Universiẗat, Diss., 2008--Giessen.

Techn. Hochsch., Diss., 2000--Aachen.

Universiẗat, Diss., 2005--Giessen.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / O cÃncer à uma doenÃa grave que atinge milhares de pessoas no mundo, possuindo altos Ãndices de morbidade e mortalidade. Atualmente existem diversos alvos terapÃuticos no tratamento do cÃncer, um deles reside no fato de inibir a replicaÃÃo do DNA impossibilitando a cÃlula tumoral de se duplicar como à o caso do 5-fluoruracil (5-FU).O objetivo do trabalho foi avaliar o efeito antiinflamatÃrio do peptÃdeo mimÃtico da ApoE (COG 133) em camundongos Swiss desafiados pela mucosite intestinal induzida pelo 5-FU. Utilizamos camundongos Swiss machos com peso de 30g, sendo desafiados com injeÃÃo (dose Ãnica) intraperitoneal de 5-FU (450 mg/kg).Alguns animais foram tratados com diferentes doses (0,3, 1,0 e 3,0 ÂM) do peptÃdeo mimÃtico da apolipoproteÃna E (ApoE) COG133 por via intra-peritoneal, apÃs o desafio com 5-FU. Animais tratados com PBS foram usados como controles. Os camundongos foram sacrificados com soluÃÃo de deslocamento cervical 3 dias apÃs o desafio com 5-FU.Algumas amostras de intestino foram congeladas imediatamente em nitrogÃnio lÃquido e em seguida armazenadas em freezer a -80ÂC para biologia molecular. Outras amostras foram fixadas em formaldeÃdo para processamento histolÃgico.Monitoramos o peso corporal dos animais e fizemos leucometria a partir de coleta retrorbital de sangue para avaliaÃÃo do efeito citotÃxico mielosupressor do 5-FU nos grupos experimentais.Avaliamosos parÃmetros morfomÃtricos de altura de vilo e profundidade de cripta em segmentos do duodeno. Para a detecÃÃo de proteÃnas de interesse, utilizamos o Western blot com a utilizaÃÃo dos seguintes anticorpos IL-1&#946;, TNF-&#945; e iNOS. Para o RT-PCR, avaliamos os seguintes primers IL-1&#946;, TNF-&#945; e iNOS. No ensaio de citocinas por ELISA, utilizamos os seguintes anticorpos IL-1&#946;, IL-10, TNF-&#945;. Nas anÃlises morfomÃtricas de duodeno, encontramos uma grande reduÃÃo na altura de vilos e profundidade de criptas nosanimais tratados com 5-FU, que foi reduzida com com a utilizaÃÃo do peptÃdeo COG133 na dose 3,0 ÂM (p<0,001).Nas anÃlises histopatolÃgicas, observamos um intensoinfiltrado inflamatÃrio nos animais do grupo que foi injetadocomo 5-FU, que foi parcialmente revertido pela administraÃÃo do peptÃdeo COG133.Os dados de ELISA evidenciaram um aumento da quantidade de citocinas prÃ-inflamatÃrias IL-1&#946; (p<0,05), TNF-&#945; (p<0,05) e uma reduÃÃo da citocina antiinflamatÃria IL-10 (p<0,05), quando comparados ao controle salina, em duodenos de animais que foram injetados com 5-FU. A administraÃÃo do peptÃdeo COG 133 reduziu os nÃveis de IL-1&#946; de maneira estatisticamente significante (p<0,05 1&#956;M; p<0,001 3&#956;M), TNF-&#945; (p<0,001 na dose de 1,0 &#956;M); p<0,001 na dose de 3&#956;M) e para IL-10 (p<0,001 na dose de 3&#956;M).Dados de Western blot evidenciam que o peptÃdeo na dose de 3&#956;M reduziu de maneira estatisticamente significante a expressÃo de citocinas como IL-1&#946; (p<0,001), TNF-&#945; (p<0,05) e do mediador inflamatÃrio iNOS (p<0,05) no duodeno. Pelo protocolo de RT-PCR,encontramos uma elevaÃÃo na expressÃo do transcrito para TNF-&#945; e iNOS nos animais injetados com 5-FU, o que foi parcialmente diminuÃdo pela administraÃÃo do peptÃdeo na dose de 3&#956;M para TNF-&#945; (p<0,05) e iNOS (p<0,05)pelo ANOVA.A partir dos nossos achados, podemos concluir que o peptÃdeo mimÃtico da ApoE (COG 133) possui uma aÃÃo anti-inflamatÃria, especialmente no segmento duodeno, visto que reduz a expressÃo de citocinas prÃ-inflamatÃrias de animais desafiados, dessa forma sugerindo sua utilizaÃÃo em humanos com mucosite intestinal sob quimio/radioterapia.

