Dissertations / Theses on the topic 'IL-1ra'

Le malattie autoinfiammatorie sistemiche si manifestano quando il sistema immunitario viene attivato in maniera incontrollata. Un sottogruppo cardinale di queste malattie comprende rare febbri periodiche caratterizzate dalla produzione disregolata della citochina proinfiammatoria interleuchina-1 (IL-1). Non esiste una cura per queste condizioni. Anakinra, il farmaco rappresentante la ricombinante dell'antagonista del recettore IL-1 (IL-1RA), è la terapia principale per questi pazienti. Tuttavia, l’anakinra ha una breve emivita e una scarsa distribuzione dei tessuti. I pazienti gravi rispondono in modo inadeguato e i loro sintomi non migliorano. Pertanto, è necessaria una terapia duratura che oltrepassi la somministrazione continua di farmaci e garantisca una risoluzione soddisfacente dell'infiammazione dei tessuti. In questo progetto di dottorato, abbiamo affrontato l'urgenza di sviluppare un trattamento efficace per queste malattie, basato su cellule staminali ematopoietiche che producono costitutivamente la proteina umana IL-1RA utilizzando un approccio di trasferimento genico mediato da vettori lentivirali (LV). Abbiamo dimostrato che le cellue staminali trasdotte producono in maniera efficace I-1RA. Il protocollo di trasduzione associato alla sovraespressione di IL-1RA non hanno alterato la vitalità cellulare, il potenziale clonogenico e l’abilità delle cellule staminali di differenziazione nel topo. Successivamente, abbiamo studiato se le cellule staminali esprimenti IL-1RA, una volta trapiantate nei topi potessero migliorare l'infiammazione acuta e cronica. Sono stati impiegati tre modelli di topi. L'espressione ectopica di IL-1RA da parte delle cellule immunitarie derivate dalle cellule staminali trapiantate ha soppresso il reclutamento dei neutrofili nel sito dell'infiammazione nei topi con peritonite indotta da cristalli di urato monosodico (MSU), noti attivatori del complesso NLRP3 dell’inflammasoma. Questo effetto protettivo era paragonabile a quello ottenuto da anakinra. Successivamente, abbiamo sfruttato un modello murino inducibile esprimente la mutazione dominante Nlrp3A350V associata alle sindromi periodiche associate alla criopirina (CAPS). Il trapianto singenico di cellule staminali trasdotte con IL-1RA in topi Nlrp3A350V+CreT ha efficacemente impedito ai topi l'insorgenza e la progressione della malattia associata a perdita di peso, leucocitosi e livelli sierici elevati dell’interleuchina 6. Infine, i nostri dati preliminari indicano che il nostro approccio di terapia genica potrebbe migliorare il tasso di mortalità e la gravità della malattia in un modello murino di encefalomielite autoimmune sperimentale. Complessivamente, i nostri risultati hanno dimostrato che la somministrazione di IL-1RA mediata da un vettore lentivirale nelle cellule staminali è un approccio sicuro ed efficiente per controllare l'infiammazione mediata da IL-1. Questi risultati hanno posto le basi per studi futuri per valutare la potenziale applicazione clinica della tarapia genica con IL-1RA per le malattie autoinfiammatorie sistemiche mediate da IL-1.
Systemic autoinflammatory diseases (SAIDs) delineate a group of diseases that manifest when the immune system is activated uncontrollably. One cardinal subgroup of SAIDs includes rare periodic fevers characterized by the dysregulated production of the proinflammatory cytokine interleukin-1 (IL-1). There is no cure for these conditions. Anakinra, the recombinant form of IL-1 receptor antagonist (IL-1RA), is the mainstay therapy for these patients. However, anakinra has a short half-life and poor tissue distribution. Severe patients respond inadequately and do not experience improvement in symptoms. Therefore, there is a need for a durable therapy that bypasses continuous drug administration and ensures a satisfactory resolution of tissue inflammation. In this PhD project, we addressed the urgency to develop an effective treatment for IL-1 induced SAIDs based on haematopoietic stem and progenitor cells (HSPCs) producing constitutively human IL-1RA using a lentiviral vector (LV)-mediated gene transfer approach. Human and mouse HSPCs transduced with an LV encoding human IL-1RA efficiently released this cytokine. Transduction procedure and IL-1RA over-expression did not alter HSPCs viability, clonogenic and differentiation potential in vivo. Next, we investigated whether, once transplanted in mice, IL-1RA-expressing HSPCs could ameliorate acute and chronic inflammation. Three mouse models were employed. The ectopic expression of IL-1RA by HSPC-derived immune cells suppressed neutrophil recruitment to the site of inflammation in mice with peritonitis induced by monosodium-urate crystals (MSU), well-known activators of the NLRP3-IL-1 axis. This protective effect was comparable to that obtained by anakinra. Next, we exploited an inducible mouse model of the cryopyrin-associated period syndrome (CAPS) carrying the dominant Nlrp3A350V mutation. Syngeneic transplant of IL-1RA-transduced HSPCs in Nlrp3A350V+CreT mice effectively prevented mice from disease onset and progression manifested as weight loss, leucocytosis, and high serum IL-6 level. Finally, preliminary data indicate that our gene therapy approach could improve mortality rate and disease severity in a mouse model of experimental autoimmune encephalomyelitis. Altogether, our results demonstrated that LV-mediated IL-1RA delivery in HSPCs is safe and efficient approach to controlling IL-1-mediated inflammation. These findings set the stage for future studies to evaluate the potential LV-mediated IL-1RA GT clinical application for IL-1-mediated systemic autoinflammatory diseases.

Research regarding the development of cardiovascular disease (CVD) is important because CVD is the leading cause of death in the United States (US) and many countries abroad. Risk factors, such as obesity and psychological stress, should be studied in order to understand contributing factors for CVD and the cellular mechanisms which link risk factors with the development of disease. Specifically, the combined influence of multiple risk factors on inflammation is of interest because many individuals have more than one risk factor, which additively increases an individual’s risk for CVD. Obesity is already characterized by disordered inflammation, which suggests that the additional burden of a psychological stressor could elicit a greater inflammatory response and a greater risk for CVD than either stressor alone. The documents included within this thesis include the significance and specific aims of the study in addition to a review of the relevant literature related to the effects of obesity and acute mental stress (AMS) on endocrine and inflammatory markers. Specifically, this study aims to address IL-1β, IL-1Ra, and leptin following an AMS task in non-obese and obese individuals. Additionally, a manuscript is included which evaluates the change in IL-1β, IL-1Ra, and leptin following a 20 minute AMS task. Variables were examined between groups at baseline and at two time points following AMS. Additionally, the relationships among the changes in the markers following AMS were examined. Appendices include expanded methodology and all questionnaires used.

