GUERRA, Priscila Valera. Efeito protetor de CXCL10 em infecção experimental por Leishmania braziliensis. 2013. 68 f. Dissertação (Mestrado em Patologia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2013. / Submitted by denise santos (denise.santos@ufc.br) on 2013-07-09T11:23:12Z No. of bitstreams: 1 2013_dis_pvguerra.pdf: 1062693 bytes, checksum: cbcefa56a6fe8c511929d08079c527fe (MD5) / Approved for entry into archive by Erika Fernandes(erikaleitefernandes@gmail.com) on 2013-07-10T10:48:55Z (GMT) No. of bitstreams: 1 2013_dis_pvguerra.pdf: 1062693 bytes, checksum: cbcefa56a6fe8c511929d08079c527fe (MD5) / Made available in DSpace on 2013-07-10T10:48:55Z (GMT). No. of bitstreams: 1 2013_dis_pvguerra.pdf: 1062693 bytes, checksum: cbcefa56a6fe8c511929d08079c527fe (MD5) Previous issue date: 2013 / Leishmania braziliensis is the more common etiologic agent of the cutaneous leishmaniasis in Brazil. BALB/c mice are more susceptible to L. braziliensis infection, although develop small and not ulcerated lesions. The lesions in BALB/c mice infected by L. braziliensis are accompanied by expression of a broad chemokines spectrum, including CXCL10, which is able to attract and activate NK and Th1 cells with IFN-γ production. The aim of this study was to evaluate whether the chemokine CXCL10, administered in early stages of infection could influence the course of disease, leading to a faster and more effective time of healing. Groups of BALB/c mice (n=24) were infected by L. braziliensis and divided into two groups: one group (n=12) received CXCL10 (100 ng/5μL) and the other (n=12) received PBS (5μL), at 1, 3, 5 and 7 days postinfection. Some parameters were evaluated: thickness of the lesions every three days; parasite burden in both the footpad and the draining lymph node, NO concentration, cytokines production (IFN-γ, IL-12p40, TNF-α, IL-4, IL-10 and TGF-β), at 15 and 30 days after infection; and histopathological analysis of lesions. Animals receiving CXCL10 showed reduced disease duration, since the lesions started to regress earlier (12 days), whereas the control animals developed the disease for longer (15 days). CXCL10 induced statistically significant differences in parasite load in the draining lymph nodes compared with control in both times evaluated (15 days: p=0.0353; 30 days: p=0.0292). In the animals receiving CXCL10 was not observed change in the number of parasites in the site of inoculation, even with reduced thickness of the lesion, showing no correlation between lesion size and parasite load. There was no difference in NO concentration between groups with 15 days of infection, and this concentration remained the same in the group receiving CXCL10 at day 30, although it was lower than that observed in the control group. CXCL10 also induced higher levels of IFN-γ and IL-12, and reduced IL-4, IL-10, and TGF-β at 30 days postinfection, when compared to control animals. Histopathology analyses of lesions after 15 days postinfection showed a very mild inflammation in both groups, differing only for the presence of neutrophils, which were practically not observed in lesions of animals receiving CXCL10. Taken together, the data from this study showed CXCL10 chemokine presented a protective effect on L. braziliensis infection in BALB/c mice, with a reduction in the progression of the disease, associated with a higher concentration of IFN-γ and lesser of IL-4 and IL-10. / Leishmania braziliensis é o agente etiológico mais comum da leishmaniose cutânea no Brasil. Camundongos BALB/c são os mais susceptíveis à infecção por L. braziliensis, embora desenvolvam lesões pequenas e não ulceradas. As lesões em BALB/c infectados com L. braziliensis são acompanhadas pela expressão de um amplo espectro de quimiocinas, entre elas CXCL10, que é capaz de atrair a ativar células NK e Th1 com produção de IFN-γ. O objetivo deste trabalho foi avaliar se a quimiocina CXCL10, administrada nos estágios iniciais da infecção, poderia alterar o curso da doença, dimunindo o tempo de cura. Para isso, camundongos BALB/c (n=24) foram infectados com L. braziliensis e divididos em dois grupos: um grupo (n=12) recebeu CXCL10 (100 ng/5µL) e o outro (n=12) PBS (5µL), nos dias 1, 3, 5 e 7 dias de infecção. Alguns parâmetros foram avaliados: a espessura das lesões a cada três dias; a carga parasitária na pata e no linfonodo de drenagem, a concentração de NO, IFN-γ, IL-12p40, TNF-α, IL-4, IL-10 e TGF-β, 15 e 30 dias após a infecção; e análise histopatológica das lesões. Os animais que receberam CXCL10 apresentaram uma redução no tempo de duração da doença, uma vez que as lesões começaram a regredir mais precocemente (12 dias), enquanto que nos animais controle (PBS) a doença evoluiu por mais tempo (15 dias). CXCL10 induziu diferenças estatisticamente significativas na carga parasitária do linfonodo de drenagem nos dois pontos avaliados, quando comparado com o controle (15 dias: p=0,0353; 30 dias: p=0,0292). Nos animais que receberam CXCL10, não foi observada mudança no número de parasitos no sítio de inoculação, mesmo com redução da espessura da lesão, mostrando que não houve correlação entre o tamanho da lesão e a carga parasitária. Não houve diferença na concentração de NO entre os grupos, no 15º dia de infecção, e esta concentração manteve-se igual no grupo que recebeu CXCL10 no 30º dia, embora tenha sido menor do que a observada no grupo controle. CXCL10 também induziu concentrações mais elevadas de IFN-γ e IL-12 e mais reduzidas de IL-4, IL-10, TGF-β 30 dias pós-infecção, quando comparados aos animais controle. A análise histopatológica das patas mostrou uma inflamação muito leve em ambos os grupos, com diferença apenas quanto à ausência de neutrófilos nas lesões dos animais que receberam CXCL10. Em conjunto, os dados mostraram que CXCL10 apresentou um efeito protetor na infecção por L. braziliensis em camundongos BALB/c, com redução no tempo de evolução da doença, associada a maior concentração de IFN-γ e menor de IL-4 e IL-10.

