The mare experiences a transient innate immune response to breeding, the resolution of which is crucial for optimal fertility. The majority of mares are able to modulate this inflammation in a timely fashion, but a subpopulation exists which fail to do so and are considered susceptible to persistent breeding-induced endometritis (PBIE). Seminal plasma has been shown to modulate aspects of this inflammation. Recently, two seminal plasma proteins have garnered interest for their immune modulating properties: cysteine-rich secretory protein-3 (CRISP-3) and lactoferrin. These proteins have been found to alter the binding between sperm and neutrophils based on sperm viability in vitro, but minimal work has evaluated their effect on endometrial mRNA expression of cytokines and inflammation in response to breeding. Experiments were performed to analyze the expression of equine CRISP-3. Found to be primarily synthesized in the ampulla of the vas deferens and to a lesser extent in the vesicular gland, CRISP-3 expression was only seen in the postpubertal stallion. Due to the effect of sperm viability on protein function in vitro, varying sperm populations were analyzed for their effect on gene expression in the uterus. It was determined that viable sperm suppressed the gene expression of the inflammatory modulating cytokine interleukin-6 (IL-6) in comparison to dead sperm. Next, the effect of CRISP-3 and lactoferrin on endometrial gene expression in the normal and susceptible mare was investigated. Neither protein had a significant effect on the mRNA expression of inflammatory cytokines in the normal mares at six hours post-breeding. In contrast, lactoferrin was found to significantly suppress the expression of the pro-inflammatory cytokine tumor necrosis factor (TNF)-α in susceptible mares. Due to this, lactoferrin was further analyzed as an immunomodulant for the treatment of PBIE. Susceptible mares were infused with varying doses of lactoferrin at six hours post-breeding. Although not in a dose-dependent fashion, lactoferrin was found to decrease both fluid retention and neutrophil migration, in addition to suppressing the expression of the pro-inflammatory cytokine interferon gamma (IFNγ) and increasing the gene expression of the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1RN). In conclusion, CRISP-3 expression occurs in secretory aspects of the male reproductive tract, and appears to be up regulated after sexual maturation. Viability of spermatozoa affects the immune response to breeding and should be taken into consideration for experimental design and interpretation of data. The seminal plasma proteins CRISP-3 and lactoferrin have minimal effect on endometrial gene expression in normal mares, but lactoferrin suppresses the expression of TNF in susceptible mares. Finally, lactoferrin was found to function as a potent anti-inflammatory for the persistent inflammation seen in susceptible mares when administered post-breeding. This protein should be further investigated as a potential therapeutic for the treatment of persistent breeding-induced endometritis.

Bislang ist ungeklärt, warum PNPs teils schmerzhaft und teils schmerzlos verlaufen. Die in der vorliegenden Arbeit untersuchte Hypothese lautete, dass ein Ungleichgewicht zwischen pro- und anti-inflammatorischen Zytokinen der unterschiedlichen Schmerzausprägung zugrunde liegt.Es wurden 32 Patienten mit schmerzhafter PNP, 20 Patienten mit schmerzloser PNP und 44 Kontrollpersonen auf die Expression und Produktion ausgewählter pro- und anti-inflammatorischer Zytokine untersucht. Zur Messung der Schmerzhaftigkeit wurden etablierte Schmerzfragebögen verwendet. Zusätzlich wurden nahezu alle Patienten mit der Allgemeinen Depressionsskala befragt. Die Diagnose, Ätiologie, Dauer, klinische Manifestation der PNP sowie die Medikation der Patienten wurde auf standardisierten Erhebungsbögen dokumentiert. Zur Messung der Zytokine wurde morgens Blut in EDTA- und