O cavalo tem sido há milhares de anos um dos animais de maior utilidade para o homem, tanto no trabalho quanto no esporte. A integridade de sua estrutura física é determinante para a qualidade de seu desempenho, sendo alvo importante das estratégias terapêuticas e preventivas da atualidade. Diversos estudos têm esclarecido parte da cascata de eventos que ocorre em ambiente intra-articular, revelando os principais mediadores nocivos e ampliando as opções para tratamentos mais eficientes. Experimentos utilizando a citometria de fluxo foram capazes de demonstrar que a adição de plasma às células de líquido sinovial (LS) previamente inflamadas diminui a produção de espécies reativas de oxigênio durante o burst oxidativo, conferindo capacidade antioxidante a este hemoderivado. Pouco se sabe, no entanto, sobre seu potencial pró ou anti-inflamatório, pois não existem relatos na literatura investigando tal atividade. Assim, desenvolvemos este estudo com o objetivo de acompanhar os efeitos exercidos pelo plasma autólogo condicionado (ACP) sobre os tecidos articulares, reportando as alterações clínicas e no LS das articulações metacarpofalangeanas hígidas que receberam este tratamento. Foram administrados 4 ml de ACP em 10 articulações metacarpofalangeanas de equinos saudáveis, enquanto as articulações contralaterais receberam 4 ml de solução fisiológica, como controle. LS foi coletado antes de cada aplicação e nos momentos 3, 6, 24, 48 e 168 horas após. Foi realizada avaliação física diária, e as amostras coletadas foram submetidas imediatamente a análise física (volume, cor, aspecto e viscosidade), teste da qualidade do precipitado de mucina, contagem total e diferencial das células nucleadas. O sobrenadante foi congelado para dosagem posterior de ureia, proteína total, ácido hialurônico (AH), condroitim sulfato (CS), prostaglandina E2 (PGE2) e das citocinas fator de necrose tumoral alfa (TNF-α), interleucina 1 beta (IL-1β) e antagonista do receptor de IL-1 (IL-1ra). A avaliação física dos animais detectou presença de claudicação nas articulações metacarpofalangeanas tratadas com ACP nos momentos 24 e 48 horas após sua administração. A análise física do LS das mesmas articulações revelou maior contaminação das amostras com sangue às 6 horas e maior contagem de células nucleadas às 3, 6, 24 e 48 horas, com predomínio de leucócitos polimorfonucleares. Ainda, o ACP induziu maiores concentrações de proteína total e PGE2 às 3 e 6 horas, e maior concentração de CS às 24 horas. Houve também tendência ao aumento de TNF-α às 24 horas. Às 168 horas, todavia, nenhuma alteração foi observada em quaisquer dos itens avaliados. Não ocorreram alterações nos demais aspectos físicos do LS nem na qualidade do precipitado de mucina, tampouco nas concentrações de ureia e AH. Estes resultados indicam que em articulações hígidas o ACP possui ação pró-inflamatória transitória, com maiores concentrações de PGE2 induzindo o catabolismo dos PGs da matriz, aumentando particularmente o CS.
History has connected human and equine species, for work and pleasure purposes and in both scenarios, horses\' physical integrity is of paramount importance for adequate performance. Soundness has been the target of many studies in orthopedic therapeutics and preventive equine medicine. Several of these studies have thrown light on the sequence of deleterious intra-articular events that take place after an insult, revealing key mediators of joint destruction and widening treatment options. Experiments evaluating the effects of plasma on reactive oxygen species production by chemically stimulated synovial fluid (SF) cells revealed a potent antioxidant effect. Little is known, however about plasma´s anti or pro inflammatory activities, still an unexplored property of plasma. This study was to designed to observe the effects of autologous conditioned plasma (ACP) on articular components, reporting findings of serial SF analysis and clinical evaluations, before and after its administration in healthy metacarpophalangeal joints. Four mililiters of ACP were injected in 10 healthy metacarpophalangeal joints, and the contralateral joints were injected with 4 ml of saline, serving as controls. SF was obtained for analysis before, and then 3, 6, 24, 48 and 168 hours after treatment injection. Horses were subjected to daily clinical evaluations and synovial fluid samples were immediately analyzed for color, viscosity, volume, aspect, quality of mucin clot and total and differential nucleated cell counts. The supernatant was frozen and stored for posterior dosages of urea, total protein, hyaluronic acid (HA), chondroitin sulphate (CS), prostaglandin E2 (PGE2), tumor necrosis factor alpha (TNF-α), interleukin 1 beta (IL- 1β), and interleukin 1 receptor antagonist (IL-1ra). Physical evaluation of ACP treated subjects detected mild lameness at 24 and 48 hours observation points. SF analysis of ACP treated joints revealed blood contamination and higher total nucleated cell counts at 3, 6, 24 and 48 hours, with predominance of polymorphonuclear cells. ACP treatment has also induced higher protein concentrations and PGE2 levels at 3 and 6 hours and higher CS levels at 24 hours in synovial fluid analysis. At 24 hours, TNFα concentrations were higher, although not significantly. At 168 hours post ACP treatment, however, no change was observed in any parameter of synovial fluid analysis. No alterations were detected in the remaining items evaluated, nor in the quality of the mucin clot, urea concentration or HA. These results indicate that, when injected into healthy joints, ACP elicits a transient inflammatory response, characterized by higher PGE2 concentrations, matrix catabolism, with particular increase in CS.