Background: Respiratory tract infections are responsible for millions of deaths annually. Interferon-stimulated genes (ISGs) play a significant role in fighting off viral respiratory tract infections in the antiviral defence system. Measuring extracellular protein products of ISGs could be potential biomarkers of viral infection. Although, the feasibility and performance of ISGs as functional and robust clinical biomarkers from a non-invasive sample format remains unknown. Methods: Three ISGs, CXCL10, CXCL11, and TNFSF10, were examined in in-vivo and in-vitro gene expression datasets (RNA-sequencing and microarray) infected with common respiratory tract infections (Rhinovirus, Respiratory syncytial virus, influenza A and SARS-CoV-2) samples and compared to negative controls. Using qualitative selection criteria of 1) elevated presence in at least one dataset with viral infection, 2) secreted protein product, and 3) commercially available antibodies for detection, CXCL10, CXCL11 and TNFSF10 gene expression levels were assessed. A correlation analysis was performed with SARS-CoV-2 infection severity and gene expression kinetics. CXCL10 was subsequently validated at the protein level in saliva as a prerequisite for developing a host-response LFA. Results: CXCL10 and CXCL11 upregulation were positively correlated with RSV compared to control (p < 0.05). CXCL10/CXCL11/TNFSF10 were not different between samples collected from RV infected subjects relative to controls (p > 0.05). No significant association was found with influenza A for all three genes. CXCL10/CXCL11/TNFSF10 upregulation was positively correlated with SARS-CoV-2 infection compared to control (p < 0.001). CXCL10 expression correlated with COVID-19 viral load. CXCL10 was chosen as a lead biomarker candidate based on these analyses that included different virus infections, time-courses, and measures of severity. CXCL10 was not detected at the protein level in healthy saliva but was elevated in saliva from COVID-19 patients. A CXCL10 LFA was developed with a sensitivity of 2 ng/ml in a buffer and artificial saliva. Conclusion: We establish and validate the potential of developing rapid test techniques to examine host immune response from a bioinformatic approach to developing a prototype rapid test with capabilities to be used in point-of-care settings. / Thesis / Master of Science (MSc) / Respiratory tract infections are a leading cause of death and one of the main reasons to seek primary care. Both historically and in the present day, respiratory tract infections remain a massive socioeconomic burden. Current diagnostics fail to quickly identify a respiratory tract infection's etiology, and prognosis, leading to suboptimal patient care and the over prescription of antibiotics. Advanced tools used in academia and research, including next-generation -omics sequencing and metagenomics, have capabilities to identify all nucleic acid material in a sample - including host RNA- which offers potential to improve the diagnosing of respiratory tract infections. However, these technologies have not been integrated into routine care due to economic, technical, and logistical barriers. We explored host RNA (transcriptomics), looking at antiviral interferon-stimulated genes for their potential as a biomarker of viral infection amenable to point-of-care testing platforms from non-invasive sample types.

Die Grundlage für diese Arbeit bildete ein Modell mit CFA-(komplettes Freundsches Adjuvant) induziertem Entzündungsschmerz in Ratten, bei denen eine zweimalige Behandlung mit Elektroakupunktur zu einer langanhaltenden Antinozizeption führte, welche abhängig von peripheren Opioiden war. In einem nächsten Schritt sollten nun die durch Akupunktur vermittelten Zytokin- und Chemokinveränderungen untersucht und deren Beitrag zu den antinozizeptiven und anttiinflammatorischen Mechanismen geklärt werden. Mittels ELISA und PCR wurden die Protein- und mRNA-Level der klassischen Zytokine und des Chemokins CXCL10 bestimmt. CXCL10, welches durch Elektroakupunktur sowohl auf Transkriptions- als auch auf Translationsebene hochreguliert wurde, ist notwendig für die Rekrutierung β-Endorphin haltiger Makrophagen in das entzündete Gewebe und für die antinozizeptive Wirkung der Akupunkturbehandlung. Ein antiinflammatorischer Effekt der Akupunkturbehandlung äußerte sich durch die Reduktion von TNF-α und IL-1β und ein erhöhtes IL-13. Das einzige hochregulierte proinflammatorische Zytokin war IFN-γ. Ein Teil der entzündungshemmenden Wirkung, die Reduktion der proinflammatorischen Zytokine TNF-α und IL-1β, wird durch Adenosin-2B-Rezeptoren vermittelt, welche bekannt sind für ihre Rolle in der „Deaktivierung“ IFN-γ-stimulierter Makrophagen. Diese Ergebnisse verweisen auf die bisher unbekannte Verbindung zwischen chemokinvermittelter peripherer, opioidabhängiger Antinozizeption durch Elektroakupunktur. Sie erweitern das Verständnis für das Zusammenspiel von Immunzellen, Adenosin und Akupunktur. Weitere Untersuchungen sind notwendig, um neuroimmunologische Verbindungen zu klären und die Wirkungen durch die Nadelinsertion mit Effekten in der entfernten Rattenpfote besser zu verstehen. / This work is based on a rat model with complete Freund's adjuvant (CFA)-induced hind paw inflammation. Animals were treated twice with electroacupuncture eliciting long-term antinociception, which depended on peripheral opioids. In a next step we wanted to study acupuncture mediated changes in cytokine and chemokine profiles and their contribution to antinociceptive and anti-inflammatory mechanisms. For the measurement of protein and mRNA levels of classical cytokines and the chemokine CXCL10 ELISA and real-time PCR were used. CXCL10, which was upregulated on transcriptional and translational level, increased infiltrating β-endorphin containing macrophages within the inflamed tissue and was necessary for the antinociceptive effect of acupuncture treatment. Anti-inflammatory effects were seen in changed cytokine profiles with decreased TNF-α and IL-1β and increased IL-13. The only pro-inflammatory cytokine which was upregulated was IFN-γ. The anti-inflammatory effect caused by the changed cytokines may partly be mediated by Adenosine-2B Receptors, known for their deactivation of IFN-γ stimulated macrophages. In summary these results show a novel connection of chemokine-mediated, peripheral, opoid-dependent antinociception in electroacupuncture. They expand our understanding of the interaction of immune cells, adenosine and acupuncture. More research is needed to examine neuroimmunological mechanisms and the connection between needle insertion and detected effects in the rat paw.

