenuated strains of BVDV (BVDV-1 and BVDV-2). Cows previously vaccinated with the experimental vaccine (n=19) and non-vaccinated controls (n=18) were mated and challenged between days 30 and 90 of gestation by intranasal inoculation of four heterologous BVDV-1 and BVDV-2 isolates. The antibody response was evaluated by serum-neutralization tests performed at different intervals after vaccination (days 34, 78 and 138 post-vaccination [pv]). Fetal protection was monitored by ultrassonographic and clinical examination of the dams and fetuses conducted during the rest of gestation; and through virological and serological examination of pre-colostral blood obtained from aborted and/or recently born fetuses/calves. At the day of challenge (day 138 pv), all vaccinated cows had neutralizing antibodies in high titers against BVDV-1 (1.280- >10.240), and with one exception (titer 20), presented moderate to high titers to BVDV-2 (80-1.280). At the end of the experiment only three out of 18 control cows (16.6%) delivered healthy, virus-free calves. Fifteen non-vaccinated cows (83.3%) presented signs of fetal infection and/or had reproductive losses. Seven of these cows (38.8%) delivered virus-positive calves; five were healthy and survived; two were premature or weak and lasted three and 15 days, respectively. The other eight cows (44.4%) aborted between day 30 post-challenge and the parturition; or delivered premature or stillbirth calves. In contrast, 17 out of 19 (89.4%) vaccinated cows delivered virus-free, healthy calves. One vaccinated cow aborted around day 130 post-challenge, yet the aborted fetus could not be examinated for the presence of virus. Another cow delivered a virus-positive calf. These results showed that the experimental vaccine induced adequate antibody titers in most animals and that the immunological response was able to prevent fetal infection and reproductive losses upon challenge with a pool of heterologous BVDV isolates. / O principal objetivo das vacinas contra o vírus da diarréia viral bovina (BVDV) é conferir proteção fetal e assim reduzir as perdas reprodutivas e a produção de bezerros persistentemente infectados (PI). Essa dissertação relata a avaliação de resposta sorológica e proteção fetal conferida por uma vacina experimental contendo duas amostras atenuadas do BVDV-1 (BVDV-1) e 2 (BVDV-2). Vacas previamente imunizadas com a vacina experimental (n=19) e controles não-vacinadas (n=18) foram colocadas em cobertura e desafiadas, entre os dias 30 e 90 de gestação, pela inoculação intranasal de quatro amostras heterólogas de BVDV-1 e BVDV-2. A resposta sorológica foi avaliada por testes de soro-neutralização realizados a diferentes intervalos após a vacinação (dias 34, 78 e 134 pós-vacinação [pv]). A proteção fetal foi monitorada por exames ultra-sonográficos e clínicos realizados durante o restante da gestação; e pela pesquisa de vírus e anticorpos no sangue pré-colostral coletado dos fetos abortados e/ou dos bezerros recém nascidos. No dia do desafio (dia 138 pv), todas as vacas vacinadas apresentavam anticorpos neutralizantes em títulos altos contra o BVDV-1 (1.280 ≥ 10.240) e, com exceção de uma vaca (título 20), todas apresentavam títulos moderados a altos contra o BVDV-2 (80 - 1.280). O monitoramento da gestação revelou que, dentre as 18 vacas não-vacinadas apenas três (16,6%) pariram bezerros saudáveis e livres de vírus. As 15 restantes (83,3%) apresentaram indicativos de infecção fetal e/ou falhas reprodutivas. Sete dessas vacas (38,8%) pariram bezerros positivos para o vírus, sendo que cinco eram saudáveis e sobreviveram; e dois apresentavam sinais de prematuridade ou fraqueza e morreram três e 15 dias após o nascimento respectivamente. As oito vacas controle restantes (44,4%) abortaram entre o dia 30 pós-desafio e às proximidades do parto, ou deram a luz a bezerros prematuros, inviáveis ou natimortos. Por outro lado, 17 de 19 vacas vacinadas (89,4%) pariram bezerros saudáveis e livres de vírus. Uma vaca vacinada abortou 130 dias pós-desafio, mas o produto não pode ser examinado para a presença de vírus. Outra vaca vacinada pariu um bezerro positivo para o vírus. Esses resultados demonstram que a vacina experimental induziu títulos adequados de anticorpos na maioria dos animais; e que a resposta imunológica produzida foi capaz de conferir proteção fetal e prevenir as perdas reprodutivas frente ao desafio com um pool de amostras heterólogas de BVDV.