Purpose: The aim of our study is to develop a long lasting biotech drug delivery system for intraartcular (IA) treatment of rheumatic diseases as rheumatoid arthritis and osteoarthritis. The study was focused on preparation of microparticles formulated with biocompatible/biodegradable polymers (PLGA and PLA). Recombinant Human IL-1Ra was used as biotechnological drug model. Introduction: Polymeric microparticles have been widely studied as drug delivery systems for biotech drugs. These formulation can guarantee long term stability of the drug and sustained release allowing for therapeutic protocol optimisation. Nevertheless, protein formulation is usually complicated by the low stability of these fragile molecules which easily undergo denaturation and inactivation under harsh manipulation conditions. The set up of proper procedures which preserve the protein activity is essential to achieve effective products. Experimental: In the present research work, the preparation of biodegradable polymeric microparticles was investigated by using several techniques: emulsion/evaporation, double emulsion/evaporation, suspension/evaporation, nanoprecipitation, spray-drying. In a systematic study, various excipient combinations (PLA, PLGA, PLGAH, PEG, Tristearin, Hyaluronic Acid, Poloxamers etc.) and operative conditions (polymer and protein concentration, instrument set up etc.) were evaluated in order to point out the main critical parameters which dictate the physicochemical properties of the preparations. The products were characterised for their morphological and dimensional properties, drug loading and release were assessed. In vitro release of Il-1Ra from microparticles was evaluated by suspension in buffer and synovial fluid using RP-HPLC and ELISA methods. Pharmacokinetic of IL-1Ra was studied in mice using ELISA methods. Therapeutic efficacy of IL-1Ra loaded microspheres was tested using an animal model of collagen inducted arthritis. Results: Many kind of formulations were obtained using different kind of biocompatible polymers, different ratios between components, different polymers concentrations and procedures of preparation. Spray-drying techniques were the most efficient as they achieved for product yeld, drug loading and narrow dimensional polydispersivity. The preparation of protein suspensions in organic solvent solution of PLA or PLGA and PEG yielded microparticles with size ranging from 1 to 30 m which were suitable for IA administration. The main critical parameters affecting the biopharmaceutical properties of the formulations were: PEG molecular weight and content in the organic solution, PLA or PLGA concentration in the organic solution, type of PLGA and spray feed rate. Under optimized conditions, spherical and homogeneous microparticles in a size range between 2 and 15m were obtained with about 70% drug loading; the microspheres were formed by PLGA (75%), Epikuron 200SH (5%), PEG 5kDa (10%) and IL-Ra (10%). Pharmacokinetic studies demonstrated that microsphere formulation permitted a prolonged in vivo release of IL-1Ra. The use of animal model of collagen inducted arthritis underlined that the injection of IL-1Ra loaded microspheres permits the reduction of Anakinra treatment rate. Conclusion: Spray Drying is a suitable method to obtain drug delivery systems for the prolonged release of biotechnological products such as cytokines and monoclonal antibodies. The physicochemical properties of the products can be tailored by changing different process conditions and formulation composition.
Scopo: Lo scopo di questo studio è stato quello di sviluppare un sistema a rilascio prolungato per il delivery di farmaci biotecnologici per il trattamento intraarticolare (IA) delle patologie infiammatorie croniche come l’artrite reumatoide e l’osteoartrosi. Lo studio è stato focalizzato sulla preparazione di microparticelle costituite da polimeri biodegradabili e biocompatibili (PLGA, PLA e derivati dell’acido ialuronico). IL-1Ra umano ricombinante (anakinra) è stato utilizzato come modello di farmaco biotecnologico ad azione antiinfiammatoria per il suo ruolo fondamentale di antagonista del recettore per l’IL-1, citochina della quale è noto il ruolo chiave proinfiammatorio nelle patologie reumatiche croniche. Introduzione: Le microparticelle polimeriche sono state ampiamente studiate come sistemi di drug delivery per i farmaci biotecnologici. Questo tipo di formulazioni può garantire la stabilità nel tempo del farmaco e un lento rilascio che consente di ottimizzare il protocollo terapeutico. Tuttavia, la formulazione di proteine è solitamente complicata dalla scarsa stabilità di queste fragili molecole che vanno incontro a denaturazione ed inattivazione se sottoposte a condizioni operative drastiche. La messa a punto di opportune procedure che conservino l’attività delle proteine è essenziale per ottenere prodotti efficaci. Materiali e Metodi: La preparazione di microparticelle a base di polimeri biodegradabili è stata studiata utilizzando diverse tecniche: nanoprecipitazione, emulsione ed estrazione della fase interna, emulsione ed evaporazione, doppia emulsione ed evaporazione, spray drying. Varie combinazioni di eccipienti (PLA, PLGA, PLGA-H, PEG, tristearina, acido ialuronico e derivati, Polossamero, fosfatidilcolina) e diverse condizioni operative (concentrazione del polimero e della proteina, settaggio della strumentazione ecc) sono state valutate al fine di evidenziare i principali parametri critici che determinano le proprietà chimico-fisiche della preparazione. I prodotti sono stati caratterizzati per le loro proprietà morfologiche e dimensionali, e sono stati valutati il caricamento e il rilascio del farmaco. Il rilascio in vitro di IL-1RA dalle microparticelle in tampone fisiologico o liquido sinoviale è stato valutato utilizzando tecniche come RP-HPLC ed ELISA. La cinetica di rilascio in vivo di IL-1Ra dalle microsfere è stata valutata mediante metodi ELISA. Sono stati effettuati degli studi di efficacia terapeutica della formulazione utilizzando un modello animale di artrite da collagene (C.I.A.); i diversi gruppi di animali sono stati trattati con diverse dosi di microsfere o di Kineret, e con frequenze diverse di somministrazione. Sono stati valutati il paw score, il peso, diametro dell’articolazione della caviglia e la tumefazione del femore. Risultati: Sono stati ottenute tipologie differenti di formulazioni utilizzando diversi tipi di polimeri biocompatibili, diversi rapporti tra i componenti, diverse concentrazioni di polimero e differenti procedure di preparazione. La tecnica di spray drying è risultata la più efficace in termini di resa, di caricamento del farmaco e di polidispersività dimensionale. La preparazione mediante spray drying di microsfere a partire da sospensioni di liofilizzati di IL-1Ra/PEG in soluzioni organiche di PLA o PLGA ha permesso l’ottenimento di microparticelle con dimensioni comprese tra 1 e 30m, compatibili con l’uso iniettabile. Si è verificato che i principali parametri critici che possono influenzare le proprietà biofarmaceutiche delle formulazioni sono: peso molecolare del PEG utilizzato e rapporto PEG/IL-1Ra nel liofilizzato, concentrazione di PLA o PLGA nel solvente organico, tipo di PLGA e velocità di alimentazione dell’ugello dello strumento. L’ottimizzazione di questi parametri ha permesso di ottenere microsfere di dimensioni adatte all’iniezione intraarticolare (2-15m) e con un elevata efficienza di caricamento del farmaco (50-70%); queste microparticelle sono costituite da 75% p/p di PLGA (in soluzione organica al 4%), 5% p/p di Epikuron 200SH (fosfatidilcolina), 10% p/p di PEG 5kDa e 10% p/p di IL-1Ra. Gli studi di farmacocinetica in topi Balb/c hanno evidenziato che, negli animali trattati con microsfere caricate con citochina, la concentrazione plasmatica di IL-1Ra decresce molto più lentamente che negli animali trattati con il prodotto commerciale Kineret® (anakinra); dopo 24h dalla somministrazione di Kineret, infatti, non vi è più traccia rilevabile di IL-1Ra nel plasma, mentre, dopo somministrazione di microsfere caricate con IL-1Ra, si rileva presenza di citochina per tempi superiori alle 48h. L’utilizzo del modello animale di artrite sperimentale ha permesso di valutare l’efficacia terapeutica delle microsfere: la somministrazione di microsfere caricate con IL-1Ra consente di ridurre la frequenza di trattamento ottenendo risultati confrontabili ad una iniezione giornaliera di Kineret. Conclusione: Il metodo di sospensione e spray drying sviluppato è adatto all’ottenimento di sistemi per il rilascio prolungato di prodotti biotecnologici come citochine, anticorpi monoclonali e proteine di fusione. Le caratteristiche chimico-fisiche dei prodotti possono essere modificate e adattate allo scopo desiderato variando le condizioni di processo e la composizione della formulazione.

El trasplantament d'illots pancreàtics és una teràpia emergent per la curació de la diabetis mellitus. Una de les limitacions radica en la baixa disponibilitat d'òrgans i l'elevada demanda existent, que queda agreujada amb l'elevat nombre d'illots que són necessaris per restablir la normoglucèmia del pacient. Estudis recents del nostre grup, han mostrat que en els primers dies després del trasplantament hi ha un augment de l'expressió d'IL-1beta en els empelts d'illots.

La hipòtesi de treball és que la citocina proinflamatòria, IL-1, està implicada en la fallada del trasplantament. Designant la sobreexpressió d'IL-1Ra com l'estratègia a seguir per millorar el pronòstic del trasplantament singènic d'illots pancreàtics. Per tant, l'objectiu general de l'estudi va ser determinar si la sobreexpressió d'IL-1Ra en els illots pancreàtics protegeix les cèl·lules beta pancreàtiques dels efectes deleteris d'IL-1 en els illots i millora el pronòstic del trasplantament.

L'estudi dels efectes d'IL-1beta i de la sobreexpressió d'IL-1Ra in vitro es va realitzar amb un cultiu primari d'illots de rata que van ser exposats durant 48h a 5.5 o 22.2 mM de glucosa en presència o absència de 50U/ml d'IL-1beta. I la inserció del gen exogen a les cèl·lules dels illots es va fer utilitzant un adenovirus V recombinant.

La proliferació de les cèl·lules beta (determinada per incorporació de BrdU) va disminuir dràsticament quan es van exposar els illots a 50 U/ml d'IL-1beta, tant a 5.5 mM com a 22.2 mM de glucosa. Aquest efecte d'IL-1beta va quedar completament abolit per la sobreexpressió d'IL-1Ra en els illots que havien estat infectats amb l'adenovirus que codificava per l'antagonista, a les dues concentracions de glucosa utilitzades.

L'apoptosi de les cèl·lules beta (determinada per immunohistoquímica mitjançant la tècnica del TUNEL i per citometria de flux, marcant les cèl·lules amb anexina V i iodur de propidi) estava significativament augmentada en els illots exposats a IL-1beta, però no en els illots que sobreexpressaven IL-1Ra.

L'estudi dels efectes de la sobreexpressió d'IL-1Ra en els illots trasplantats es va realitzar utilitzant un model de trasplantament singènic. Grups de 500 illots control (no infectats) o que sobreexpressaven IL-1Ra van ser trasplantats sota la càpsula renal de rates Lewis diabètiques. 500 illots són una massa beta clarament insuficient per restablir la normoglucèmia, així doncs els animals d'ambdós grups es van mantenir hiperglucèmics durant tot l'estudi. Els empelts es van recuperar després de 3, 10 i 28 dies del trasplantament i es van processar per fer estudis histològics.

La sobreexpressió d'IL-1Ra en els illots trasplantats va fer augmentar significativament la proliferació de les cèl·lules beta dels empelts de 3, 10 i 28 dies i va protegir parcialment les cèl·lules beta de l'increment d'apoptosi detectat després del trasplantament, tant a curt com a llarg termini. L'àrea individual de les cèl·lules beta estava augmentada de manera similar tant en els empelts d'illots control com en els illots que sobreexpressaven IL-1Ra als 10 i 28 dies d'evolució. Finalment, la sobreexpressió d'IL-1Ra resultà en una recuperació de la massa beta inicialment trasplantada.