Serummonovetten asserviert und entsprechend der Messmethodik weiterverarbeitet. Die relative Genexpression wurde aus Gesamt-RNA mittels reverser Transkription und quantitativer real-time PCR, die Serumproteine mittels enzyme-linked immunosorbant assay gemessen. Die Patienten mit schmerzhafter PNP hatten in der Mehrzahl Neuropathie-typische Plussymptome und mittelstarke Schmerzen, die eine starke bis sehr starke Behinderung darstellten. Die hier untersuchten Zytokinmuster bei Patienten mit schmerzhafter und schmerzloser PNP zeigten eine Verschiebung zu pro-inflammatorischen Zytokinen bei Patienten mit schmerzhafter PNP. Die Zytokinexpression der Patienten mit schmerzhafter PNP war im Vergleich zu Patienten mit schmerzloser PNP und Kontrollen bezüglich der IL-2 und TNF Expression und Produktion signifikant erhöht. Umgekehrt lagen bei Patienten mit schmerzloser PNP die Produktion und die Expression des IL-4 im Vergleich zu Patienten mit schmerzhafter PNP und Kontrollen höher. Die Expression des IL-10 lag bei Patienten mit schmerzloser PNP ebenfalls höher als bei Patienten mit schmerzloser PNP und Kontrollen, unterschied sich aber auf Proteinebene nicht in den drei Gruppen. Die einleitend gestellte Hypothese, dass der schmerzhafte oder schmerzlose Verlauf einer PNP durch unterschiedliche Zytokinprofile bedingt ist, kann durch die vorliegenden Ergebnisse gestützt werden. In Zusammenschau mit den Daten aus der Grundlagenforschung scheint einem pro-inflammatorischen Zytokinmuster eine entscheidende Rolle an der Entstehung und Aufrechterhaltung neuropathischer Schmerzen zuzukommen. Für TNF sind entsprechende pathophysiologische Wirkungen bekannt. Anti-inflammatorische Zytokine, wie IL-4 und IL-10 zeigten analgetische Wirkungen im Tierversuch. Die Mitwirkung des IL-2 an peripheren Opioid-Rezeptoren lässt eine endogene periphere Analgesie vermuten. Hieraus lassen sich Folgerungen für zukünftige Diagnostik und Therapie neuropathischer Schmerzen ziehen. Durch Erkennung von Zytokin-Imbalancen wären schmerzhafte PNPs früher einer adäquaten Therapie zuzuführen. Durch die Modulation von Zytokinprofilen im Rahmen schmerzhafter PNPs könnten sich zusätzlich therapeutische Möglichkeiten eröffnen. / BACKGROUND: Pain is a common symptom in peripheral neuropathies. The factors determining why some peripheral neuropathies are painful and others are not are incompletely understood. Pro-inflammatory cytokines have been implicated to play a crucial role in the generation of pain. OBJECTIVE: To investigate whether cytokine profiles differ between patients with painful or painless neuropathy. METHODS: In this prospective study, we analyzed blood mRNA and protein levels of the pro-inflammatory cytokines interleukin-2 (IL-2) and tumor necrosis factor-alpha (TNF) and the anti-inflammatory cytokines IL-4 and IL-10 in 32 patients with painful neuropathy, 20 patients with painless neuropathy, and 38 healthy control subjects, using quantitative real-time PCR and ELISA. RESULTS: Patients with a painful neuropathy had about twofold higher IL-2 mRNA (p = 0.001) and TNF mRNA (p < 0.0001) and protein levels (p = 0.009) than healthy control subjects and about twofold higher IL-2 and TNF mRNA (p = 0.03; p = 0.001) and protein levels (p = 0.04; p = 0.04) than patients with painless neuropathy. In contrast, mRNA levels of the anti-inflammatory cytokine IL-10 were about twofold higher in patients with painless neuropathy than in patients with painful neuropathy (p = 0.001) and controls (p = 0.004). IL-4 protein levels were 20-fold higher in patients with painless neuropathy (p < 0.0001) and 17-fold higher in patients with painful neuropathy (p < 0.0001) than in healthy control subjects. CONCLUSIONS: A pro-inflammatory cytokine profile seems to be associated with pain in the setting of a peripheral neuropathy, corroborating findings in animal models with experimental painful neuropathies. This may have implications for future treatment strategies.