Protein-protein interactions play an important role in nearly all processes of the living cells and the function of many proteins is dependent on their specific interactions with other biomolecules. A reliable tool to modulate these interactions would be invaluable for the development of molecules suitable for diagnostics, medicine, and biotechnology. In this work, we aimed to study the specificity of interactions in the model system of Interferon gamma receptor 1 (IFNgR1) and its natural ligand Interferon gamma (IFNg), important in innate immunity. We searched for mutations within the interferon receptor molecule IFNgR1 to modulate (increase as well as decrease) its affinity to IFNg by in silico analysis of the existing crystal structures of the complex between IFNgR1 and IFNg. We modeled amino acid substitutions and gauged how they influenced the interaction using empirical force field implemented in software FoldX. All selected promising IFNgR1 variants were expressed in Escherichia coli, purified to homogeneity, characterized, and kinetics of their interactions with IFNg was measured by Surface Plasmon Resonance (SPR). The first set of IFNgR1 variants included mutations on the interface of the IFNg/IFNgR1 complex. According to our SPR measurements, the affinity of most of these receptor...

Interleukin-7 (IL-7) increases lymphocyte numbers, a critical feature of immune reconstitution, through mechanisms that are still poorly understood. Part of the problem is that IL-7 is produced in limited amounts by non-lymphoid cells, making in vivo studies of the cytokine's activity a challenge. To overcome this, we developed an in vitro system by which lymphocytes from secondary immune organs could be cultured to produce IL-7 responsive cells. Using this method, we showed that CD8hiCD44hi T cells accumulate in culture with IL-7 from a population of lymph node or splenic cells. These results were validated when a similar lymphocyte subset was found in mice expressing a constitutively active form of STAT5b, a key transducer of IL-7 signals. Interestingly, IL-7-expanded cells also up regulated the activation marker, CD69. The IL-7-derived CD44hiCD69hi cells were not generated from naïve cells, but expanded from an existing population, since culture in IL-7 of naïve lymphocytes from OT-1/Rag1-/- mice did not produce CD44hiCD69hi cells. Using the in vitro culture system to study lymphocytes from mice deficient in the apoptotic protein, BIM, we were able to attribute the expansion of CD8hiCD44hiCD69hi T cells to the proliferative and not survival activity of IL-7. The in vitro culture system provides an important new methodology to examine the activities of this essential as well as immunotherapeutic cytokine.