Per tal d'estudiar si els efectes beneficiosos de la sobreexpressió d'IL-1Ra aconseguien reduir el nombre d'illots necessaris per restablir la normoglucèmia, es va trasplantar una massa beta marginal (800 illots) d'illots control i Ad-IL-1Ra a animals diabètics. El 100% dels animals trasplantats amb illots Ad-IL-1Ra eren normoglucèmics després de 14 dies del trasplantament i només un 40% dels animals trasplantats amb illots control assoliren l'euglucèmia en aquest dia.

En aquest treball es mostra que la citocina proinflamatòria IL-1beta indueix clarament apoptosi a les cèl·lules beta dels illots de rata en cultiu i inhibeix dràsticament la replicació d'aquestes cèl·lules. La sobreexpressió d'IL-1Ra protegeix les cèl·lules beta dels efectes deleteris d'aquesta citocina i amplifica la resposta replicativa de les cèl·lules beta exposades a concentracions altes de glucosa. La sobreexpressió d'IL-1Ra en els illots augmenta la replicació de les cèl·lules beta trasplantades, les protegeix de l'apoptosi induïda després del trasplantament, i preserva la massa beta inicialment trasplantada. Els efectes beneficiosos de la sobreexpressió d'IL-1Ra observats en els illots trasplantats permeten reduir el nombre d'illots necessaris per restablir la normoglucèmia dels animals diabètics.

Aquests resultats suggereixen que la IL-1 juga un paper important en l'evolució dels empelts d'illots, ja que el seu bloqueig implica una millora dels illots trasplantats.
BACKGROUND AND AIMS:

IL-1beta could contribute to the dramatic beta cell loss that takes place after islet transplantation. It is known that exposure to sustained hyperglycemia has a deleterious effect on transplanted islets. Moreover, it has been recently reported that IL-1beta expression is increased in islets exposed to high glucose levels. IL-1Ra is a naturally occurring inhibitor of IL-1 action and its overexpression protects pancreatic islets from the deleterious effects of IL-1â on beta cell replication, apoptosis and function. The aim of this study was to determine whether viral gene transfer of the IL-1Ra gene into rat islets ex vivo could have a beneficial effect on beta cell replication and mass of transplanted islets.

METHODS:

Lewis rat islets were infected for 2h with 6.25 × 106 pfu of Ad-IL-1Ra and streptozotocin-diabetic Lewis rats were transplanted with 500 Ad-IL-1Ra infected islets (Ad-IL-1Ra group) or 500 uninfected islets (control group) under the kidney capsule. Grafts were removed 3 (n = 12), 10 (n = 12) and 28 (n = 12) days after transplantation and beta cell replication, apoptosis and mass were determined.

RESULTS:

500 islets is an insufficient mass to restore normoglycemia and therefore, all animals but one (IL-1Ra group) remained hyperglycemic until the end of the study. Beta cell replication (determined by BrdU incorporation) was significantly increased in Ad-IL-1Ra group on days 3 (0.78 ± 0.23%), 10 (1.15 ± 0.16%) and 28 (1.22 ± 0.2%) after islet transplantation compared to beta cell replication in normal pancreas (0.24 ± 0.04%; p< 0.05). In contrast, in control group, beta cell replication was not increased on day 3 after transplantation (0.41 ± 0.11%), and although it increased on day 10 (0.89 ± 0.18%; p< 0.01) it was reduced again on day 28 (0.59 ± 0.10%) in agreement with previous reports of limited beta cell replication with persistent hyperglycemia. Beta cell apoptosis (determined by TUNEL method) was significantly increased in transplanted islets from both groups compared to pancreas. Although Ad-IL-1Ra group showed lower beta cell apoptotic levels than control group, differences did not reach statistical significance. The initially transplanted â-cell mass (1.34 ± 0.03 mg) was similarly reduced in both control (0.32 ± 0.06 mg) and Ad-IL-1Ra groups (0.45 ± 0.10 mg) (p<0.001) on day 3 after transplantation. In Ad-IL-1Ra islet grafts, beta cell mass increased after 10 (1.04 ± 0.091 mg; p< 0.010) and 28 (0.8 ± 0.24 mg) days of transplantation. In contrast, beta cell mass of control group was also increased on day 10 after transplantation (0.69 ± 0.12 mg), but it dropped again on day 28 (0.41 ± 0.05 mg) paralleling with the evolution of beta cell replication in this group.


CONCLUSIONS:

Islets overexpressing IL-1Ra showed an increased beta cell replication and a preserved beta cell mass after transplantation, that was maintained even after longterm exposure to hyperglycemia.

The goal of this thesis was to develop a new drug-delivering material to deliver anti-inflammatory protein for treating OA. Our central hypothesis for this work is that a controlled release/presentation system will more effectively deliver anti-inflammatory protein therapies to the OA joint. The primary goal of this work was to synthesize a block copolymer that could self-assemble into injectable, sub-micron-scale particles and would allow an anti-inflammatory protein, IL-1ra, to be tethered to its surface for efficient protein delivery. The block copolymer incorporated an oligo-ethylene monomer for tissue compatibility and non-fouling behavior, a 4-nitrophenol group for efficient protein tethering, and cyclohexyl methacrylate, a hydrophobic monomer, for particle stability. We engineered the copolymer and tested it in both in vitro culture experiments and an in vivo model to evaluate protein retention in the knee joint. The rationale for this project was that the rational design and synthesis of a new drug- and protein-delivering material can create a modular polymer particle that can deliver multi-faceted therapies to treat OA. This work characterizes the in vitro and in vivo behavior of our polymer particle system. The protein tethering strategy allows IL-1ra protein to be tethered to the surface of these particles. Once tethered, IL-1ra maintains its bioactivity and actively targets synoviocytes, cells crucial to the OA pathology. This binding happens in an IL-1-dependent manner. Furthermore, IL-1ra-tethered particles are able to inhibit IL-1beta-induced NF-kappaB activation. These studies show that this particle system has the potential to deliver IL-1ra to arthritic joints and that it has potential for localizing/targeting drugs to inflammatory cells of interest as a new way to target OA drug treatments.

Immunological stressors, in the form of clinical and sub-clinical disease are currently controlled using both prophylactic antibiotics in-feed, and therapeutic antibiotic treatment. Respiratory disease, primarily Actinobacillus pleuropneumoniae (App) infection, is recognised as a major factor causing reduced productivity in pigs. This thesis reports investigations into the use of novel immunomodulators in particular Interleukin 4 (IL-4) and Interleukin 1 receptor antagonist (IL-1ra) as alternatives to antibiotics to treat App infection. Immunological and molecular biological assays were used to investigate and accumulate data. An in vitro study undertaken to find potential anti-inflammatory substances, revealed that Interleukin 8 (IL-8) mRNA production stimulated by PMA or LPS in whole pigs' blood was suppressed by IL-4. IL-1ra also suppressed stimulated IL-8 mRNA production by heat killed App bacteria (KB) in vitro. An acute LPS challenge in pigs in vivo however, showed no variation in illness or weight loss between pigs treated prophylactically with anti-inflammatory substance (IL-4 and IL-1ra) and saline treated pigs. The use of plasmids as a delivery system for anti-inflammatory substance did not show promise since it did not enhance growth or prolong the expression of the substances in the pigs. However, in the chronic App challenge model IL-4 and IL-1ra administered prophylactically in vivo showed an ability to improve growth. The therapeutic administration of IL-4 and IL-1ra to App challenged pigs showed no difference in pigs' growth, regardless of the treatment or control administered. To conclude, IL-4 and IL-1ra showed promise when administered prophylactically and improved growth and abrogated disease under conditions of App challenge. However when IL-4 and IL-1ra where administered therapeutically they did not perform as well. Moreover these compounds have potential as a commercial application to reduce the growth reduction caused by disease such as App.

