Aachen, Techn. Hochsch., Diss., 2002. / Computerdatei im Fernzugriff.

Aachen, Techn. Hochsch., Diss., 2002. / Computerdatei im Fernzugriff.

Diseases of the eye are common problems in dogs and can be painful, therapeutically challenging and distressing to both patient and owner. Ocular disease can result in visual impairment, vision loss or, in severe cases, enucleation. Much of the tissue damage that occurs during ocular disease results from the activity of secreted proteins that control processes such as inflammation, blood vessel growth, cellular proliferation and cellular death. These proteins are called growth factors and cytokines. The purpose of this study was to examine the expression and effects of one such cytokine, interleukin 11, in the canine eye. Interleukin 11 was found to be constitutively expressed in all layers of the canine cornea at both the protein and message level. Treatment of primary corneal cell cultures with TGF-β1 resulted in a statistically significant increase in IL-11 expression in the corneal epithelium, fibroblasts and endothelium. In order to study the biological effects of IL-11 on the canine cornea, a presumptive corneal epithelial cell line (DCE39R) was created. Such a cell line represents an important, and previously unavailable, tool to study the effects of cytokines on the corneal epithelium itself as well as create corneal constructs for research and therapeutic work. Research done using this cell line demonstrated that IL-11 has a pro-migratory effect on the corneal epithelial cells and provides a cytoprotective effect in the case of nutrient deprivation. It however, does not induce proliferation of the canine corneal epithelial cells. This study serves as an important building block for future research on the effect of IL-11 in the canine cornea, and it also provides an important tool for future research: the cell line DCE39R. / Pet Trust

Techn. Hochsch., Diss., 2002--Aachen.

Intravenous immunoglobulin (IVIg) has been used to treat a variety of autoimmune disorders including multiple sclerosis (MS); however, its mechanism of action remains elusive. Our results demonstrate a novel finding wherein IVIg treatment induces a dramatic surge (>1000-fold increase) in the levels of IL-11 in the circulation and that the liver is the organ of increased IL-11 transcription. Furthermore, we show that IL-11Rα knockout mice, although initially protected, become resistant to protection by IVIg during experimental autoimmune encephalomyelitis (EAE) and develop disease with a similar incidence and severity as control-treated IL-11Rα-/- mice. The inability of IVIg to prevent EAE in IL-11Rα-/- mice correlated with a failure of this agent to decrease IL-17 production by myelin-reactive T-cells in the draining lymph nodes. Finally, we show that IL-11 can directly inhibit IL-17 production by lymph node cells in culture. Together, these results implicate IL-11 as an important immune effector of IVIg in the amelioration of EAE.

Intravenous immunoglobulin (IVIg) has been used to treat a variety of autoimmune disorders including multiple sclerosis (MS); however, its mechanism of action remains elusive. Our results demonstrate a novel finding wherein IVIg treatment induces a dramatic surge (>1000-fold increase) in the levels of IL-11 in the circulation and that the liver is the organ of increased IL-11 transcription. Furthermore, we show that IL-11Rα knockout mice, although initially protected, become resistant to protection by IVIg during experimental autoimmune encephalomyelitis (EAE) and develop disease with a similar incidence and severity as control-treated IL-11Rα-/- mice. The inability of IVIg to prevent EAE in IL-11Rα-/- mice correlated with a failure of this agent to decrease IL-17 production by myelin-reactive T-cells in the draining lymph nodes. Finally, we show that IL-11 can directly inhibit IL-17 production by lymph node cells in culture. Together, these results implicate IL-11 as an important immune effector of IVIg in the amelioration of EAE.

Techn. Hochsch., Diss., 2003--Aachen.