Die Pathophysiologie der PNP wie auch die Entstehung der oft assoziierten neuropathischen Schmerzen ist unklar. Gleichzeitig gibt es bislang keine geeigneten Biomarker, die die oft komplizierte Differentialdiagnose vereinfachen können. Einige Tiermodelle und klinische Studien lieferten bereits Hinweise auf die entscheidende Rolle pro- und anti-inflammatorischer Zytokine in diesen Prozessen. Ziel unserer Studie war es, die systemische Genexpression pro- und anti-inflammatorischer Zytokine in einer großen Kohorte von Patienten mit PNP verschiedener Ätiologie zu charakterisieren. Insgesamt konnten 111 PNP-Patienten und 38 gesunde Kontrollpersonen prospektiv rekrutiert werden. Nach Isolation von PBMC aus Blutproben von 97 Patienten wurde die Genexpression der pro-inflammatorischen Zytokine TNF, IL1, IL2, IL6, IL8 und der anti-inflammatorischen Zytokine IL4 und IL10 mittels qRT-PCR bestimmt. Bei 47 Patienten und 12 Kontrollen wurde zudem die IL6-, IL-8- und TNF-Zytokinproduktion von PBMC in vitro nach Stimulation durch LPS mittels ELISA untersucht. Hauptbefund war ein pro-inflammatorisches Zytokinprofil der PNP-Patienten mit höherer Genexpression von IL1, IL2, IL8 und TNF im Vergleich zu den gesunden Kontrollen. Im Falle der entzündlichen Neuropathien konnte zudem eine niedrigere Genexpression von IL10 im Vergleich zu Gesunden nachgewiesen werden. Sowohl schmerzhafte als auch schmerzlose Verlaufsformen wiesen ein pro-inflammatorisches Zytokingenexpressionsprofil im Vergleich zu Gesunden auf, das bei schmerzhaften PNP deutlich mehr beteiligte pro-inflammatorische Zytokine umfasste; relevante Unterschiede zwischen den PNP-Patienten mit und ohne Schmerz sowie der diagnostischen Subgruppen fanden sich nicht. Eine niedrigere Stimulationsschwelle der PBMC lag bei PNP-Patienten im Vergleich zu Gesunden nicht vor. Insgesamt erscheint die Rolle einzelner Zytokine als systemische Biomarker für die Differenzierung verschiedener PNP-Formen bzw. bezüglich neuropathischen Schmerzes aufgrund einer niedrigen Spezifität deutlich eingeschränkt. Dennoch sprechen unsere Ergebnisse für eine mögliche Rolle eines pro-inflammatorischen Milieus bei der Entstehung bzw. des Verlaufes verschiedener entzündlicher und nicht-entzündlicher Neuropathien und neuropathischen Schmerzes. / Distinct cytokine expression patterns have been reported in biomaterial of patients with polyneuropathies (PNP). We investigated gene expression profiles of pro- and anti-inflammatory cytokines in peripheral blood mononuclear cells (PBMC) of patients with neuropathies of different etiologies. We prospectively studied 111 patients with neuropathies and compared data between diagnostic subgroups and healthy controls. Gene expression of a panel of pro- and anti-inflammatory cytokines was analyzed (interleukin-1 [IL-1], IL-2, IL-6, IL-8, and tumor necrosis factor-alpha [TNF], IL-4 and IL-10) in PBMC samples of 97 patients and 38 healthy controls. Furthermore, protein levels of IL-6, IL-8, and TNF were measured in supernatant of PBMC stimulated with lipopolysaccharide (LPS). PNP were associated with higher PBMC gene expression of IL-1 (p<0.05), IL-2 (p<0.05), IL-8 (p<0.001), and TNF (p<0.01) compared to healthy controls. Inflammatory neuropathies were associated with higher gene expression of IL-8 (p<0.001) and TNF (p<0.05) and lower gene expression of IL-10 (p<0.05) compared to healthy controls. More pro-inflammatory cytokines were elevated in painful neuropathy (IL-1, IL-2 [p<0.05], IL-8 [p<0.001] and TNF [p<0.05]) than in painless neuropathy (IL-8 [p<0.01] and TNF [p<0.01]) compared to healthy controls. Disease duration positively correlated with IL-6 gene expression (p<0.01). Supernatant protein levels of IL-6, IL-8, and TNF did not differ between groups. Conclusion: Systemic gene expression of pro-inflammatory cytokines is increased in patients with neuropathies and may be influenced by the presence of neuropathic pain.

Interleukin-7 (IL-7) is an essential lymphocyte growth factor required for the survival and proliferation of mature T-cells. As a therapeutic agent, IL-7 has the potential to restore T-cell numbers following immune depletion and to promote immunity against cancers. While the survival function of IL-7 is well established, less is known about how it supports T-cell expansion, a critical feature of the immune response. To study the biological effects of IL-7 on T-cell growth, we developed an in vitro culture technique to expand T-cells ex vivo. A significant finding from our studies is that IL-7 did not induce the expansion of all T-cells, indicating that there are inherent differences in the response of individual T-cell subsets to IL-7. Culture with high doses of IL-7 ( > 150 ng/ml) preferentially expanded CD8 T-cells, but lead to the dramatic loss of CD4 T-cells which favored growth in lower dosages of IL-7 ( > 10 ng/ml). This effect was due to the regulation of LCK, a kinase predominantly associated with the CD4 co-receptor. We found that transgenic expression of the CD4 co-receptor onto CD8 T-cells promoted their growth in lower concentrations of IL-7. Conversely, inhibition of LCK activity in CD4 T-cells restored their responsiveness to high doses of IL-7 as indicated by the activation of the transcription factor STAT5, in a manner similar to CD8 T-cells. Interestingly, not all CD8 T-cells expanded in high doses of IL-7 and this effect was specific to CD8 T-cells that expressed an activated memory phenotype. We found that IL-7 promoted the proliferation of CD8 T-cells through Cdc25A, a phosphatase required for cell cycle progression. Expression of a constitutively active Cdc25A could maintain T-cell survival and proliferation in the absence of IL-7, demonstrating that Cdc25A is a crucial transducer of IL-7 growth signals. Inhibition of Cdc25A was sufficient to decrease proliferation and down-regulate the expression of activation/ memory markers on CD8 T-cells in the presence of IL-7. Upon further study, we identified a novel role for IL-7 through Cdc25A in the regulation of CD62L, an adhesion molecule required for lymph node entry. Culture with high doses of IL-7 down-regulated the expression of CD62L, suggesting that high doses of IL-7 could affect the ability of T-cells to enter or re-enter the lymph nodes. Collectively, our findings demonstrate that IL-7 administration at the supraphysiological doses currently used in the clinical trials could have a negative impact on the growth of CD4 T-cells and the homing of CD8 T-cells to the lymph nodes, effects which can impede the generation of an effective immune response.

No description available.

No description available.