Zytotoxische CD8+ T-Zellen spielen eine bedeutende Rolle in der Immunantwort gegen HIV-1. Trotz allem kommt es jedoch im Verlauf der Infektion bei den meisten Betroffenen zum Anstieg der Viruslast und Abfall der CD4 Zellzahl, obwohl auch in diesem fortgeschrittenen Stadium der Infektion virusspezifische CD8+ T-Zellantworten mittels INF-&#947;-Produktion nachgewiesen werden können. Ziel der vorliegenden Arbeit war es daher, weitere funktionelle und phänotypische Merkmale von CD8+ T-Zellen zu untersuchen, um eine mögliche Ursache für die im Verlauf der Infektion ineffizient werdende Immunantwort zu finden. Einen statistisch signifikanten Unterschied der INF-&#947; Produktion CD8+ T-Zellen zwischen Progressors und Controllers ließ sich mittels INF-&#947; Elispot bei den von uns untersuchten Patienten nicht nachweisen. Es ist also davon auszugehen, dass CD8+-T-Zellen im fortgeschrittenen, chronischen Stadium der Infektion funktionelle Lücken aufweisen, die sich nicht durch INF&#61543;-basierte Untersuchungsmethoden nachweisen lassen. Anhand intrazellulärer Zytokinfärbung ließ sich unabhängig vom Schweregrad der Infektion eine erhaltene Produktion der Zytokine INF&#947; und TNF&#945;, nicht hingegen eine Produktion von IL-2 nachweisen. Bei der Untersuchung HIV spezifischer CD8+ T-Zellen mit Hilfe von Tetramerfärbungen zeigte sich in Bezug auf den Aktivierungsgrad, gemessen an der CD38-Expression, ein deutlicher Unterschied zwischen Progressors und Controllers, der innerhalb der HIV-spezifischen CD8+ T-Zellen im Vergleich zur gesamten CD8+ T-Zellpopulation noch ausgeprägter zu erkennen war. Hier gab es eine signifikante positive Korrelation zur Viruslast und eine signifikante negative Korrelation zur CD4-Zellzahl der HIV-Infizierten. Anhand des Aktivierungsmarkers HLA-DR ließ sich dieser Unterschied nicht nachweisen. Im Bezug auf die Proliferationsfähigkeit, Apoptoseempfindlichkeit und lytische Funktion HIV-spezifischer Zellen konnte kein statistisch signifikanter Unterschied zwischen Progressors und Controllers ausgemacht werden. Schlüsse für die Gesamtheit der HIV-spezifischen Zellen eines HIV-Infizierten kann man natürlich nicht ziehen. Man darf nicht außer Acht lassen, dass uns lediglich eine begrenzte Anzahl von Epitopen bei den durchgeführten Untersuchungen zur Verfügung stand. Es wurde jedoch deutlich, dass es feine Unterschiede und Trends zu geben scheint, die eine gezielte weiterführende Untersuchung erfordern. Weiterhin ist auch zu bedenken, dass möglicherweise andere Faktoren, als die von uns untersuchten, für den unterschiedlichen Krankheitsverlauf verantwortlich sein können. Welche Faktoren nun tatsächlich die Ursache sind dafür, dass das menschliche Immunsystem gegen das HI-Virus in der Regel früher oder später verliert, bleibt weiterhin offen. Sehr wahrscheinlich handelt es sich hierbei um das Zusammenspiel mehrerer Mechanismen. Die gezielte Untersuchung HIV-spezifischer Zellen ist bei der weiteren Erforschung der genauen Zusammenhänge unerlässlich.

