Die vorliegende Arbeit befaßt sich mit T-Helferzellen und ihren Interaktionen mit Gewebszellen, wie sie im gesunden Organismus und in Autoimmunerkrankungen auftreten. Es werden Fragen der Toleranzinduktion durch orale Gabe von Antigenen, speziell der oralen Verabreichung von Collagen II bei Patienten mit rheumatoider Arthritis diskutiert. Eine Immundeviation als Mittel, inflammatorische Th1-Zellantworten in anti-inflammatorische Th2-Zellantworten zu verwandeln, kann durch Eingriffe in die T-Zell-Signaltransduktion erreicht werden. Es werden neue Ansätze zu Mechanismen diskutiert, die das Immunprivileg des Zentralnervensystems gewährleisten. Die hirnresidenten Immunzellen, zu denen Mikrogliazellen und Astrozyten zählen, besitzen Eigenschaften, die eine Entzündung unwahrscheinlich machen. Sie müssen aktiviert werden, um Antigene präsentieren zu können. In organtypischen entorhinal-hippocampalen Schnittkulturen konnte gezeigt werden, dass Mikrogliazellen durch Th1-Zellen aktiviert, von Th2-Zellen hingegen deaktiviert werden. Die Möglichkeit, dass die Costimulation über CD80 oder CD86 differentielle Effekte auf den Charakter der Immunantwort hat, wird diskutiert. Der Einfluß von pro-inflammatorischen Zytokinen auf Mikrogliaaktivierung und den Erhalt von Nervenfasern wurde ebenfalls in Hirnschnittkulturen untersucht. Astrozyten sind wesentlicher Bestandteil der Blut-Hirn-Schranke. Diese kann jedoch von aktivierten T-Zellen überwunden werden. In dieser Arbeit wird gezeigt, dass Astrozyten über eine Expression von CD95L in aktivierten T-Zellen Apoptose induzieren können. Davon sind jedoch nicht alle T-Zellen betroffen. Andererseits wird eine T-Zellproliferation unterdrückt, indem T-Zellen unter Astrozyteneinfluß verstärkt CTLA-4 exprimieren, was einen Zellzyklusarrest zur Folge hat. Darüber hinaus ist eine verstärkte Produktion von Nervenwachstumfaktor (NGF; nerve growth factor) nach antigenspezifischer Interaktion von Astrozyten mit Th1- und Th2-Zellen als zusätzliches Mittel, eine Neuroinflammation einzudämmen, anzusehen. Die Arbeit stellt diese Ergebnisse in fünf Kapiteln dar, welche gleichzeitig eine Einführung in die als Anlagen enthaltenen zehn Publikationen geben. / This thesis deals with T helper cells and their interactions with tissue cells as they occur in the healthy organism and in autoimmune diseases. Questions of tolerance induction by oral application of antigens are discussed especially oral treatment with type II collagen in patients with rheumatoid arthritis. In order to transform inflammatory Th1 responses into anti-inflammatory Th2 responses, immune deviation can be reached by interference with T-cell signal transduction. New approaches towards the different ways that the immune privilege of the central nervous system is maintained are discussed. The resident immune cells, i.e. microglia and astrocytes possess properties that make inflammation unlikely. They have to be activated in order to present antigens. It has been shown in organotypic entorhinal-hippocampal slice cultures that Th1 cells activate whereas Th2 cells deactivate microglial cells. The possibility is discussed as to whether costimulation via CD80 or CD86 differentially influences the character of the immune response. The influence of pro-inflammatory cytokines on microglial activation and preservation of nerve fibers has also been studied in brain slice cultures. Astrocytes are an essential part of the blood-brain barrier, which can be crossed by activated T cells. The thesis shows that astrocytes can induce apoptosis in activated T cells via expression of CD95L. However, not all T cells are affected. T cell proliferation is suppressed by increased CTLA-4 expression in T cells under the influence of astrocytes, resulting in a cell cycle arrest. An additional mechanism of confining neuroinflammation is increased production of the nerve growth factor (NGF) following antigen-specific interaction of astrocytes and Th1 and Th2 cells, respectively. These results are presented in five chapters that also introduce the ten attached publications.