FIGUEIREDO, Webertty Mayk Eufrásio de. Efeito da quimiocina CXCL10 na infecção por Leishmania infantum chagasi em camundongos BALB/c. 2012. 65 f. Dissertação (Mestrado em Patologia) - Universidade Federal do Ceará. Faculdade de Medicina, Fortaleza, 2012. / Submitted by denise santos (denise.santos@ufc.br) on 2013-05-10T14:16:18Z No. of bitstreams: 1 2012_dis_wmefigueiredo.pdf: 442833 bytes, checksum: 07c3a09e8e63fe5a99d0e9bcbcbb337f (MD5) / Approved for entry into archive by Erika Fernandes(erikaleitefernandes@gmail.com) on 2013-05-16T13:41:11Z (GMT) No. of bitstreams: 1 2012_dis_wmefigueiredo.pdf: 442833 bytes, checksum: 07c3a09e8e63fe5a99d0e9bcbcbb337f (MD5) / Made available in DSpace on 2013-05-16T13:41:11Z (GMT). No. of bitstreams: 1 2012_dis_wmefigueiredo.pdf: 442833 bytes, checksum: 07c3a09e8e63fe5a99d0e9bcbcbb337f (MD5) Previous issue date: 2012 / Visceral leishmaniasis caused by Leishmania infantum chagasi is characterized by the loss of the ability of host to generate an effective immune response. In this study it was investigated the role of CXCL10 chemokine in controlling L. infantum chagasi infection in vivo. Groups of BALB/c mice were treated or not with recombinant CXCL10 chemokine (5 μg/kg) with 1, 3 and 7 days of infection and after 1, 7 and 23 days of treatment, some parameters were evaluated: parasite load, levels of IFN-g, IL-4, TGF-β and IL-10, and the histopathological alterations in the liver. After 23 days of treatment, CXCL10 induced in the spleen a significant reduction on the number of parasites as compared to control group. In the liver, parasite load decreased in treated group between the 7th and 23th day post treatment. However, the antileishmanial effect of CXCL10, in this work, does not seem to be mediated by NO, since there was no difference in the NO production among the groups. IFN-γ was induced most significantly in treated group than in controls, and reached its maximum production (100 pg/mL) on day 23 after treatment, correlating with the reduction in parasite burden in target organs. IL-4 was produced in low doses, in both groups, although treated animals had shown higher levels than control group. Regarding to anti-inflammatory cytokines, after the 23th day of treatment, IL-10 levels in treated animals were smaller than in control group. Production of TGF-β after 7 days of treatment was 2 times lower in treated group when compared to control, and after the 23th day of treatment, this cytokine remained with lower levels than those observed in control. In the histopathological analysis of the liver after the 1st day of treatment, were found in both groups more immature granulomas (GI) than non-granulomatous infiltrate (NG), and some few mature granulomas (GM) were only observed in treated group. After 7 days of treatment, the amount of NG infiltrates was lower, and GI were still the most frequent in both groups, besides a slight increase of GM was observed in treated group. In summary, at the light of the found results, it is possible to suggest an important leishmanicidal role to CXCL10 in BALB/c mice infected by L. infantum chagasi, which seems to be mediated by a significant IFN-g production, and suppression of immunoregulatory cytokines, IL-10 and TGF-β, opening the hypothesis that this would be associated to a decrease in the frequency of regulatory T cells induced by CXCL10 in these animals. / A leishmaniose visceral causada por Leishmania infantum chagasi é caracterizada pela perda da habilidade do hospedeiro gerar uma resposta imunológica eficaz. Neste estudo, foi investigado o papel da quimiocina CXCL10 no controle da infecção por L. infantum chagasi in vivo. Grupos de camundongos BALB/c foram tratados ou não com CXCL10 (5 μg/kg) com 1, 3 e 7 dias de infecção e após 1, 7 e 23 dias do tratamento, alguns parâmetros foram avaliados: a carga parasitária, os níveis de IFN-, IL-4, TGF-β e IL-10, e as alterações histopatológicas no fígado. Após 23 dias de tratamento, CXCL10 induziu, no baço, uma redução expressiva no número de parasitos, quando comparado ao grupo controle. No fígado, a carga parasitária mostrou uma queda no grupo tratado, entre o 7º e 23º dia após o tratamento. Entretanto, o efeito leishmanicida de CXCL10, neste trabalho, não parece ser mediado por NO, uma vez que não houve diferença na produção de NO entre os grupos. IFN-γ foi induzida de maneira mais significativa no grupo tratado do que nos controles, e atingiu sua produção máxima (100 pg/mL) no 23º dia após o tratamento, correlacionando-se com a queda da carga parasitária nos órgãos-alvo. IL-4 foi produzida em baixas concentrações, em ambos os grupos, embora os animais tratados com CXCL10 tenham mostrado níveis mais elevados do que os controles. Em relação às citocinas antiinflamatórias, após 23 dias do tratamento, os níveis de IL-10 nos animais tratados foram menores do que os do controle. A produção de TGF-β após 7 dias do tratamento foi 2 vezes menor no grupo tratado quando comparado ao controle, e após 23 dias do tratamento, essa citocina continuou com níveis mais baixos do que aqueles observados no controle. Na análise histopatológica do fígado após o 1º dia do tratamento, foram encontrados, em ambos os grupos, mais granulomas imaturos (GI), do que infiltrados não granulomatosos (NG) e alguns poucos granulomas maduros (GM) apenas no grupo tratado. Após 7 dias do tratamento, a quantidade de infiltrados NG estava menor e os GI ainda foram os mais encontrados, em ambos os grupos, além disso, foi observado um pequeno aumento de GM no grupo tratado. Em resumo, diante dos resultados encontrados, é possível sugerir um importante papel leishmanicida de CXCL10 em camundongos BALB/c infectados por L. infantum chagasi, que parece ser mediado por uma expressiva produção de IFN-g e supressão das citocinas imunorreguladoras, IL-10 e TGF-β, abrindo a hipótese se isto não estaria associado a uma diminuição na frequência de células T regulatórias, induzida por CXCL10, nesses animais.

The tumor-selective viral replication capacity and pro-apoptotic effects of oncolytic reovirus have been reported to be dependent on the presence of an activated RAS pathway in several solid tumor types. However, the mechanisms of selective anticancer efficacy of the reovirus-based formulation for cancer therapy (Reolysin, pelareorep) have not been rigorously studied in soft tissue sarcomas (STS). Here we report that Reolysin triggered a striking induction of the anti-angiogenic chemokine interferon-gamma-inducible protein 10 (IP-10)/CXCL10 (CXC chemokine ligand 10) in both wild type and RAS mutant STS cells. Further analysis determined that Reolysin treatment possessed significant anti-angiogenic activity irrespective of RAS status. In addition to CXCL10 induction, Reolysin dramatically downregulated the expression of hypoxia inducible factor (HIF)-1 alpha, HIF-2 alpha and inhibited vascular endothelial growth factor (VEGF) secretion. CXCL10 antagonism significantly diminished the anti-angiogenic effects of Reolysin indicating that it is a key driver of this phenomenon. Xenograft studies demonstrated that Reolysin significantly improved the anticancer activity of the anti-angiogenic agents sunitinib, temsirolimus, and bevacizumab in a manner that was associated with increased CXCL10 levels. This effect was most pronounced following treatment with Reolysin in combination with temsirolimus. Further analysis in additional sarcoma xenograft models confirmed the significant increase in CXCL10 and increased anticancer activity of this combination. Our collective results demonstrate that Reolysin possesses CXCL10-driven anti-angiogenic activity in sarcoma models, which can be harnessed to enhance the anticancer activity of temsirolimus and other agents that target the tumor vasculature.