Etude du rôle de « Liver Receptor Homolog-1 » (LRH-1 ; NR5A2) dans la régulation de la réponse inflammatoire hépatique et dans l’homéostasie du cholestérol Les récepteurs nucléaires sont des facteurs de transcription impliqués dans des processus biologiques cruciaux tels que la reproduction, le développement et la différenciation. Le « liver receptor homolog-1 » (LRH-1 ; NR5A2) est un facteur de transcription, constitutivement actif, appartenant à la famille des récepteurs nucléaires. Il est essentiellement exprimé dans les tissus d’origine endodermique tels que le foie, le pancréas et l’intestin. LRH-1 joue un rôle majeur dans le développement, la stéroïdogénèse et l’homéostasie du cholestérol via la régulation du transport réverse du cholestérol et la biosynthèse des acides biliaires. Récemment, nous avons démontré le rôle de LRH-1 dans la régulation de la réponse de phase aiguë au niveau hépatique. En effet, la sur-expression de LRH-1dans des hépatocytes résulte en l’inhibition de l’induction de l’expression d’haptoglobine et SAA par les cytokine IL1 et IL6. De plus, l’induction de la réponse inflammatoire est significativement exacerbée dans des cellules hépatiques déficientes pour LRH-1. Des études de promoteur, d’ARN interférence et de chromatine immuno-précipitation révèlent que LRH-1 régule la réponse inflammatoire d’une part en antagonisant la voie de signalisation C/EBP et d’autre part en induisant l’expression de IL-1RA (« Interleukin-1 Receptor Antagonist »). L’activité anti-inflammatoire de LRH-1 est démontrée in vivo dans les souris hétérozygote pour LRH-1. LRH-1 est décrit comme régulateur du métabolisme lipidique en contrôlant l’expression des gènes impliqués dans la régulation de la synthèse des acides biliaires et dans l’homéostasie du cholestérol. Les particules HDL sont considérées comme des particules anti-anthérogène par leur capacité à promouvoir le transport réverse du cholestérol. Des études récentes rapporte que ApoM semble important dans la formation des particules prè-HDL et dans l’efflux cholestérol induit par les particules HDL. Par ailleurs, ApoM inhibe la progression de l’athérosclérose dans les souris LDLr KO. Nous avons étudié le rôle de LRH-1 dans la régulation du gène codant ApoM. En utilisant des hépatocytes déficient pour LRH-1 ou sur exprimant LRH-1, nous avons démontré que LRH-1 régule l’expression de ApoM en se liant à un LRH-1 RE localisé dans le promoteur du gène. Nous démontrons également que le répresseur transcriptionnelle SHP inhibe l’expression de ApoM en inhibant l’activité transcriptionnelle de LRH-1 in vitro et in vivo. Les propriétés anti-inflammatoires de LRH-1 et son rôle dans l’homéostasie du cholestérol sont confirmés par la caractérisation des souris LRH-1 conditionnelles KO. L’ensemble de ces résultats démontre que LRH-1 est un régulateur physiologique de la réponse de phase aiguë et joue un rôle majeur dans le métabolisme du HDL-cholestèrol.

Immunological stressors, in the form of clinical and sub-clinical disease are currently controlled using both prophylactic antibiotics in-feed, and therapeutic antibiotic treatment. Respiratory disease, primarily Actinobacillus pleuropneumoniae (App) infection, is recognised as a major factor causing reduced productivity in pigs. This thesis reports investigations into the use of novel immunomodulators in particular Interleukin 4 (IL-4) and Interleukin 1 receptor antagonist (IL-1ra) as alternatives to antibiotics to treat App infection. Immunological and molecular biological assays were used to investigate and accumulate data. An in vitro study undertaken to find potential anti-inflammatory substances, revealed that Interleukin 8 (IL-8) mRNA production stimulated by PMA or LPS in whole pigs' blood was suppressed by IL-4. IL-1ra also suppressed stimulated IL-8 mRNA production by heat killed App bacteria (KB) in vitro. An acute LPS challenge in pigs in vivo however, showed no variation in illness or weight loss between pigs treated prophylactically with anti-inflammatory substance (IL-4 and IL-1ra) and saline treated pigs. The use of plasmids as a delivery system for anti-inflammatory substance did not show promise since it did not enhance growth or prolong the expression of the substances in the pigs. However, in the chronic App challenge model IL-4 and IL-1ra administered prophylactically in vivo showed an ability to improve growth. The therapeutic administration of IL-4 and IL-1ra to App challenged pigs showed no difference in pigs' growth, regardless of the treatment or control administered. To conclude, IL-4 and IL-1ra showed promise when administered prophylactically and improved growth and abrogated disease under conditions of App challenge. However when IL-4 and IL-1ra where administered therapeutically they did not perform as well. Moreover these compounds have potential as a commercial application to reduce the growth reduction caused by disease such as App.

Doctor of Philosophy (PhD)
Asthma is a chronic inflammatory disease of the airways. Corticosteroid medication is the most effective currently available treatment. Complications of corticosteroid therapy are dose-dependent, however, the clinical efficacy of varying doses of inhaled corticosteroids has been studied with mixed results. A randomized, double-blind, parallel group study was used to evaluate the inhaled corticosteroid dose-response relationship for clinical endpoints and in vitro parameters of underlying airway inflammation and remodelling. The mannitol provocation test with Forced Oscillation Technique (FOT) was used to derive potential dose-differentiating endpoints. In vitro inflammatory markers were measured in alveolar macrophages from bronchoalveolar lavage. Basement membrane thickness was measured from bronchial biopsies. Eleven nonasthmatic subjects were enrolled for comparison. This thesis addresses the null hypothesis that there is no significant difference in clinical and biological effects between low dose (200mcg/day, n=11) and high dose (1000mcg/day, n=11) treatment (for 6-7 weeks) with inhaled fluticasone propionate (FP) for a range of clinical outcomes and in vitro markers of airway inflammation and remodelling. Significant changes after FP included increased FEV1, reduced airway hyperresponsiveness (AHR) (by FOT and FEV1), exhaled nitric oxide and Juniper symptom score. In addition, significant reductions occurred in expression of GM-CSF, TNF-alpha and IL-1ra in macrophages. A lower baseline FOT-derived respiratory system conductance was predictive of a greater degree of improvement in symptoms. No statistically significant differences in the changes after treatment between low and high dose FP were found in spirometry, exhaled nitric oxide, symptom scores, AHR, alveolar macrophage cytokine levels (GM-CSF, TNF-alpha, IL-1ra, IL-10) and basement membrane thickness, although there were trends towards greater improvements in many of the parameters after high dose FP. Basement membrane thickness appeared to be reduced by high dose FP, although this reduction was not statistically significant. There was a weak, but statistically significant, negative correlation between basement membrane thickness and FOT-derived conductance (r2=0.135, p=0.042). With the recognition of the limitations in the interpretation of these data, the results suggest that, in previously steroid naïve mild to moderate asthmatics, there may be only minimal benefit derived from an additional 800µg/day of inhaled fluticasone above the low dose of 200µg/day.