A morfina é um analgésico eficaz e muitas vezes opioide usado para aliviar a dor moderada a grave durante o período neonatal precoce. A exposição repetida de morfina no início da vida tem implicações duradouras para o desenvolvimento do sistema nervoso, tais como alterações neuroquímicas e comportamentais a longo prazo em ratos. O exercício físico vem sendo utilizado como uma alternativa não farmacológica para tratamento de quadros dolorosos. Deste modo nosso objetivo foi avaliar o efeito da exposição a morfina no período neonatal nas respostas nociceptiva (térmica e mecânica) e bioquímicas (citocinas e neurotrofinas) em ratos de P30 e P60 antes e após a exposição ao exercício físico. Ratos Wistar com 7 dias foram divididos em dois grupos: salina (SA) e morfina (MO) e submetidos a 5 mg / dia / 7 dias P8 para P14 a soro fisiológico ou MO respectivamente. Nas idades de P16, P30 e P60 a resposta nociceptiva térmica foi avaliada através do teste da placa quente (PQ), a resposta mecânica por Von Frey (VF) e Randal e Selitto (RS). Ainda foram medidos os níveis basais de BDNF, NGF, IL-6 e IL-10 em córtex cerebral e tronco encefálico. Após a sessão de exercício foram realizados em P30 e P60 o teste de PQ, 1h e 24h após o exercício, o teste de VF foi realizado 24h após e os níveis de BDNF, NGF, IL-6 e IL-10 também foram medidos em córtex cerebral e tronco encefálico após a exposição ao exercício. Nossos resultados demonstram que os animais que receberam morfina no período neonatal apresentam diminuição do limiar nociceptivo térmico e mecânico em P30 e P60. Os níveis de BDNF, NGF, IL-6 e IL-10 apresentaram relação direta com a idade em tronco encefálico, aumento ao longo do tempo. Em córtex cerebral os níveis de BDNF e NGF demonstraram uma interação entre os fatores grupo e idade, onde os animais do grupo MO têm diminuição desses com a idade. A IL-10 teve efeito somente da idade, enquando a IL-6 não se mostrou alterada por nenhum fator. Após a exposição ao exercício no teste da PQ no P30 e P60 os animais SAE tiveram uma diminuição do limiar nociceptivo se igualando aos grupos que receberam morfina. No teste de VF em P30 os grupos que receberam morfina são diferentes do grupos salina. Em P60 o grupo SAE mostra mais uma vez diminuiçao do limiar nociceptivo se igualando as grupos morfina. Nos níveis de BDNF e NGF em tronco encefálico ocorreu interação entre idade e grupo, onde o grupo MOE demonstra diminuição. Em tronco encefalico a IL-6 e Il-10 só tiveram efeito da idade. Em córtex cerebral os níveis de BDNF tiveram interação entre idade e grupo, o grupo MOE teve diminuição destes níveis em comparação aos demais grupos. Nos níveis de NGF se observou efeito do tempo e do grupo onde os grupos que recebem morfina têm níveis menores do que os que recebem salina em P60. O grupo MOS teve níveis menores de IL-6 em cortex cerebral do que os demais grupos, enquanto que os níveis de IL-10 só tiveram efeito da idade. Portanto a morfina no período neonatal leva a diminuição no limiar nociceptivo térmico e mecânico em ratos e que o exercício físico melhora os níveis BDNF, NGF e IL-6 em animais expostos a morfina no período neonatal. Porém o exercício físico não foi capaz de reverter a hiperalgesia e alodínia induzida pela morfina nos animais de P30 e P60. Sendo assim nossos dados mostram a necessidade de mais estudos sobre a dor em recém-nascidos e o sobre o uso de opioides neste período. Também se mostra necessário mais estudos sobre tratamentos não farmacológicos como o exercício físico. / Morphine is an effective analgesic often