Die Restenose stellt ein zentrales Problem der interventionellen Kardiologie dar und ist häufigste Komplikation nach perkutanen Angioplastieverfahren. Hauptursache dieser Wiederverengung des Gefäßes ist die Bildung einer Neointima durch die Proliferation transdifferenzierter vaskulärer glatter Muskelzellen und die Sekretion extrazellulärer Matrix. Die Entstehung reaktiver Sauerstoffspezies (ROS) und die Entzündungsreaktion nach der Gefäßverletzung werden als frühe, die Neointimabildung induzierende Prozesse diskutiert. Im Rahmen dieser Arbeit wurden mehrere Projekte bearbeitet, die Aufschluss über die während der Neointimabildung statt findenden Prozesse geben sollen. Mit Hilfe eines Verletzungsmodells der murinen Femoralarterie wurde der Einfluss der Entzündung und der ROS-Bildung auf die Neointimabildung in der Maus untersucht. Die Behandlung mit dem mitochondrialen Superoxiddismutase-Mimetikum MitoTEMPO verminderte die Bildung der Neointima besser, als die Behandlung mit dem globalen ROS-Fänger N-Acetylcystein. Die stärkste Hemmung der Neointimabildung wurde jedoch durch die Immunsuppression mit Rapamycin erreicht. Interferon-γ (INFγ) ist ein wichtiges Zytokin der Th1-Immunantwort, das in Folge der Gefäßverletzung freigesetzt wird und die proinflammatorischen Chemokine CXCL9 (MIG, Monokine Induced by INF), CXCL10 (IP-10, INF inducible Protein of 10 kDa) und CXCL11 (I-TAC, Interferon inducible T cell-Chemoattractant) induziert. CXCL9, CXCL10 und CXCL11 sind Liganden des CXC-Chemokinrezeptors 3 (CXCR3) und locken chemotaktisch CXCR3 positive Entzündungszellen zum Ort der Gefäßverletzung. Daher wurde die spezielle Bedeutung des Chemokins CXCL10 in der Restenose untersucht. Dazu wurden CXCL10-defiziente Mäuse dem Femoralisverletzungsmodell unterzogen und die Gefäße nach 14 Tagen morphometrisch und immunhistologisch untersucht. CXCL10-Defizienz führte in Mäusen zu einer verminderten Neointimabildung, die mit einer verringerten Inflammation, Apoptose und Proliferation im verletzten Gefäß korrelierte. Neben der Inflammation beeinflusst aber auch die Reendothelialisierung der verletzten Gefäßwand die Restenose. Interessanterweise war im Vergleich zu Wildtyp-Mäusen in den CXCL10-Knockout-Mäusen auch die Reendothelialisierung erheblich verbessert. Offensichtlich ist das CXCR3-Chemokinsystem also in völlig unterschiedliche biologische Prozesse involviert und beeinflusst nicht nur die Bildung der Neoimtima durch die Förderung der Entzündung, sondern auch die Unterdrückung der Reendothelialisierung der verletzten Gefäßwand. Tatsächlich wird der CXCR3 nicht nur auf Entzündungszellen, sondern auch auf Endothelzellen exprimiert. Zur separaten Untersuchung der Rolle des CXCR3 in der Inflammation und der Reendothelialisierung wurde im Rahmen dieser Arbeit damit begonnen konditionelle CXCR3-Knockout-Mäuse zu generieren, in denen der CXCR3 entweder in Entzündungszellen oder in Endothelzellen ausgeschaltet ist. Zum besseren Verständnis der molekularen Mechanismen, mit denen der CXCR3 seine Funktionen vermittelt, wurde zudem untersucht ob dieser mit anderen G-Protein-gekoppelten Rezeptoren (GPCR) interagiert. Die Analyse von Coimmunpräzipitaten deutet auf eine Homodimerisierung der beiden CXCR3 Splicevarianten CXCR3A und CXCR3B, sowie auf die Heterodimerbildung von CXCR3A und CXCR3B mit sich, sowie jeweils mit CCR2, CCR3, CCR5 und den Opioidrezeptoren MOR und KOR hin. Die getestete Methode des Fluoreszenz-Resonanz-Energietransfers (FRET) erwies sich jedoch als ungeeignet zur Untersuchung von CXCR3, da dieser in HEK293T-Zellen nicht korrekt transient exprimiert wurde. Insgesamt deuten die Ergebnisse dieser Arbeit darauf hin, dass das CXCR3-Chemokinsystem eine zentrale Rolle in unterschiedlichen, die Neointimabildung beeinflussenden Prozessen