Dissertação para obtenção do grau de Mestre no Instituto Universitário Egas Moniz
A peri-implantite consiste numa condição patológica que ocorre nos tecidos em redor dos implantes, sendo caracterizada por inflamação na mucosa peri-implantar e subsequente perda progressiva de osso de suporte, devido à acumulação microbiana e/ ou fatores iatrogénicos. Contudo, a doença é provavelmente o resultado de vários fatores que podem influenciar a resposta inflamatória do hospedeiro, como o tabaco, o stress e variações genéticas, polimorfismos, em determinados genes relevantes para a reação imunológica. Os principais objetivos desta revisão narrativa são caracterizar a peri-implantite, descrevendo a sua etiologia, os seus principais fatores de risco, prevalência, parâmetros de diagnóstico e principais formas de tratamento atuais. Também se pretende reunir a mais recente evidência científica acerca da possível relação entre a existência de polimorfismos nos genes IL-1A, IL-1β e IL-1RN e o desenvolvimento da peri-implantite. Também serão identificadas e descritas as principais técnicas laboratoriais genéticas e bioquímicas utilizadas para averiguar esta possível relação. Para a realização desta revisão narrativa, foi efetuada uma pesquisa bibliográfica recorrendo às plataformas informáticas de literatura científica Pub-Med, SciELO, Medline e Cochrane. A pesquisa foi efetuada em Português e Inglês, recorrendo a termos chave como: “Peri-implantite/Peri-implantitis”, “diagnóstico peri-implantite/ Peri implantitis diagnostic” “tratamento peri-implantite /treatment”, “polimorfismos IL-1/ Il-1 Gene polymorphisms”, “Marcadores enzimáticos Peri-implantite/Peri-implantitis Biomarkers”.
Peri-implantitis consists in a pathologic condition that occurs in tissues surrounding the implants, characterized by inflammation in the peri-implant mucosa and subsequent progressive bone loss, caused by microbial accumulation and/or iatrogenic factors. However, the disease is likely to be the result of several factors that can influence the inflammatory host response, such as tobacco, stress and genetic variations, polymorphisms, in specific genes which are relevant for the immune response. The main objectives of this narrative review are to caracterize peri-implantitis, describing its etiology, main risk factors, prevalence, diagnostic parameters and its current main treatment pathways. It is also intended the gathering the most recent scientific evidence regarding the possible relation between the existence of IL-1A, IL-1β and IL-1RN gene polymorphisms and the development of peri-implantitis. The main genetic and biochemical laboratorial techniques used to verify this relation will also be identified and described. To develop this narrative review, a bibliographic search was conducted by resorting to the scientific literature computer platforms Pub-Med, SciELO, Medline and Cochraine, in order to scientifically support this work. The search was conducted in portuguese and english, by using keywords such as: “Peri-implantite/Peri-implantitis”, “diagnóstico peri-implantite/ Peri-implantitis diagnostic” “tratamento Peri-implantite/ Peri implantitistreatment”, “polimorfismos IL-1/ Il-1 Gene polymorphisms”, “Marcadores enzimáticos Peri-implantite/Peri-implantitis Biomarkers”.

碩士
長庚大學
生化與生醫工程研究所
94
Primary non-function is the common effect that is involved in xeno-, allo-, auto- and iso-transplant rejection. Activation of macrophages is thought to be the underlying mechanism of primary non-function. We hypothesized that IL-1β plays an important role on primary non-function. In this proposal, we will use a 6-liter bioreactor to prepare high amount of GST-IL-1ra fusion protein (IL-1 receptor antagonist fused to glutathione-S-transferase) in a large-scale cell lysate. The purified GST-IL1ra was loaded in silicagel to sustainedly release IL-1ra. The effect of GST-IL-1ra xerogel on islet function was evaluated in order to study the protective role of IL-1ra and the adverse effect of IL-1β on islets. As a result, a higher amount of recombinant GST-IL1ra fusion protein was obtained from a 5-l bioreactor at three hours after IPTG induction. We found that the xerogel prepared with higher pH value of Tris-HCl resulted in smaller particle size but there is no obvious disparity in the releasing profiles. In the toxicity study, we co-incubated islets with empty silicagel nanoparticles in vitro and found that the glucose stimulated insulin secretion of islets were not influenced significantly. Following 7-day incubation with GST-IL1ra xerogel, in vitro islets secreted significantly more insulin to glucose stimulation than control islets. In conclusion, sustainedly release of IL1-ra from GST-IL1ra xerogel protects islets against the suppressive effect of IL-1β in vitro. In the future, the effect of GST-IL1ra xerogel on islet function will be evaluated in xenogenic, allogenic, and syngeneic transplantation models. This findings also underline the fact that immobilization of protein could be a effective tool for therapy.

Dissertação para obtenção do grau de Mestre no Instituto Universitário Egas Moniz
Objetivos: Avaliar a possível associação dos polimorfismos genéticos IL-1A-889, IL-1B+3954, IL-1RN (intrão 2) e o desenvolvimento de peri-implantite, através de um estudo piloto efetuado numa população de pacientes caucasianos da Clínica Dentária Egas Moniz. Materiais e Métodos: Foram selecionados 20 pacientes, 10 com saúde peri-implantar e 10 com peri-implantite. As amostras contendo células da mucosa jugal foram armazenadas a -20ºC e posteriormente submetidas ao processo de extração de DNA. A análise genética foi realizada através da técnica de PCR (Polymerase chain reaction), RFLP (Restriction fragment length polymorphism) e através de análise eletroforética. A análise estatística foi realizada através do programa Statistical Package for the Social Sciences (SPSS). Resultados e Discussão: Para o gene da IL-1A-889, observou-se que o alelo mutado foi observado em maior percentagem no grupo peri-implantite comparativamente ao grupo controlo (30% vs 15% respetivamente). Para o gene IL-1B+3954, observou-se também que o alelo alterado estava presente em maior percentagem no grupo doença comparativamente ao grupo controlo (35% vs 10% respetivamente). O genótipo positivo (presença de pelo menos um alelo T em ambos os polimorfismos IL-1A-889 e IL-1B+3954), foi detetado em 6 pacientes, sendo que 5 pertenciam ao grupo doença e 1 ao grupo saúde. Para o IL-1RN observou-se a presença do alelo 1 em todos os pacientes, à exceção de um paciente pertencente ao grupo saúde, que apresentava o alelo 1 (VNTR de 412pb) e o alelo 3 (VNTR de 326 pb). Não se observou uma diferença estatisticamente significativa quanto à presença dos polimorfismos nos genes da IL-1 e do IL-1RN entre os grupos. Conclusão: Para o polimorfismo do gene IL-1RN não se verificaram diferenças entre os grupos. Relativamente aos polimorfismos dos genes da IL-1 não se observou uma diferença estatisticamente significativa entre o grupo saúde e doença, contudo uma tendência deve ser realçada mostrando uma potencial ligação do genótipo da IL-1 e a peri-implantite.
Objective: To evaluate the possible association of genetic polymorphisms IL-1A-889, IL-1B+3954, IL-1RN (Intron 2) and the development of peri-implantitis, through a pilot study carried out in a population of Caucasian patients from the Egas Moniz Dental Clinic. Materials and Methods: 20 patients were selected, 10 with peri-implant health and 10 with peri-implantitis. Samples containing cells from the buccal mucosa were stored at -20ºC and later submitted to the DNA extraction process. Genetic analysis was performed using the PCR (Polymerase chain reaction), RFLP (Restriction fragment length polymorphism) and electrophoretic analysis. Statistical analysis was performed using the Statistical Package for Social Sciences program (SPSS). Results and Discussion: For the IL-1A-889 gene, it was observed that the mutated allele was observed in a higher percentage in the peri-implantitis group compared to the control group (30% vs 15% respectively). For the IL-1B+3954 gene, it was also observed that the altered allele was present in a higher percentage in the disease group compared to the control group (35% vs 10% respectively). The positive genotype was detected in 6 patients, 5 belonging to the disease group and 1 to the health group. For IL-1RN, the presence of allele 1 was observed in all patients, except for one patient belonging to the health group, which had allele 1 and allele 3. There was no statistically significant difference in the presence of polymorphisms in the IL-1 and IL-1RN genes between the groups. Conclusion: For the IL-1RN gene polymorphism there were no differences between groups. Regarding IL-1 gene polymorphisms, there was no statistically significant difference between the health and disease group, however a trend should be highlighted, showing a potential link between the IL-1 genotype and peri-implantitis.