Die Knochenhomöostase erfolgt durch das Zusammenspiel mehrerer Zelltypen. Während die Osteoblasten für den Knochenaufbau verantwortlich sind, resorbieren die Osteoklasten Knochengewebe. Beide Vorgänge werden durch die Osteozyten streng reguliert. Eine Störung im strikt regulierten Gleichgewicht zwischen Knochenabbau und Knochenaufbau kann daher zu Knochenkrankheiten wie Osteoporose führen. Auf molekularer Ebene erfolgt die Kommunikation zwischen den einzelnen Zelltypen über zwei wichtige Signalwege, den der „Bone Morphogenetic Protein“-Superfamilie (BMPs) und den der Wnt-Proteine. Die Signalübertragung wird hierbei durch sekretierte Faktoren induziert, die an Rezeptoren auf der Zelloberfläche binden. Deren Aktivierung führt zu einem intrazellulären Signal, welches letztlich die Expression von Zielgenen reguliert. Beide Signalwege werden auf mehreren Ebenen, extrazellulär, membranständig und intrazellulär reguliert. Das 2003 identifizierte Sclerostin ist ein Vertreter der extrazellulären Regulatorproteine und wurde aufgrund seiner Zugehörigkeit zur DAN-Familie zunächst fälschlicherweise als direkter Inhibitor des BMP-Signalwegs eingestuft. Mittlerweile wird allerdings davon ausgegangen, dass Sclerostin den Wnt-Signalweg negativ reguliert, indem es die Wnt Ko-Rezeptoren LRP5 und LRP6 bindet, die beide zu der Familie der „Low-density lipoprotein receptors“ gehören. Über den molekularen Inhibitionsmechanismus von Sclerostin war jedoch zum Startpunkt dieser Dissertationsarbeit wenig bekannt. Daher wurde Sclerostin im Rahmen dieser Arbeit biophysikalisch und biochemisch charakterisiert. Die Aufklärung mittels NMR-Spektroskopie ergab für Sclerostin eine Struktur, die sich in drei Regionen gliedert: den Cystinknoten, sowie einen „Loop“-Bereich und die Fingerregion. Vom zentralen Cystinknoten gehen drei Peptid-Schleifen in zwei entgegengesetzte Richtungen aus. Schleife eins und drei bilden eine definierte ß-Faltblattstruktur und ähneln zwei Fingern einer Hand. Die zweite Schleife, welche vom Cystinknoten isoliert in die entgegengesetzte Richtung verläuft („Loop“), ist wie die beiden langen N- und C-Termini flexibel und unstrukturiert. Die in Zusammenarbeit mit der Firma AbD-Serotec entstandenen Fab-Fragmente ermöglichten die Bestimmung des Bindeepitops der Sclerostin/LRP5-Interaktion im Bereich der unstrukturierten dritten Schleife von Sclerostin. Die Struktur von Sclerostin und die Identifikation des Bindeepitops auf Sclerostinseite geben nun erste Einblicke in den molekularen Mechanismus der Sclerostin/LRP5-Interaktion. Diese Kenntnis kann für die Entwicklung von Kleinmolekülinhibitoren mittels rationalem Drugdesign genutzt werden, welche, wie auch der in Kooperation entwickelte die Sclerostinaktivität neutralisierende Antikörper AbD09097, hochinteressante Ansätze für neuartige anabole Therapien von Krankheiten mit Knochenschwund darstellen. / Different cell types like osteoblasts, osteoclasts and osteocytes maintain bone homeostasis. While osteoblasts build up bone, osteoclasts resorb bone tissue and both actions are tightly regulated by the osteocytes. Imbalance between bone formation and resorption will lead to various bone diseases, e.g. osteoporosis. On a molecular level communication between these cell types occurs through two major signalling pathways, i.e. the bone morphogenetic proteins (BMPs) and the Wnt-factors. In both pathways signal transduction is induced by secreted factors, which bind to cell surface receptors. This activation leads to an intracellular signal that finally regulates expression of target genes. Both pathways are tightly regulated at various cellular levels, extracellular, at the membrane as well as intracellular. Sclerostin, which was identified in 2003, is a member of the extracellular modulator proteins. Initially it was wrongly classified as a direct inhibitor of the BMP-signalling pathway due to its classification as a member of the DAN-family. Meanwhile it became apparent that sclerostin targets the Wnt-pathway by binding to the Wnt co-receptors LRP5 and LRP6, which belong to the family of low-density lipoprotein receptors. At the beginning of this work very little was known about the molecular mechanism how sclerostin inhibits the Wnt-pathway. The structure analysis of sclerostin employing NMR-spectroscopy revealed in a modular architecture, which can be divided into three regions: the central, characteristic cystine knot, the loop-region and the two fingers. From the cystine knot three loops emanate in two opposite directions. Loop one and loop three form defined ß-sheet structures resembling two fingers of a hand. Loop two, which runs into the opposite direction, is unstructured and highly flexible like the long N- and C-termini. Antibody fab-fragments, which were generated in collaboration with AbD-Serotec, facilitated the mapping of the binding-epitop of sclerostin to LRP5/6, highlighting an extended area of the unstructured loop region of sclerostin as the LRP5/6 binding site. The high-resolution structure of sclerostin and the identification of the LRP5-binding-epitop yield first insights into the molecular mechanism of sclerostin-LRP5 interaction. This knowledge can now be used to develop small-molecule inhibitors by rational drug design, which are, like the sclerostin activity neutralising fab-fragment AbD09097, highly interesting targets for new bone-anabolic therapies of diseases characterised by bone loss.