spielt. Damit könnten der CXCR3 und insbesondere das Chemokin CXCL10 interessante Zielmoleküle in der Entwicklung neuer verbesserter Therapien zur Verhinderung der Restenose darstellen. / Restenosis represents a central problem after coronary angioplasty procedures and is caused by intimal hyperplasia, also called neointima, as a result of transdifferentiation, proliferation of vascular smooth muscle cells and secretion of extracellular matrix. Formation of reactive oxygen species (ROS) and inflammation after vascular injury caused by angioplasty are discussed as early inducers of neointima formation. In several projects the processes causing the development of intimal hyperplasia were investigated. First of all, the impact of inflammation and ROS in neointima formation was investigated using the mouse femoral injury model. The mitochondrial superoxide dismutase mimetic mitoTEMPO could reduce neointima formation better than the global ROS scavenger N-acetylcystein. However, the strongest reduction of neointima formation was achieved by the treatment with the immunosuppressant rapamycin. Interferon-γ(INFγ) is a major cytokine of the Th1 immune response. It is released as a result of vessel injury and induces the proinflammatory chemokines CXCL9 (MIG, Monokine Induced by INF), CXCL10 (IP-10, INF inducible Protein of 10 kDa) and CXCL11 (I-TAC, Interferon inducible T-cell-Chemoattractant), which are ligands of the CXC chemokine receptor 3 (CXCR3) and by this chemotactically recruit CXCR3 positive cells to the site of vessel injury. In this work the special role of CXCL10 in restenosis was investigated. Therefore, CXCL10 decient mice underwent the mouse femoral injury model. The vessels were analysed morphometrically and immunohistologically 14 days after injury. CXCL10 deciency lead to decreased neointima formation that correlated with a reduced recruitment of inflammatory cells as well as diminished numbers of apoptotic and proliferating cells at the site of vessel injury. In addition to inflammation the reconstitution of the endothelium has also impact on the development of restenosis. Interestingly reendothelialisation was strongly improved in CXCL10 decient mice compared to wildtype mice. Obviously the CXCR3 chemokine system is involved in different biological prosesses and impairs neointima formation on one hand by the advancement of inflammation and on the other hand by the suppression of reendothelialisation. In fact the CXCR3 is not only expressed on inflammatory cells but also on endothelial cells. To investigate the role of CXCR3 in inflammation and reendothelialisation separatly the generation of conditional CXCR3 knockout mice with a CXCR3 knockout in T-cells or endothelial cells was started in an additional project. For a better understanding of the molecular mechanisms on which the CXCR3 mediates its biological functions the protein-protein interactions of the CXCR3 with other G-protein coupled recteptors (GPCR) was analysed. Coimmunoprecipitation showed homodimerization of the CXCR3 splice variants CXCR3A and CXCR3B, as well as heterodimerization of CXCR3A and CXCR3B with each other and with the chemokine receptors CXCR4, CCR2, CCR3, CCR5 and the opioid receptors MOR and KOR. The additional tested Fluorecence resonance energy transfer (FRET) method proved to be not suitable to measure interactions of CXCR3, since this receptor could not be expressed correctly on the cell surface after transient transfection. To summarise, the results indicate that the CXCR3 chemokine system plays a central role in different processes that mediate neointima formation. Thus, the CXCR3 and especially the chemokine CXCL10 could be interesting therapeutic targets in the development of new or improved treatments to reduce the risk of restenosis.