used to relieve moderate to severe pain during the early neonatal period. In our previous study, repeated morphine exposure in early life triggered persistent implications for the development of the nervous system, such as neurochemical and behavioral alterations in rats at long-term. The exercise has been used as a non-pharmacological alternative for treating painful conditions. Thus our aim was to evaluate the effect of repeated morphine exposure during the neonatal period upon nociceptive responses (thermal and mechanical) and biochemical markers (cytokines and neurotrophins) before and after unique physical exercise session in rats at P30 and P60. Seven-day-old male Wistar rats were divided into two groups: saline and morphine and subjected to saline and morphine (5 μg/day/7 days) from P8 to P14, respectively. At P16, P30 and P60, the thermal nociceptive response was assessed using the hot plate test (HP), while the mechanical response by Von Frey (VF) and Randal and Selitto (RS) tests. The basal levels of BDNF, NGF, IL-6 and IL-10 were measured in brainstem and cerebral cortex. One hour and 24h after exercise, the HP was conducted in P30 and P60, the VF test was only performed 24 ho after exercise, as well as the levels of BDNF, NGF, IL-6 and IL-10 were also measured in cerebral cortex and brainstem. Our results show that rats that received morphine in the neonatal period presented decreased thermal and mechanical nociceptive threshold in P30 and P60. And, BDNF, NGF, IL-6 and IL-10 levels presented a direct relationship with age in brainstem, increase their levels when the age increased. In cerebral cortex, BDNF and NGF levels showed an interaction between age and treatment group, where the morphine group showed decreased levels when the age increased. There was age effect upon IL-10 levels and no effects upon IL-6 levels in cerebral cortex. After 24h of exercise, saline group subjected to exercise presented decreased nociceptive threshold in using HP at P30 and P60, with similar threshold presented by morphine group. In VF test, both morphine groups presented decreased threshold in relation to both saline groups at P30. However, at P60, saline group subjected to exercise presented decreased nociceptive threshold, matching the morphine groups. In brainstem, we found interaction between age and group in BDNF and NGF levels, where morphine-exercise group showed decreased levels; and we observed only age effect upon IL-6 and IL-10 levels. In cerebral cortex, we observed interaction between age and group upon BDNF levels, where morphine-exercise group showed decreased levels compared to other groups. In relation to NGF levels, we observed effect of age and group, where morphine groups presented lower levels than saline groups in P60. The morphine-sedentary group presented lower IL-6 levels in the cerebral cortex than the other groups, while only age effect was observed on IL-10 levels. Our data lead us to conclude that morphine exposure in the neonatal period triggers a decrease in thermal nociceptive and mechanical thresholds in rats. And, the physical exercise improves BDNF, NGF and IL-6 levels in rats exposed to morphine in the neonatal period. However, on session of exercise was not able to revert the hyperalgesia and allodynia induced by morphine in rats at P30 and P60. Therefore, our data highlight the need of more studies about pain in newborns and neonates and the effect of the opioid use in this period. And, it is necessary more studies about non-pharmacological treatments, for example exercise.