O câncer cervical é o segundo mais comum em mulheres em países em desenvolvimento, sendo causado por infecção persistente por Papilomavírus Humano (HPV). Quando esta persistência ocorre, entre outros fatores, está relacionada a mecanismos de evasão do sistema imune apresentados pelo vírus. A frequência de macrófagos aumenta com a progressão da lesão cervical e há aumento de células mielóides no baço de camundongos com tumor. Este trabalho tem como objetivo observar os efeitos sistêmicos de tumores associados ao HPV sobre a proliferação e recrutamento de células do sistema imune e identificar fatores que tenham papel nesses mecanismos. Utilizando modelos de tumor in vivo, observou-se que tumores associados ao HPV recrutam mais células para o tumor e induzem maior proliferação celular. Também avaliamos o perfil de expressão de citocinas nas linhagens tumorais e o perfil geral de expressão de proteínas através de eletroforese 2D. Com isto, demonstramos que linhagens tumorais positivas para HPV apresentam maior expressão de IL-6, IL-8, CXCL1, sICAM e Serpina E1. / Cervical cancer is the second most common type of cancer in women in developing countries. Its main etiologic factor is persistent infection with high risk human papillomavirus (HPV). This persistence occurs only in some cases and, among other factors, is related to mechanisms of immune evasion displayed by the virus. There is an increase in the frequency of macrophages proportional to cervical intraepithelial lesion grade and an increase of myeloid cells in the spleen of tumor bearing mice. This work aims to observe the systemic effects of HPV associated tumors on the proliferation and recruitment of immune cells, and identify factors that have a role in these mechanisms. Using in vivo tumor models, we found that HPV positive tumors recruit a higher percentage of cells and induce cellular proliferation. We also studied cytokine expression profiles of tumor cell lines, and performed proteomic assay with tumor cells transduced with HPV oncogenes. Our data shows that HPV associated tumor cell lines display higher expression of IL-6, IL-8, CXCL1, sICAM and Serpin E1.

Nos últimos anos, vários estudos têm demonstrado que durante o desenvolvimento da insuficiência cardíaca (IC) ocorre uma ativação no sistema imunológico, notadamente através de alterações nos níveis plasmático de citocinas pro e antiinflamatórias. Desta forma, estratégias terapêuticas têm sido utilizadas com o intuito de modular a ação destas citocinas e neste caso, o treinamento físico aeróbio parece promissor. Em nosso estudo, avaliamos o efeito de oito semanas de um programa de treinamento aeróbio em esteira para ratos com IC secundária a infarto do miocárdio (IM). Apesar da produção e expressão dos genes (TNF-<font face=\"symbol\">a, IL-6 e IL10) estarem aumentados nos músculo sóleo dos animais com IC, o programa de treinamento aeróbio foi capaz de reverter este quadro, demonstrando valores próximos aos animais normais (sem IC). Desta forma, nos estudo sugere que o treinamento aeróbio moderado demonstrou efeito antiinflamatório em animais com IC secundária a (IM), podendo exercer um importante papel como terapêutica em programas de reabilitação de doenças cardiovasculares. / Recently, several studies has demonstrated immune activation during heart failure (HF) development, notably through changes in plasmatic levels pro and anti-inflammatory cytokines. For this reason, therapeutic interventions have been used with targeting to modulate this cytokine action and in this way, endurance training may be promising. In our study, we evaluated the effect of 8 weeks of endurance training program in a treadmill for post-myocardial (MI) HF rats. Despite increased of levels and gene expression (TNF-<font face=\"symbol\">a, IL-6 e IL10) in soleus muscle of post-MI HF rats, endurance training was able to reverse this change, showing similar values that as founded in control group (without HF). In this way, our study suggests that moderate endurance training demonstrated anti-inflammatory effect in post-MI HF rats, and it may play an important role as therapeutic in rehabilitation programs of cardiovascular disease.