La pemphigoïde bulleuse (PB) est la plus fréquente et la plus grave des dermatoses bulleuses auto-immunes caractérisée par une cascade inflammatoire impliquant plusieurs cytokines et cellules inflammatoires, avec la libération de protéases (MMP-9, élastase) conduisant à la formation de la bulle. L’objectif de cette étude est de caractériser cette cascade inflammatoire en analysant la contribution de la chimiokine CXCL10 et des cellules inflammatoires comme les macrophages dans le mécanisme physiopathologique associé à la maladie. Nos résultats ont montré que CXCL10 est présent à des taux importants dans le sérum des patients et dans le liquide de bulle, de plus cette chimiokine reste fortement présente dans le sérum des patients qui rechutent. D’autre part nous avons montré pour la première fois que l’orientation des macrophages dans la PB est contrôlée dans un premier temps par les sérums de patients, qui préactivent les macrophages dérivés de monocytes, puis par le liquide de bulle qui différencient les macrophages vers une polarisation M2. Nous avons également montré que seulement les neutrophiles et les monocytes isolés de patients atteints de PB, mais pas leur lymphocytes, répondaient à une stimulation par CXCL10 par une augmentation de leur sécrétion en MMP-9 via l’activation des voies de signalisation ERK1/2, P38 et PI3K. Cette expression de la MMP-9 a été associée à une polarisation de type M2 dans les macrophages issus de monocytes de patients. Enfin, nous avons montré l’importance des activateurs sélectifs des récepteurs des glucocorticoïdes, tels que le composé A, qui était aussi efficace que le traitement avec la méthylprénisolone dans l'inhibition de la MMP-9, mais avec des effets distincts sur l'expression des marqueurs phénotypiques de macrophages. / Bullous pemphigoid (BP) is the most common and serious autoimmune bullous dermatosis characterized by inflammatory cascade involving many cytokines and inflammatory cells, with the release of proteases (MMP-9, HLE) leading to the formation of the blister. The objective of this study is to characterize the inflammatory cascade by analyzing the contribution of the CXCL10 chemokine and inflammatory cells such as macrophages in the pathophysiology associated with the disease. Our results showed that CXCL10 is present at significant levels in the serum of patients and in the blister fluid, this chemokine remains strongly present in the serum of patients who relapse. On the other hand we have shown for the first time that macrophages polarization in BP is controlled by the sera of patients, which activate macrophages derived from monocytes, and then blister fluid differentiate macrophages in an M2 polarization. We also showed that only neutrophils and monocytes isolated from patients, but not the lymphocytes responding to stimulation by CXCL10 by an increase in secretion of MMP-9 via the activation of signaling pathways ERK1 / 2, P38 and PI3K. This expression of MMP-9 has been associated with an M2 macrophages polarization derived from monocytes of patients. Finally, we have demonstrated the importance of selective glucocorticoid receptor activators, such as Compound A, which was as effective as treatment with methylprednisolone in inhibiting MMP-9, but with distinct effects on the expression of phenotypic markers of macrophages.