O sarcoma de Ewing (SE) é um dos mais agressivos tipos de câncer pediátrico. Apesar dos significativos avanços no tratamento dessa doença, ainda há uma grande necessidade no aumento das taxas de cura, redução da toxicidade quimioterápica e redução da resistência ao tratamento. Tem sido proposto que SE provém de precursores neuronais, podendo ter sua fisiologia afetada, pois, por neurotrofinas (NTs). Examinamos a influência de receptores de NTs (Trks) em SE. Foram avaliadas a expressão proteica de NTs (NGF e BDNF) e seus receptores (TrkA e TrkB, respectivamente) em amostras de tumores de pacientes com SE, e a expressão de mRNA nas linhagens celulares RD-ES e SK-ES-1. O tratamento das linhagens com o pan-inibidor de Trks (K252a) modificou a morfologia celular e diminuiu a expressão de mRNA de NGF, TrkA, BDNF e TrkB. Ainda, a inibição de Trks diminuiu drasticamente a proliferação e capacidade clonogênica celular. Efeitos sinérgicos foram observados quando as células foram tratadas em conjunto com baixas doses de quimioterápicos, tanto em células selvagens de SE, quanto nas quais induzimos quimiorresistência. Esse estudo sugere, pela primeira vez, que a inibição de Trks reduz a proliferação e sobrevivência celular em SE, além de aumentar a sensibilidade ao tratamento quimioterápico. / Ewing's sarcoma (ES) is one of the most aggressive types of pediatric cancer. Despite significant advances in the treatment of this disease, there is still a great need in increasing cure rates, reducing chemotherapy toxicity and treatment resistance. It has been proposed that ES might derive from neuronal precursors and may be influenced, therefore, by neurotrophins (NTs). We have examined the influence of Trk neurotrophin receptors in ES. Protein expression of NTs (NGF and BDNF) and their receptors (TrkA, and TrkB, respectively) was detected in tumor samples from patients with ES, and mRNA expression was analyzed in the RD-ES, SK-ES-1 cell lines. Treating cells with a Trk Pan-inhibitor (K252a) altered cell morphology and decreased the mRNA expression of NGF, TrkA, BDNF, and TrkB. In addition, Trk inhibition dramatically decreased cell proliferation and clonogenic capacity. Synergistic effects were observed when cells were treated in combination with low doses of cytotoxic chemotherapeutics, both in normal ES cells and cells in which chemoresistance was induced. The results suggest for the first time that Trk inhibition can reduce the proliferation and survival of ES cells and sensitize them to cytotoxic chemotherapy.

Spinal cord injury (SCI) remains a significant cause for morbidity and mortality in North America. Bladder dysfunction following SCI is very common and could lead to severe complications including renal failure and autonomic dysreflexia (AD). AD involves life threatening episodes of hypertension in patients with SCI above T6 level. Current management protocols for AD are symptomatic and usually ineffective. Botulinum toxin-A (BTX-A), has been successfully used recently in SCI patients because it reduces the detrusor contractility via inhibiting acetylcholine release from efferent nerve endings. Recent evidence, however, suggests a sensory involvement via modulation of sensory neuropeptides, neurotransmitters, and receptors. It is still, however, unclear whether BTX-A can affect putative spinal neurons involved in AD. In this study we demonstrated that intravesical BTX-A treatment has blocked AD in rats with T4-SCI, and also provided a novel mechanism for the control of autonomic dysreflexia via a minimally invasive treatment modality.

Spinal cord injury (SCI) remains a significant cause for morbidity and mortality in North America. Bladder dysfunction following SCI is very common and could lead to severe complications including renal failure and autonomic dysreflexia (AD). AD involves life threatening episodes of hypertension in patients with SCI above T6 level. Current management protocols for AD are symptomatic and usually ineffective. Botulinum toxin-A (BTX-A), has been successfully used recently in SCI patients because it reduces the detrusor contractility via inhibiting acetylcholine release from efferent nerve endings. Recent evidence, however, suggests a sensory involvement via modulation of sensory neuropeptides, neurotransmitters, and receptors. It is still, however, unclear whether BTX-A can affect putative spinal neurons involved in AD. In this study we demonstrated that intravesical BTX-A treatment has blocked AD in rats with T4-SCI, and also provided a novel mechanism for the control of autonomic dysreflexia via a minimally invasive treatment modality.

At the plasma membrane, both NGF receptors have been shown to localized to lipid rafts, specific subdomains that are enriched in cholesterol, sphingolipids and the presence of caveolin proteins (Cav1 and/or Cav2). The focus of this work is on this membrane microenvironment mediated modulation of NGF signaling which via two receptors: p75NTR and TrkA. In the present work we found that overexpression of Cav-1 in mouse dorsal root ganglia neurons significantly impacted neurite extension. Similarly, overexpression of Cav-1 in PC12 cells strongly inhibits their ability to grow neurites in response to NGF. It inhibits NGF signaling without, impairing transient MAPK pathway activation. Rather, it does so by sequestering NGF receptors in lipid rafts, which correlates with the cell surface localization of downstream effectors, and phosphorylated-Rsk2, resulting in the prevention of the phosphorylation of CREB. By contrast, overexpression of Cav-2 potentiates NGF induced differentiation, which is accompanied by sustained activation of downstream effectors, and standard internalization of the receptors. This differential effect could be due to the different localization of Caveolins, that modifies the microenvironment, thereby affecting NGF signaling. Furthermore, PC12 cells expressing the non-phosphorylatable Cav-1 mutant (S80V), neither TrkA trafficking or CREB phosphorylation are inhibited and the response resembles that observed in Cav-2 expressing PC12 cells. These studies underline the interplay between caveolins and NGF signalling, offering insight into the potential impact of Caveolin-1 and mutations thereof in certain cancers where NGF signaling is involved.