A erliquiose monocitotrópica canina é caracterizada como uma infecção persistente, podendo evoluir para doença fatal. Mecanismos imunopatogênicos estão implicados no desenvolvimento da doença, porém não é completamente compreendido o papel da resposta imune celular nas infecções causadas por Ehrlichia canis transmitida pelo carrapato Rhipicephalus sanguineus. A infecção do vetor ocorre somente durante o repasto em cães riquetsêmicos, porém não é reconhecido o nível de riquetsemia infectante durante o curso da infecção nos cães. O objetivo desse estudo foi verificar os níveis de riquetsemia relativa capazes de infectar o vetor (R. sanguineus) durante o curso da erliquiose canina; e avaliar a resposta imune celular induzida por exposição de cães a carrapatos infectados com Ehrlichia canis. Um cão foi utilizado para produção do inoculo e infecção de ninfas de R. sanguineus. Subseqüentemente, os carrapatos adultos infectados, oriundos das ninfas que se alimentaram nos cães infectados, foram utilizados para infecção dos cães dos grupos I (n=3) e II (n=3). Cães do grupo III (n=3) foram inoculados com sangue infectado com E. canis, por via intravenosa. Os grupos IV (n=3) e V (n=3) foram compostos por cães não infectados. Os grupos II e IV foram reinfestados periodicamente com ninfas não infectadas de R. sanguineus. Foram observadas, alterações clínicas como febre, apatia e disorexia nos cães infectados, durante a fase aguda da infecção, porém de forma mais intensa e precoce nos cães infectados por inoculação intravenosa (grupo III). As alterações hematológicas mais evidentes foram redução do número de plaquetas, leve redução na série vermelha e branca. Todos os animais infectados soroconverteram aos 14 dias após inoculação ou infestação com carrapatos infectados (dpi), mantendo títulos entre 10240 e 81920. Os níveis de riquetsemia foram variáveis durante o curso da infecção, persistindo até 364 dpi, porém mais constante na fase aguda da doença. Um pequeno número de carrapatos alimentados nos cães infectados apresentou amplificação de DNA de E. canis, porém foram demonstrados até 308 dpi. Foi observada uma redução na relação CD4:CD8 nos animais infectados, no 28° dpi, mantendo-se de forma mais branda até 252 dpi. Uma maior proporção de linfócitos T CD4+ produtores de IFN-&gamma; e IL-4 foi observada no tempo zero de cães que não adquiriram infecção após infestação com carrapatos infectados. Os níveis séricos de citocinas mostraram de forma mais evidente a elevação de IL-10 e TNF-&alpha; em um cão que desenvolveu doença fatal, podendo indicar a participação de mecanismos imunes na apresentação clínica e na persistência do agente no organismo hospedeiro. / The canine monocytic ehrlichiosis is characterized as a persistent infection, which can develop fatal disease. Immunopathogenic mechanisms are implicated in the development of the disease, however it is not completely understood the role of cellular immune in the infection caused by Ehrlichia canis transmitted by the tick Rhipicephalus sanguineus. The vector becomes infected only while feeding on rickettsemic dogs, however the level of rickettsemia is not recognized during the course of the infection in the dogs. The objective of this study was to verify the level of relative rickettsemia capable to infect the vector (R. sanguineus) during the course of the canine ehrlichiosis; and to evaluate the cellular immune induced in dogs exposed to Ehrlichia canis-infected ticks. A dog was used for production of the inoculum and infection of nymphs of R. sanguineus. Infected adult ticks that had fed as nymphs on the infected dog were used to feed on and transmit the infection to dogs of groups I (n=3) and II (n=3). Group III dogs (n=3) were intravenously inoculated with E. canis-infected dogs. Group IV (n=3) and V (n=3) dogs were the control groups, never exposed to E. canis. Dogs of groups II and IV were reinfested periodically with uninfected R. sanguineus nymphs. Clinical alterations such as fever, apathy and dysorexia were observed in the infected dogs during the acute phase of the infection, however in a more intense and precocious form in the dogs infected by intravenous inoculation (group III). The more evident hematological alterations were reduction of the number of platelets, mild reduction in the red and white cells series. All infected animals soroconverted by 14 days after inoculation or infestation by infected ticks (dpi), showing titers between 10240 and 81920 during the study. The rickettsemia levels were variable during the course of the infection, persisting up to 364 dpi, however more constant in the acute phase of the disease. A small number of ticks that fed on infected dogs presented amplification of E. canis DNA, however they were demonstrated up to 308 dpi. A reduction in the relationship CD4:CD8 in the infected animals was observed in 28th dpi, keeping in a mild form up to 252 dpi. A larger proportion of lymphocytes T CD4+ producing IFN-&gamma; and IL-4 it was observed in the zero time of dogs that did not acquire infection after infestation with infected ticks. The cytokine seric levels showed the elevation in a more evident form of IL-10 and TNF-&alpha; in a dog that developed fatal disease, what could indicate the participation of immune mechanisms in the clinical presentation and in the persistence of the agent in the organism host.

Changes in the microenvironment have been implicated in the transition of pre-invasive ductal carcinoma in-situ (DCIS) to invasive breast cancer. Normal myoepithelial cells have a tumour suppressor phenotype but they are altered in DCIS and ultimately lost with transition to invasive cancer. A consistent change in DCIS is up-regulation of the integrin αvβ6 in myoepithelial cells. Preliminary observations identified a correlation between myopeithelial αvβ6 and an increased peri-ductal inflammatory infiltrate. The hypothesis of this study is that the altered myoepithelial phenotype influences the peri-ductal inflammatory environment, which in turn mediates a pro-apoptotic effect on myoepithelial cells contributing to their loss. To investigate this, the inflammatory infiltrate was characterised in a series of DCIS tissue in relation to αvβ6 status. This demonstrated significantly higher levels of CD4+ve and FOXP3+ve T cells around αvβ6+ve DCIS ducts compared to αvβ6-ve ducts (P=<0.01), suggesting an increase in Treg cells. In-vivo, Matrigel plugs containing injected into the flanks of female C57/Blk6 normal mice generated influx of higher levels of CD4+ve cells (p=0.005) and FOXP3+ T cells (p=0.007) in the presence of αvβ6+ve myoepithelial cells compared to αvβ6-ve cells, supporting the findings in human tissue samples. Since Treg cells produce TRAIL that can induce apoptosis, we investigated the influence of αvβ6 on myoepithelial cells on the levels of TRAIL in T cells and the hypothesis that αvβ6-positive myoepithelial ells may be more susceptible to TRAIL-induced apoptosis, leading to loss of the myoepithelial barrier. Firstly, levels of TRAIL in Jurkat and primary T cell populations co-cultured with β4 (ii) or β6 myoepithelial cells were measured. This demonstrated a higher level of TRAIL in primary T cells co-cultured β6 myoepithelial cells compared to those co-cultured with β4 myoepithelial cells. β6+ve and β6-ve myoepithelial cells were exposed to TRAIL, and this demonstrated that TRAIL enhanced apoptosis, measured by cleaved PARP, in β6+ve cells. Furthermore, these cells showed loss of the anti-apoptotic protein Galectin-7, and knockdown of Galectin-7 in normal β6-ve myoepithelial cells rendered them more susceptible to TRAIL-induced apoptosis. In DCIS tissues, an inverse relationship between αvβ6 and Galectin-7 in myoepithelial cells was demonstrated, and Cytokine Array analysis showed that αvβ6+ve myoepithelial cells express higher levels of IL-16, which has a role in Treg cell recruitment. Taken together these results suggest that expression of αvβ6 by myoepithelial cells in DCIS generates a tumour-promoter peri-ductal inflammatory infiltrate through altered cytokine release, is associated with reduced galectin-7 expression and enhances myoepithelial cell apoptosis in response to TRAIL. This provides a potential mechanism by which myoepithelial cells may be lost during evolution of DCIS and so contribute to progression to invasive disease.