A precious treasure in traditional Chinese medicine (TCM), acupuncture played a vital and irreplaceable role in contributing to people’s health in the thousands of years of Chinese history, and in 2010 was officially added to the “Representative List of the Intangible Cultural Heritage of Humanity” by the United Nations. Because of the side-effects of long-term drug therapy for pain, and the risks of dependency, acupuncture has been widely accepted as one of the most important alternative choice therapies for treating varieties of acute and chronic pain-related disorders. The clinical application and scientific mechanism research of acupuncture have therefore increased intensively in the last few decades. Besides hand acupuncture, other treatment approaches e.g. electroacupuncture (EA) have been widely accepted and applied as an important acupuncture-related technique for acupuncture analgesia (AA) research. The involvement of opioid peptides and receptors in acute AA has been shown via pre-EA application of opioid receptor/peptide antagonists. However, existing publications still cannot illuminate the answer to the following question: how does sustained antinociception happen by EA treatment? The hypothesis of opioid peptide-mediated tonic AA might be able to answer the question. In the first part of this thesis, the institution of a reproducible acupuncture treatment model as well as the endogenous opioid-related mechanisms was demonstrated. An anatomically-based three-dimensional (3D) rat model was established to exhibit a digital true-to-life organism, accurate acupoint position and EA treatment protocol on bilateral acupoint GB-30 Huantiao. The optimal EA treatment protocol (100 Hz, 2-3 mA, 0.1 ms, 20 min) at 0 and 24 h after induction of inflammatory pain by complete Freund’s adjuvant (CFA) on conscious free-moving rats was then established. EA elicited significant sustained mechanical and thermal antinociception up to 144 h. Post-EA application of opioid receptors (mu opioid receptor, MOR; delta opioid receptor, DOR) antagonists naloxone (NLX) and naltrindole (NTI), or opioid peptide antibodies anti-beta-endorphin (anti-END), met-enkephalin (anti-ENK) or -dynorphin A (anti-DYN) could also block this effect at a late phase (96 h) of CFA post-EA, which suggested opioid-dependent tonic analgesia was produced by EA. Meanwhile, EA also reduced paw temperature and volume at 72-144 h post CFA indicating anti-inflammatory effects. Nociceptive thresholds were assessed by paw pressure threshold (Randall-Sellito) or paw withdrawal latency (Hargreaves) and an anti-inflammatory effect was evaluated by measurement of plantar temperature and volume of inflamed paw. The second part of the thesis further suggests the correlation between the chemokine CXCL10 (= interferon-gamma inducible protein 10, IP-10) and opioid peptides in EA-induced antinociception. Based on a comprehensive Cytokine Array of 29 cytokines, targeted cytokines interleukin (IL)-1alpha, interleukin (IL)-1beta, tumor necrosis factor (TNF)-alpha, interleukin (IL)-4, interleukin (IL)-13, interferon (IFN)-gamma as well as CXCL10 were selected and quantified by enzyme-linked immunosorbent assay (ELISA), and real time reverse transcription-polymerase chain reaction (RT-PCR) quantification confirmed upregulation of CXCL10 mRNA at both 72 and 96 h. The following hyperalgesic assessment suggested the antinociceptive effect of CXCL10. The double immunostaining localizing opioid peptides with macrophages expressed the evident upregulation of CXCR3-receptor of CXCL10 in EA treated samples as well as the significant upregulation or downregulation of opioid peptides by repeated treatment of CXCL10 or antibody of CXCL10 via behavioral tests and immune staining. Subsequent immunoblotting measurements showed non-alteration of opioid receptor level by EA, indicating that the opioid receptors did not apparently contribute to AA in the present studies. In vitro, CXCL10 did not directly trigger opioid peptide END release from freshly isolated rat macrophages. This might implicate an indirect property of CXCL10 in vitro stimulating the opioid peptide-containing macrophages by requiring additional mediators in inflammatory tissue. In summary, this project intended to explore the peripheral opioid-dependent analgesic mechanisms of acupuncture with a novel 3D treatment rat model and put forward new information to support the pivot role of chemokine CXCL10 in mediating EA-induced tonic antinociception via peripheral opioid peptides. / Als wertvoller Schatz in der traditionellen chinesischen Medizin (TCM) spielt die Akupunktur eine wichtige und unersetzliche Rolle für die Gesundheit der Menschen in der über tausendjährigen Geschichte von China und wurde im Jahr 2010 offiziell in das "Weltkulturerbe" der Vereinten Nationen aufgenommen. Aufgrund der Nebenwirkungen von Langzeittherapien zur Schmerzbehandlung und dem Risiko der Abhängigkeit wird Akupunktur weithin als eine wichtigste Alternative für die Behandlung von akuten und chronischen Schmerzen eingesetzt. Die klinische Anwendung und Forschung in der Akupunktur wurden in den letzten Jahrzehnten intensiv vorangetrieben. Neben Handakupunktur gibt es noch andere Behandlungsmöglichkeiten, wie z.B. die Elektroakupunktur (EA). EA ist vor allem eine allgemein akzeptierte und wichtige akupunkturbezogene Technik für die Akupunkturanalgesie (AA) in der Forschung. Die Beteiligung von Opioidpeptiden und Opioidrezeptoren in der akuten AA wurde mittels Anwendung von Opioidrezeptorantagonisten/Opioidpeptidantikörpern appliziert vor EA gezeigt. Nach dem aktuellen Forschungsstand kann man jedoch nicht die Frage beantworten, wie die längerfristige (tonische) Antinozizeption nach EA-Behandlung funktioniert. Mit einer Hypothese zur Opioidpeptid vermittelten tonischen AA könnte man