La naissance prématurée (NPM) est la cause primaire de morbidité et de mortalité néonatales. Les études ont révélé que l’interleukine-1 (IL-1) possède un rôle primordial dans la pathophysiologie de la NPM, puisqu’elle induit la production de médiateurs pro-inflammatoires et des protéines activatrices de l’utérus menant au travail pré-terme. L’inflammation utéroplacentaire, néfaste pour le fœtus en voie de développement, entraine des séquelles à vie chez ces nouveau-nés. Présentement, le traitement de l’accouchement prématuré repose principalement sur l’administration d’agents tocolytiques, dont la nifédipine, qui ne permettent de prolonger la gestation que de quelques heures. De plus, les études cliniques ont démontré à maintes reprises que les agents tocolytiques ne préviennent pas la NPM, ni les morbidités néonatales. Notre groupe a développé un peptide allostérique (rytvela) qui antagonise le récepteur d'IL-1. Rytvela s'est révélé efficace à prévenir la NPM en inhibant l’inflammation via l’inhibition des voies signalétiques activant le facteur de transcription AP-1 tout en préservant l’activation de la voie NF-B, qui est impliquée dans l’immunovigilence innée et la cytoprotection. Des études antérieures ont démontré l’efficacité de rytvela en mode prophylactique à inhiber la réponse pro-inflammatoire ainsi que l’activation utérine tout en préservant le développement fœtal. Cette étude a pour but de faire progresser le développement pré-clinique de rytvela et de caractériser ses propriétés pharmacodynamiques. Nous avons évalué la dose maximale efficace et la durée de traitement minimale de rytvela pouvant inhiber l’inflammation ainsi que prévenir la NPM et les lésions tissulaires néonatales lorsqu’administré comme agent prophylactique. Nous avons ensuite déterminé le délai maximal d’administration de rytvela à la suite de l’induction du travail pré-terme. Les efficacités de rytvela et de la nifédipine permettant de prévenir les issues gestationnelles et néonatales indésirables ont été comparées. Nous démontrons que lorsqu’administré en mode prophylactique, rytvela présente une efficacité maximale à une dose de 2 mg/kg/jour à une durée minimale de traitement de 36 heures dans des modèles murins de travail pré-terme induit par une infection systémique (LPS) ou de l’inflammation stérile utéroplacentaire (IL-1). Dans le but de représenter une intervention clinique pertinente, nous avons déterminé que rytvela démontrait une efficacité maximale lorsqu’administré jusqu’à 2 heures après l’induction du travail pré-terme. De plus, rytvela possède une efficacité supérieure à celle de l’agent tocolytique, la nifédipine, puisque ce peptide a prévenu les issues gestationnelles et néonatales indésirables associées à la NPM. En somme, à ses propriétés anti-inflammatoires de rytvela, rytvela diminue l’activation utérine afin de prolonger efficacement la gestation ainsi que de prévenir la NPM et les atteintes tissulaires néonatales. Cette étude représente un développement pré-clinique majeur permettant de prévenir les complications causées par la NPM et de s’attaquer à ce besoin non-comblé, puisque les agents tocolytiques demeurent inefficaces. En conclusion, rytvela présente des propriétés désirables pour prévenir et traiter le travail pré-terme afin de promouvoir le développement néonatal. / Preterm birth (PTB) is the leading cause of neonatal morbidity and mortality and affects over 15 million births every year. Studies have shown that interleukin-1 (IL-1) plays a key role in the pathophysiology of PTB as it induces the production of pro-inflammatory mediators and uterine activating proteins leading to labor. More importantly, uteroplacental inflammation, associated with the parturition pathways of PTB, is detrimental to fragile fetal tissues and leads to long term sequalae. Clinical studies have repeatedly shown that tocolytic agents, such as nifedipine, do not prevent PTB or neonatal morbidities. Our group has developed an allosteric peptide (rytvela) that antagonizes the IL-1 receptor. Rytvela is potent and safe in preventing PTB by suppressing inflammation via the inhibition of pathways activating AP-1 while preserving the activation of the NF-B pathway, important in immune-vigilance and cytoprotection. Past work has shown that the prophylactic administration of rytvela inhibits inflammatory upregulation as well as uterine activation and preserves fetal development. This present study aimed to further pre-clinical development of rytvela and to characterize its pharmacodynamic properties. We evaluated the maximal effective dose and minimal duration of treatment of rytvela to inhibit the inflammatory cascade, prevent PTB and neonatal tissue injury when administered as a prophylactic agent. We then determined the maximal delay to administer rytvela after inducing preterm labor (PTL), to inhibit inflammatory upregulation, prolong gestation, and promote neonatal outcome. The efficacies of rytvela and nifedipine in preventing PTB as well as adverse gestational and neonatal outcomes were compared. We demonstrate that when administered prophylactically, rytvela exerts a maximal efficacy at a dose of 2 mg/kg/day at a minimal duration of treatment of 36 hours in murine models of PTL induced by systemic infection (LPS) or by intrauterine sterile inflammation (IL-1). In a clinically relevant treatment approach, rytvela exhibited a maximal efficacy when administered up to 2 hours after the induction of PTL. Moreover, rytvela displayed a superior efficacy in preventing adverse gestational and neonatal outcomes when compared to the tocolytic agent nifedipine. Altogether, through the anti-inflammatory properties of rytvela, rytvela decreases uterine activation to effectively prolong gestation and prevent PTB and neonatal tissue injury. This work represents a relevant treatment approach to prevent complications caused by PTB and would set in place a major preclinical development to address this unmet medical need as tocolytic agents are ineffective. Therefore, rytvela exhibits desirable properties for the prevention and treatment of PTL to prevent adverse gestational and neonatal outcomes.