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior / Interleukin 11 (IL-11) is a multifunctional cytokine in the IL-6 type family of long-chain helical cytokines, which modulates the proliferation, differentiation and maturation of various types of hematopoietic cells. Recombinant human interleukin-11 (rhIL-11) produced by DNA technology in Escherichia coli is currently being used worldwide for the prevention of thrombocytopenia and to reduce the need for platelet transfusions after myelosuppressive chemotherapy in patients with nonmyeloid malignancies. A stability-indicating reversed-phase liquid chromatography (RP-LC) method was validated for the assessment of recombinant human interleukin-11 (rhIL-11) in biopharmaceutical formulations. The RP-LC method was carried out on a Jupiter C4 column (250 mm x 4.6 mm i.d.), maintained at 25ºC. The mobile phase A consisted of 0.1% TFA and the mobile phase B was acetonitrile with 0.1% TFA, run as follows: time 0 to 0.1 min 40% of B; from 0.1 to 30 min linear up to 65% of B; from 30.01 to 31 min linear down to 40% of B, maintained up to 40 min. The flow rate was 1 mL/min, and using photodiode array (PDA) detection at 214 nm. Chromatographic separation was obtained with a retention time of 27.6 min, and was linear over the concentration range of 1 200 μg/mL (r2 = 0.9995). The limits of detection and quantitation were 0.34 and 1.12 μg/mL, respectively. Specificity was established in degradation studies, which also showed that there was no interference of the excipients. The accuracy was 100.22% with bias lower than 1.25%. Moreover, the in vitro cytotoxicity test of the degraded products showed non-significant differences (p>0.05). The proposed method was applied to the assessment of rhIL-11 and related proteins in biopharmaceutical dosage forms, and the results were correlated to those of a bioassay, showing a higher mean difference of the estimated content/potencies of 2.60% for the RP-LC method, aiming to establish new alternatives to monitor stability, improve quality control and thereby assure therapeutic efficacy of the biological medicine. / A interleucina 11 (IL-11) é uma citocina multifuncional que pertence a família da interleucina- 6 e estimula a proliferação, diferenciação e maturação de células hematopoiéticas. A interleucina-11 humana recombinante (rhIL-11) é produzida pela tecnologia do DNA em Escherichia coli e é usada clinicamente para a prevenção de trombocitopenia grave e redução da necessidade de transfusão de plaquetas após quimioterapia mielossupressiva em pacientes com neoplasias malignas não mielóides. No presente trabalho foi desenvolvido e validado método por cromatografia líquida em fase reversa (CL-FR) para a avaliação de rhIL-11 em formulações de produtos farmacêuticos. Utilizou-se coluna Júpiter C4 (250 mm x 4,6 mm d.i.), mantida a 25ºC. A fase móvel A foi constituída de TFA 0,1% e a fase móvel B de acetonitrila com 0,1% TFA, eluídas no seguinte gradiente: 0 0,1 min, 40% de B; 0,1 30 min, 40 65% de B; 30,01 a 31 min, 65 40% de B, mantendo-se nesta proporção até 40 min. Utilizou-se vazão de 1 mL/min e detector de arranjo de diodos (DAD) a 214 nm. A eluição cromatográfica foi obtida no tempo de 27,6 min, sendo linear na faixa de concentração de 1 200 μg/mL (r2 = 0,9995). Os limites de detecção e quantificação foram 0,34 e 1,12 μg/mL, respectivamente. A especificidade foi avaliada em estudos de degradação, que também demonstraram que não houve interferência dos excipientes. A exatidão foi 100,20%, com bias inferior a 1,25%. Além disso, realizou-se o teste de citotoxicidade in vitro das formas degradadas, as quais não apresentaram diferença significativa em relação a forma intacta (p>0,05). O método proposto foi aplicado para a avaliação da potência de rhIL-11 e de proteínas relacionadas em formulações farmacêuticas, e os resultados foram comparados com o bioensaio, observando-se diferenças das médias de conteúdo/potência 2,60% superiores para o método por CL-FR. Contribuíu-se assim para estabelecer procedimentos que aprimoram o controle da qualidade, garantindo a segurança e eficácia terapêutica do produto biotecnológico.

L’âge et le sexe biologique influencent plusieurs attributs du système immunitaire humain, tels la capacité à monter des réponses efficaces suite à la vaccination ou à une infection et le risque de développer une maladie auto-immune. Les interleukines sont des messagers moléculaires importants dans la coordination des réponses immunes et leur perception par les diverses populations de cellules mononucléées du sang périphérique (PBMC) dépend entre autres de la distribution de leurs récepteurs respectifs sur celles-ci. Nous montrons ici par cytométrie en flux réalisée sur des PBMC fraîchement isolées de sang périphérique d’adultes en santé que l’âge et le sexe sont des facteurs agissant sur la répartition des récepteurs aux interleukines 6, 11 et 27 de la famille IL-6. Les comparaisons statistiques réalisées entre quatre groupes constitués de 10 participants par sexe et par groupe d’âge (20 à 30 ans et 40 à 65 ans) indiquent plusieurs variations. Des différences ont été observées pour la présence de ces récepteurs en termes de proportion de cellules et de densité de molécules exprimées par cellule. Par exemple, les pourcentages de cellules immunes des jeunes hommes arborant la chaine IL-6 récepteur alpha étaient plus modestes pour plusieurs populations immunes en comparaison aux autres groupes. Toutefois, les proportions de cellules immunes des femmes plus âgées exprimant la chaine du récepteur IL-11 alpha étaient plus importantes en comparaison aux autres groupes. Le savoir généré par la présente étude ajoute à la connaissance grandissante de la régulation immunitaire chez l’humain par les interleukines en fonction de l’âge et du sexe en fournissant des données extensives y compris pour des cytokines (e.g. IL-11) qui ont été moins étudiées jusqu’à maintenant. Ces constats sont pertinents dans le cadre de l'étude de l'immunobiologie, par exemple en permettant de discuter du rôle de cytokines comme l'IL-6 dans l'état inflammatoire des individus ayant la capacité d'influencer entre autres la trajectoire d'un vieillissement sain. / Age and biological sex shape numerous properties of the human immune system, such as the capacity to generate an efficient response to a vaccine or an infection and the risk of developing an autoimmune disease. Interleukins are molecular messengers of importance in the coordination of immune responses and their perception by peripheral blood mononuclear cells (PBMC) depends on the distribution of their respective receptors. We show here, using flow cytometry on freshly isolated PBMC from the peripheral blood of healthy adults, that age and sex impact the distribution of the receptors of interleukins 6, 11, and 27 from the IL-6 family. Statistical comparisons executed on four groups consisting of 10 participants per sex and age group (20 to 30 years old and 40 to 65 years old) indicate that these receptors vary in expression in terms of immune cell types and density per cell. For example, the percentages of immune cells in young men displaying the IL-6 receptor alpha chain were more modest for several immune populations compared with the other groups. However, the proportions of immune cells in older women expressing the IL-11 receptor alpha chain were greater compared with the other groups. The knowledge generated in this study will add to the increasing understanding of the immune regulation mediated by interleukins in humans according to age and sex by providing extensive data, especially for cytokines (e.g. IL-11) that have been less studied so far. These findings are of importance in the area of immunobiology, by eliciting discussion about the role of cytokines such as IL-6 in the inflammatory state of an individual, which is susceptible to impact healthy ageing.