die Frage hierzu beantworten. In der vorliegenden Arbeit wurden in einem Modell der Akupunktur zum ersten Mal endogene Opioid-vermittelte Mechanismen reproduzierbar nachgewiesen. Es wurde ein dreidimensionales (3D) anatomisch-basiertes Rattenmodell entworfen, um am wachen Tier ein genaue Akupunkturbehandlung (EA) an den Akupunkten GB-30 Huantiao beidseitig durchzuführen. Darüber hinaus wurde ein optimiertes Behandlungsprotokoll von EA (100 Hz, 2-3 mA, 0.1 ms, 20 min) bei Ratten 0 und 24 h nach intraplantarer Injektion von komplettem Freunds Adjuvans (CFA) etabliert. Nozizeptive Schwellen wurden mittels Pfotendruckschwelle (Randall-Sellito) oder Pfotenrückzugslatenzzeit (Hargreaves) gemessen und die entzündungshemmende Wirkung durch Messung der Pfotentemperatur und Volumen der entzündeten Pfote ausgewertet. EA bewirkte eine signifikante mechanische und thermische Antinozizeption, welche bis zu 144 h anhielt. Die antinozizeptive Wirkung durch EA war nach Injektion von Opioidrezeptorantagonisten (mu opioid receptor, MOR; delta opioid receptor, DOR) Naloxon (NLX) und Naltrindol (NTI) oder Antikörpern gegen die Opioidpeptide beta-Endorphin (anti-END), Met-Enkephalin (anti-ENK) oder Dynorphin A (anti-DYN) während der späten Entzündungsphase (96 h) mit CFA blockierbar. Dies lässt auf eine durch EA induzierte tonische Analgesie schließen. Darüber hinaus reduzierte EA auch die erhöhte Pfotentemperatur und das erhöhte Pfotenvolumen, welche sehr typisch ist für eine 96-144 h Entzündung mit CFA. Dies spricht für eine entzündungshemmende Wirkung von EA. Nachfolgend wurde die Beteiligung von Opioidpeptiden und dem Chemokin CXCL10 (= Interferon-gamma induziertes Protein 10, IP-10) sowie die Korrelationen zwischen beiden geklärt. Basierend auf umfangreichenden Zytokinarrays mit 29 Zytokinen, wurden die Zytokine Interleukin (IL)-1alpha, Interleukin (IL)-1beta, Tumornekrosefaktor (TNF)-alpha, Interleukin (IL)-4, Interleukin (IL)-13, Interferon (IFN)-gamma und CXCL10 gezielt ausgewählt und im Enzym Immunoassay (ELISA) sowie durch eine Echtzeit Reverse Transkription-Polymerase Kettenreaktion (RT-PCR) quantifiziert. Die Quantifizierung auf mRNA-Ebene zeigte eine Hochregulation von CXCL10 bei 96 h und zum früheren Zeitpunkt von 72 h. Nachfolgende Schmerzschwellenmessungen wiesen auf eine antinozizeptive Wirkung von CXCL10 hin. Eine Doppelimmunfärbung zeigte die Lokalisation von Opioidpeptiden in Makrophagen. Nachweislich wurde die Expression des CXCR3-Rezeptor von CXCL10 in EA behandelten Pfoten erhöht. Ebenso kam es zu einer signifikanten Hochregulation von Opioidpeptiden durch wiederholte Behandlung mit CXCL10 bzw. Herunterregulation der Opioidpeptide nach wiederholter Behandlung mit anti-CXCL10 Antikörper. Dies wurde sowohl in Verhaltenstests als auch in der Immunfärbung zu beobachtet. Immunoblotting zeigte keine Veränderung der Expression von Opioidrezeptoren nach EA, was schließen lässt, dass die Menge an Opioidrezeptoren in den vorliegenden Untersuchungen keine Rolle spielen. Da CXCL10 in vitro keine direkten Effekt auf die Freisetzung des Opioidpeptides beta-END aus frisch isolierten Rattenmakrophagen hat, liegt die Vermutung nahe, dass CXCL10 in vitro eine indirekte Rolle als Mediator zukommt und indirekt die Opioidpeptidfreisetzung aus Makrophagen in entzündlichen Gewebe stimuliert wird. Zusammenfassend wurden in dem hier vorgestellten Projekt die Opioidpeptid-abhängigen analgetischen und peripheren Mechanismen der Akupunktur mit einem neuartigem 3D-Behandlungsmodell der Ratte untersucht und eine Schlüsselrolle des Chemokins CXCL10 bei der Vermittlung der EA-induzierten tonischen Antinozizeption in Abhändigkeit von peripheren Opioidpeptiden. (Translated by Dr. Dagmar Hackel and revised by Priv.-Doz. Dr. Heike Rittner)

Lymphocytic infiltration is a feature of basal breast cancer tumors. CXCL10 is a chemokine that was found expressed higher in basal tumor RNA compared to estrogen receptor positive tumor RNA. Both CXCL10 and its receptor CXCR3 were expressed in familial breast cancer tissues, a proportion of which have a basal phenotype. CXCL10 expression was associated with lymphocytic infiltration, and with CXCR3 expression. CXCL10 ligand and receptor were overexpressed individually or together in the human MCF7 cell line. Recombinant human CXCL10 was found to dose dependently decrease cell proliferation. CXCR3 and CXCL10-CXCR3 expressing cells had the potential for increased migration independent of CXCL10 concentration. Co-expression of both genes increased proMMP-2 levels, and conditioned media from one of these clones chemoattracted more of the CXCR3 clones, CXCL10-CXCR3 clones, and CD4+ T-lymphocytes. CXCL10 neutralization suggested that CXCL10 could play a role in this chemoattraction, though it is likely not the only factor involved.

Lymphocytic infiltration is a feature of basal breast cancer tumors. CXCL10 is a chemokine that was found expressed higher in basal tumor RNA compared to estrogen receptor positive tumor RNA. Both CXCL10 and its receptor CXCR3 were expressed in familial breast cancer tissues, a proportion of which have a basal phenotype. CXCL10 expression was associated with lymphocytic infiltration, and with CXCR3 expression. CXCL10 ligand and receptor were overexpressed individually or together in the human MCF7 cell line. Recombinant human CXCL10 was found to dose dependently decrease cell proliferation. CXCR3 and CXCL10-CXCR3 expressing cells had the potential for increased migration independent of CXCL10 concentration. Co-expression of both genes increased proMMP-2 levels, and conditioned media from one of these clones chemoattracted more of the CXCR3 clones, CXCL10-CXCR3 clones, and CD4+ T-lymphocytes. CXCL10 neutralization suggested that CXCL10 could play a role in this chemoattraction, though it is likely not the only factor involved.