碩士
國立陽明大學
解剖學研究所
88
ABSTRACT Neointimal hyperplasia, mainly the proliferation of smooth muscle cells and accumulation of macrophage-derived foam cells and interleukin-1 beta might play a central role in the process of atherosclerosis. In animal study, we investigated the expression of IL-1beta, IL-1ra, ICAM-1 and VCAM-1 during atherosclerosis in high cholesterol-fed rabbits by in situ hybridization, immunohistochemical staining and western blot. New Zealand White rabbits were fed with 2% cholesterol-containing diet for 2 week, 3 weeks and 6 weeks. At designated time points, artery were excised for paraffin embedding. In situ hybridization using digoxigenin-labeled IL-1beta and IL-1ra cDNA as a probe was conducted to determine the distribution of IL-1beat and IL-1ra mRNA. Immunohistochemical staining was used to study the expression of IL-1beta, IL-1ra, ICAM-1 and VCAM-1 and to identify cells in the arterial wall responsible for IL-1beta, IL-1ra, ICAM-1 and VCAM-1 expression. Western blot was used to determine the volume of IL-1beta and IL-1ra. Otherwise, we used the methods of above to determine the expression of IL-1beta, IL-1ra, ICAM-1 and VCAM-1 in human serious atherosclerosis. In cell cultured study, we observed the expression of ICAM-1 and VCAM-1 in IL-1beta-treated HASMCs by cell ELISA. The results revealed : IL-1beta mRNA expression was detected in the thickened intima in cholesterol-fed rabbit for 2 weeks, 3 weeks and 6 weeks and in human atherosclerosis. The thickened intima also showed strong IL-1beta protein reactions. IL-1ra mRNA expression was detected in the slight thickened intima in cholesterol-fed rabbit for 2 weeks and 3 weeks and IL-1ra mRNA expression was remarkable in the thickened intima in cholesterol-fed rabbit for 6 weeks and in human atherosclerosis. But the expression of IL-1ra protein was not clear and obvious. The expression of ICAM-1 and VCAM-1 protein were remarkable in the thickened intima in cholesterol-fed rabbit for 6 weeks and in human atherosclerosis. By using the antibodies against the macrophages and smooth muscle cells respectively,the cells in the thickened intima were considered to be macrophages and few smooth muscle cells in cholesterol-fed rabbits. Nevertheless, the major component of the thickened intima is smooth muscle cell in human serious atherosclerosis. The expression of ICAM-1 and VCAM-1 protein were not increase in 10ng/ml IL-1beta-treated HASMCs in this study.

碩士
國立中央大學
生命科學研究所
99
Interleukin-1 receptor antagonist (IL-1Ra) is a member of the IL-1 cytokine family that binds to IL-1 receptors and subsequently prevents IL-1 from triggering a signal in the cell. IL-1Ra can be produced by a variety of cell types in response to inflammatory stimuli. Accumulating evidences indicates that IL-1Ra functions as an anti-inflammatory cytokine that can block LPS-induced TNF-a and IL-1β production in inflammatory cells. Moreover, the human recombinant IL-1Ra is currently used as a therapeutic agent for rheumatoid arthritis. In this study, we demonstrated that stimulation of Raw 264.7 cells and mouse peritoneal macrophages with LPS increased IL-1Ra release in a time- and dose-dependent manner. The cAMP-specific phosphodiestrase PDE4 inhibitor Rolipram, a cAMP-elevating agent, significantly enhanced the LPS-stimulated IL-1Ra release with the EC50 of approx. 0.3~1 uM. This induction of IL-1Ra by LPS and Rolipram was also observed at the transcriptional level. Moreover, the increase in the IL-1Ra release was mimicked by the treatment of the cells with the PKA activator 6-Bnz-cAMP, whereas the Epac activator 8-pCPT-2’-O-Me-cAMP did not produce the similar effect. In addition, the Rolipram-enhanced IL-1Ra production was reversed by the PKA inhibitor Rp-8-CPT-cAMPS. These results indicated that the effect of Rolipram was mediated by activation of the cAMP/PKA signal pathway. To further dissect among the four PDE4 subtypes (PDE4A, 4B, 4C, 4D) which one is responsible for the regulation of the IL-1Ra production, PDE4-deficient peritoneal macrophages were employed. The results showed that ablation of PDE4B enhanced LPS-stimulated IL-1Ra release to the level similar to that produced by the WT cells treated with both LPS and Rolipram, indicating the effect of Rolipram was mediated by inhibition of PDE4B. Additionally, using anti-IL-1Ra antibody to deplete IL-1Ra in the culture medium, our results indicated that in the presence of LPS alone the TNF-a release could be suppressed by the release of IL-1Ra in the medium. However, the Rolipram-enhanced IL-1Ra release was not responsible for the inhibition of TNF-a release caused by Rolipram. Taken together, these findings suggested that ablation or inhibition of PDE4B activates cAMP/PKA signaling and leads to upregulation of LPS-stimulated IL-1Ra production, which however does not contribute to its down-regulation of TNF-a release in mouse macrophages.

博士
國立臺灣大學
生化科學研究所
102
Abstract Dendrobium huoshanense is a valuable and versatile Chinese herbal medicine with the anecdotal claims of cancer prevention and anti-inflammation. However, its immunological activities are limited to in vitro studies on a few cytokines and immune cell functions. First, we investigated the effects of polysaccharides isolated from DH (DH-PS) on inducing a panel of cytokines/chemokines in mice in vivo and human in vitro. We found that DH polysaccharides (DH-PS) induced TH1, TH2, inflammatory cytokines and chemokines in mouse in vivo and human cells in vitro. Secondly, we demonstrated that DH-PS expanded mouse splenocytes in vivo including CD4+ T cells, CD8+ T cells, B cells, NK cells, NKT cells, monocytes/macrophages, granulocytes and regulatory T cells. Notably, DH-PS induced an anti-inflammatory molecule, IL-1ra, in mouse and human immune cells, especially monocytes. The serum level of IL-1ra elicited by the injection of DH-PS was over 10 folds of IL-1β, suggesting that DH-PS-induced anti-inflammatory activities might over-ride the inflammatory ones mediated by IL-1β. The signaling pathways of DH-PS-induced IL-1ra production was shown to involve ERK/ELK, p38 MAPK, PI3K and NFκB. Finally, we observed that IL-1ra level induced by DH-PS was significantly higher than that by F3, a polysaccharide extract isolated from another popular Chinese herbal medicine, Ganoderma lucidum. These results indicated that DH-PS might have potential applications for ameliorating IL-1-induced pathogenic conditions.