No description available.

During pregnancy, 4 - 7% of women suffer from major depressive disorder. When antidepressive treatment is needed, selective serotonin reuptake inhibitors (SSRIs) are the most commonly used. Although severe complications from SSRI treatment are rare, association with a number of adverse pregnancy and fetal outcomes has been found. Also, antenatal depression per se has been shown to affect pregnancy outcomes. The overall aim of this thesis was to examine the effects of SSRIs on human placenta and embryo. In the first study, gene expression was investigated in placenta from depressed, SSRI-treated and healthy pregnant women, using microarray analysis. Antenatal depression and SSRI treatment induced alterations in gene expression, but only 20 genes in common were noted. Validation with qRT-PCR showed that six out of seven selected genes were altered in SSRI-treated women compared with controls, and two genes were altered between depressed women and controls. In study two, the protein levels in placenta from depressed, SSRI-treated and healthy pregnant women were investigated, focusing on the NGF signaling pathway. NGF, phosphorylated Raf-1, ROCK2 and phosphorylated ROCK2, were altered in both SSRI-treated and depressed women, although the proteins were regulated differently in the two groups. In the third study, human embryos were treated with fluoxetine. Embryo development and protein expression were studied. Fluoxetine had some effect on the timing of embryo developmental stages. Also, several proteins were uniquely found in fluoxetine-treated embryos compared with untreated embryos. Fluoxetine also altered the levels of proteins secreted from the embryo. In the fourth study, the human neuroblastoma cell line SH-SY5Y/TrkA was treated with TPA and NGF. The activation of Raf-1 was investigated and the involvement of Ras and PKC was studied. Both NGF and TPA activated Raf-1, but to a different extent and via different pathways. The NGF-induced activation of Raf-1 was mediated via Ras, while TPA induced signaling via PKC. In conclusion, SSRI treatment and antenatal depression influence placental gene and protein expression. These findings may affect placental development and function, which in turn could affect fetal development. Also, direct exposure of embryos to fluoxetine has some effects on embryo development and protein expression, which may affect the development of the fetus.

The survival of several neuron populations during development, including sympathetic neurons, is strictly regulated by neurotrophins such as nerve growth factor (NGF) released from innervation targets. NGF activates its receptor, TrkA, at axon terminals, to generate signals that are transmitted retrogradely to cell bodies to induce signaling cascades regulating survival. A general view of this process is that NGF generates retrograde survival signals that, when delivered to cell bodies, induce downstream survival signaling that prevents apoptosis. A retrograde survival signal proposed to be necessary for sympathetic neuron survival consists of endosomes containing NGF and phosphorylated TrkA. For this signal, phosphorylated TrkA arriving at cell bodies is required to initiate survival signaling. Studies have tested the necessity of TrkA phosphorylation in the cell bodies for survival: results from different studies contradict each other. Moreover, the Trk inhibitor, K252a, used in these studies, has reported non-specific effects. Using an alternate Trk inhibitor, Gö6976, data presented in this thesis demonstrates that NGF can promote survival by retrograde signaling that does not require TrkA phosphorylation in the cell bodies. These retrograde signals may be composed of signaling molecules activated downstream of TrkA in axons since pro-survival molecules downstream of TrkA, Akt and CREB, were found activated in the cell bodies/proximal axons. Data presented in this thesis also reveals a fundamentally different mechanism for how NGF promotes sympathetic neuron survival: a retrograde apoptotic signal that is suppressed by NGF. NGF withdrawal from axons induced the “axon apoptotic signal” that was retrogradely transmitted to cell bodies to activate a key pro-apoptotic molecule, c-jun. The axon apoptotic signal, which was blocked by the kinase inhibitors rottlerin and chelerythrine, was necessary for apoptosis in response to NGF deprivation. Evidence GSK3 is involved in generation or transmission of the axon apoptotic signal was provided by experiments with GSK3 inhibitors and siRNA. The axon apoptotic signal discovery refutes the previous view that NGF acting on axon terminals supports survival exclusively by generating retrograde survival signals. The axon apoptotic signal has broad implications for understanding nervous system development and other conditions where neuronal apoptosis occurs, such as neurotrauma and neurodegenerative diseases.