O câncer cervical é o segundo mais comum em mulheres em países em desenvolvimento, sendo causado por infecção persistente por Papilomavírus Humano (HPV). Quando esta persistência ocorre, entre outros fatores, está relacionada a mecanismos de evasão do sistema imune apresentados pelo vírus. A frequência de macrófagos aumenta com a progressão da lesão cervical e há aumento de células mielóides no baço de camundongos com tumor. Este trabalho tem como objetivo observar os efeitos sistêmicos de tumores associados ao HPV sobre a proliferação e recrutamento de células do sistema imune e identificar fatores que tenham papel nesses mecanismos. Utilizando modelos de tumor in vivo, observou-se que tumores associados ao HPV recrutam mais células para o tumor e induzem maior proliferação celular. Também avaliamos o perfil de expressão de citocinas nas linhagens tumorais e o perfil geral de expressão de proteínas através de eletroforese 2D. Com isto, demonstramos que linhagens tumorais positivas para HPV apresentam maior expressão de IL-6, IL-8, CXCL1, sICAM e Serpina E1. / Cervical cancer is the second most common type of cancer in women in developing countries. Its main etiologic factor is persistent infection with high risk human papillomavirus (HPV). This persistence occurs only in some cases and, among other factors, is related to mechanisms of immune evasion displayed by the virus. There is an increase in the frequency of macrophages proportional to cervical intraepithelial lesion grade and an increase of myeloid cells in the spleen of tumor bearing mice. This work aims to observe the systemic effects of HPV associated tumors on the proliferation and recruitment of immune cells, and identify factors that have a role in these mechanisms. Using in vivo tumor models, we found that HPV positive tumors recruit a higher percentage of cells and induce cellular proliferation. We also studied cytokine expression profiles of tumor cell lines, and performed proteomic assay with tumor cells transduced with HPV oncogenes. Our data shows that HPV associated tumor cell lines display higher expression of IL-6, IL-8, CXCL1, sICAM and Serpin E1.

This dissertation represents efforts to determine the functional consequences acquired by vascular smooth muscle cells (SMC) in response to CD40 ligation by activated CD154+ T cells, and to elucidate components of the signaling pathway(s) activated in response to CD40 signaling in both monocytes and SMC. To study the consequences of CD40 stimulation, primary human monocytes and aortic SMC were treated with plasma membranes purified from CD154 + , CD4+ T cells. The results presented in this dissertation demonstrate that SMC, like monocytes/macrophages, are capable of interacting with T cells in a manner that results in reciprocal activation events. SMC were shown to present antigen to, and activate T cells. In turn T cell stimulus resulted in the activation of proinflammatory function in SMC initiated through the CD154:CD40 interaction. CD40 stimulation of SMC resulted in the production of the chemokines interleukin 8 (IL-8) and macrophage chemotactic protein-1 (MCP-1), and the upregulation of intercellular adhesion molecule (ICAM). Examination of the intracellular signaling pathways activated through CD40 signaling revealed the involvement of MAPKs in the pathway leading to induction of proinflammatory activity. Evaluation of CD40 signaling in monocytes demonstrated the activation of the MAPK family members ERK1/2, but not the MAPK family members p38 or c-jun-N-terminal kinase (JNK). In contrast, CD40 signaling in SMC was shown to result in ERK1/2 and p38 activation, and both of these kinases were shown to play a critical role in the induction of chemokine synthesis. An examination of the ability of anti-inflammatory cytokines to modulate CD40 signaling in monocytes and SMC demonstrated that the anti-inflammatory cytokines IL-4 and IL-10 abrogate CD40-mediated induction of inflammatory cytokine production by monocytes. This inhibition was shown to be a result of a negative influence of IL-4 and IL-10 on CD40 mediated ERK1/2, activation in monocytes. However, IL-4 and IL-10 did not inhibit SMC proinflammatory responses indicating a difference in the intracellular responses to these cytokines by the two cell types. (Abstract shortened by UMI.)