The studies presented in this thesis were designed to elucidate whether the abundance of ovulation-inducing factor/nerve growth factor (OIF/NGF) in alpaca semen can be used as a biomarker to predict male fertility. The neurotrophin, OIF/NGF has been identified in camelid, cattle and human semen. It is only in camelids, however, that an ovulation-inducing role for OIF/NGF has been described. The information gathered from several studies clearly demonstrate that this protein is the stimulus responsible for initiating the ovulatory cascade in camelids. In addition, intramuscular administration of OIF/NGF resulted in a dose-dependent response in terms of ovulation rate, corpus luteum (CL) lifespan, luteinizing hormone (LH) and progesterone secretion. I hypothesized that the quantity of OIF/NGF differs among male alpacas and this abundance arbitrates ovulation and pregnancy rates as well as CL formation and function. To substantiate this hypothesis, the following questions were answered: 1) can OIF/NGF in alpaca semen be quantified using a radioimmunoassay; 2) does the concentration and total abundance of OIF/NGF in alpaca semen vary within and among male ejaculates; 3) what is the glandular source of OIF/NGF that contributes to the male ejaculate; 4) is OIF/NGF concentration or abundance related to parameters associated with male fertility; 5) can OIF/NGF concentration or total abundance in the ejaculate discriminate fertile and subfertile males using both retrospective and prospective approaches; and 6) can power Doppler ultrasonography be used to assess the luteotrophic effect of OIF/NGF in tissue vasculature of the developing CL? I discovered that the source and the amount of OIF/NGF varies among species. In llamas, OIF/NGF is produced by both the corpus and disseminate portions of the prostate gland. In rats, OIF/NGF was detected in testis interstitial cells and in the lumen of the coagulating gland (anterior prostate). Ovulation-inducing factor/NGF secretion by the ampullae and vesicular glands contributed to its presence in bull (cattle and bison) ejaculates. In elk and white tail deer, OIF/NGF was detected in the ampullae and prostate glands, respectively. To gain an understanding of the abundance of OIF/NGF in ejaculates and changes in its concentration within and among males, OIF/NGF levels in semen were quantified using the radioimmunoassay. The assay developed exhibited parallel displacement curves among recombinant NGF, OIF/NGF purified from llama seminal plasma, llama and bull (cattle) seminal plasma. Ovulation-inducing factor/NGF comprised a greater percentage of the total protein found in camelid ejaculates than in cattle. Ovulation-inducing factor/NGF concentration correlated positively with sperm concentration and negatively with pH and semen volume, while total abundance of OIF/NGF was related to total prostate area and OIF/NGF concentration. Although a correlation was found between sperm concentration, neither OIF/NGF concentration nor total abundance was associated with higher ovulation, pregnancy or live birth rates. A clear association of the quantity of OIF/NGF in the male ejaculate at breeding and CL form and function was not evident. The measurement of CL vasculature by power Doppler ultrasonography, however, was able to determine nonpregnancy in alpacas earlier than the assessment of changes in CL diameter. In summary, my results did not support the hypothesis that the measurement of OIF/NGF concentration or total abundance in alpaca semen can be used to predict fertility in male alpacas.