INTRODUCTION : Il a été démontré que le nombre de lipoprotéines apolipoprotéine B (apoB) est un prédicteur du développement du diabète de type 2 (DT2), mais le mécanisme est inconnu. La résistance à l'insuline (RI) et l'hyperinsulinémie compensatoire (HI) entraînent l’épuisement des cellules β et la progression vers le DT2. De plus, l'activation du système de l'interleukine -1β (IL- 1β) est impliquée dans la pathophysiologie du DT2. Notre objectif était donc d'étudier si l’apoB est associé à la RI et à l’HI chez les humains et si cette corrélation est médiée par l’activation du système IL-1β. MÉTHODOLOGIE : 47 femmes ménopausées, non diabétiques, obèses ou en surpoids et 28 hommes, âgés de 45 à 74 ans ont été recrutés. La sécrétion d'insuline (SI) et la sensibilité à l'insuline ont été mesurées par un clamp Botnia modifié. La 1ère et 2ème phase de SI furent mesurées lors d'un test de tolérance au glucose intraveineux (IVGTT) d’une heure, suivi d’un clamp hyperinsulinémique euglycémique (HEIC) de 3 heures (taux de perfusion d'insuline de 75 mU/m2/min) pour mesurer la sensibilité à l'insuline lors des 30 dernières minutes du clamp (état d'équilibre). La sensibilité à l'insuline est exprimée comme étant le taux de perfusion de glucose (GIR) seul ou divisé par le taux d’insuline à l’état d’équilibre (M/I). RÉSULTATS : Chez les femmes, l’apoB à jeun corrélait avec une augmentation de la 2e phase de SI, la SI totale et la sécrétion totale de C-peptide (r=0,202; r=0,168; r=0,204) et avec une diminution de la sensibilité à l'insuline (GIR r=-0,299; M/I r=-0,180) indépendamment de l'adiposité. L’IL-1Ra à jeun (indicateur de l’activation du système IL-1β) corrélait positivement avec la 2e phase, la SI totale et la sécrétion totale de C-peptide (r=0,217; r=0,154; r=0,198) et négativement avec la sensibilité à l'insuline (GIR r=-0,304; M/I r=-0,214). L’IL-1Ra était également corrélée avec l'apoB (r=0,352). Une fois corrigé pour l'IL-1Ra, toutes les associations entre l'apoB et les indices de sensibilité à l'insuline et de SI ont été perdues. Malgré des glycémies similaires, il n’y avait pas de corrélation de l’apoB avec les indices mesurés chez les hommes. CONCLUSION : L’apoB est associé à l’HI et la RI chez les femmes non diabétiques obèses et en surpoids, potentiellement via l'activation du système IL-1β. Ces différences sexuelles doivent être prises en compte dans l'exploration de la physiopathologie du DT2.
INTRODUCTION: The number of plasma apolipoprotein B lipoproteins (apoB) is reported to predict the development of type 2 diabetes (T2D); however the underlying mechanism is unknown. Insulin resistance (IR) and compensatory hyperinsulinemia (HI) are believed to promote β-cell exhaustion and progression to T2D. Moreover, the activation of the interleukin-1β (IL-1β) system is implicated in the pathophysiology of T2D. Our aim was thus to investigate whether plasma apoB associates with IR and HI in humans and whether this is mediated through the IL-1β system. METHODOLOGY: 47 non-diabetic overweight and obese postmenopausal women and 28 men, 45-74 years old were recruited. Insulin secretion (IS) and insulin sensitivity were examined by a modified Botnia clamp. 1st and 2nd phase IS were measured during a 1 hour intravenous glucose tolerance test (IVGTT), followed by a 3 hour hyperinsulinemic-euglycemic clamp (HEIC, insulin infusion rate of 75 mU/m2/min) to measure insulin sensitivity during the last 30 minutes of the clamp (steady state). Insulin sensitivity was expressed as steady state glucose infusion rate (GIR) alone or divided over steady state plasma insulin (M/I). RESULTS: In women, fasting plasma apoB correlated positively with increased 2nd phase and total IS and with total C-peptide secretion (r=0.202; r=0.168; r=0.204 respectively) and negatively with insulin sensitivity (r: GIR= -0.299 and M/I =-0.180) independent of adiposity. Similar to plasma apoB, fasting plasma IL-Ra (indicator of activated IL-1β system) correlated positively with 2nd phase and total IS and with total C-peptide secretion (r=0.217; r=0.154; r=0.198 respectively) and negatively with insulin sensitivity (GIR r=-0.304; M/I r=-0.214). Fasting plasma IL-Ra also correlated with apoB r=0.352). Once corrected for IL-1Ra, the associations between apoB and the indexes of insulin sensitivity and IS were all lost. Despite similar fasting glucose, plasma apoB did not correlate with any indices of insulin secretion or sensitivity in men. CONCLUSION: ApoB is associated with HI and IR in non-diabetic overweight and obese women, which may be mediated through activation of the IL-1β system. Gender differences may need to be considered in exploring the pathophysiology of T2D in humans.

Einleitung Zytokine bewirken maßgeblich die Kommunikation und Koordination der zellulären und humoralen Effektorsysteme der angeborenen und erworbenen Immunität. Die Immunzellen stellen selbst die Hauptproduzenten der Zytokine dar. Pro- und anti-inflammatorische Zytokine nehmen nicht nur innerhalb der Zellkommunikation ablaufender Immun- und Entzündungsreaktionen eine Schlüsselrolle ein, sondern sind somit ebenso am Ablauf von Pathogenesen zahlreicher Erkrankungen beim Pferd beteiligt. Trotz verschiedener Studien anhand unterschiedlicher Modelle existiert keine einheitliche Datenlage zu validierten, vergleichbaren in vivo-nahen Zellkultursystemen, die es erlauben die Kinetiken equiner Zytokine als Grundlage zur weiterführenden Erforschung von Zytokinwechselwirkungen sowie zur Testung potentieller Arzneimittel abzubilden. Aktuell werden insbesondere Glukokortikoide weiterhin aufgrund ihrer anti-inflammatorischen und immunmodulatorischen Eigenschaften häufig, aber in Bezug auf Zytokine unspezifisch zur Therapie equiner Erkrankungen eingesetzt. Ziele der Untersuchung Das Ziel der vorliegenden Studie bestand darin, eine einfache, schnelle, günstige und reproduzierbare Methodik zur ex vivo-Messung von Zytokinen (Tumornekrosefaktor-alpha [TNF-α], Interleukin-1 Rezeptor-Antagonist [IL-1Ra] und Interferon gamma [IFN-γ]) und deren zeit- und konzentrationsabhängige Freisetzung in der equinen Vollblutzellkultur zu entwickeln. Anhand dessen sollte weiterführend der Effekt der Glukokortikoide Dexamethason (DEX) und Hydrocortison (HC) auf die Produktion von TNF-α, IL-1Ra und IFN-γ im equinen Vollblut untersucht werden. Zurzeit sind Zytokine, ihre Freisetzung sowie das Eintreten ihrer vermittelten Effekte Gegenstand der gegenwärtigen Forschung, mit dem Ziel spezifische Wechselwirkungen aufzuzeigen und somit zielgerichtete Therapeutika etablieren zu können. Insbesondere Pferde, die eine Anfälligkeit gegenüber mit Sepsis einhergehenden Erkrankungen oder für equines Asthma aufweisen, könnten davon profitieren. Material und Methoden Hierfür wurde Pferdevollblut, in den Verdünnungen zu 10%, 20% und 50% eingesetzt und mit Lipopolysaccharid (LPS), einer Kombination aus Phytohämagglutinin, Concanavalin A, Pokeweed-Mitogen und LPS (PCPwL) oder equinem rekombinantem TNF-a (erTNF-α) zur Zytokinfreisetzung stimuliert. Zur Erstellung der Zytokinkinetiken wurden Zellkulturüberstände zu verschiedenen Zeitpunkten gesammelt und die Konzentrationen von TNF-α, IL-1Ra und IFN-γ mit spezifischen enzyme-linked immunosorbent assays (ELISA) analysiert. In weiterführenden Versuchen wurden in der etablierten equinen Vollblutzellkultur DEX und HC in Konzentrationen von 10-12 - 10-5 M eingesetzt, um die LPS- oder PCPwL- induzierte Zytokinfreisetzung zu modulieren. Statistische Analysen erfolgten über die Berechnungen der Mittelwerte mit den dazugehörigen Standardfehlern. Signifikanzen wurden über ein- und zweifaktorielle ANOVA bestimmt. Ergebnisse Die durchgeführten Untersuchungen ergaben, dass die optimale Detektion der Zytokine in equinen Vollblutzellkulturen mit einem Blutanteil von 20% durchgeführt werden kann. TNF-α, IL-1Ra und IFN-γ wurden zeitabhängig freigesetzt und zeigten unterschiedliche Freisetzungskinetiken. Die PCPwL- induzierte TNF-α- und IL-1Ra-Freisetzung stiegen jeweils kontinuierlich über 24 - 48 Stunden an. In ähnlicher Weise erreichte die LPS- stimulierte TNF-α-Konzentration ein Maximum zu Zeitpunkten zwischen 8 - 12 Stunden und begann daraufhin abzufallen, wohingegen die Konzentration von IL-1Ra 24 Stunden später gipfelte und vielmehr fortgeführt über 48 Stunden hinaus akkumulierte. Equines rekombinantes TNF-α konnte ebenso die IL-1Ra-Freisetzung induzieren. Die PCPwL-induzierte IFN-γ-Freisetzung begann zeitversetzt und verlief kontinuierlich ansteigend über 48 - 72 Stunden. In weiterführenden konzentrationsabhängigen Untersuchungen konnte anhand der equinen Vollblutzellkultur eine stärkere Suppression der LPS-induzierten TNF-α- und IL-1Ra-Produktion sowie der PCPwL-induzierten IFN-γ-Produktion durch DEX als durch HC nachgewiesen werden. DEX hemmte die Zytokinfreisetzung mit einer mittleren inhibitorischen Konzentration (IC50) von 0,09 μM (TNF-α), 0,453 μM (IL-1Ra) und 0,001 μM (IFN-γ), während HC IC50 Werte von 1,45 μM (TNF-α), 2,96 μM (IL-1Ra) und 0,09 μM (IFN-γ) aufwies. Schlussfolgerungen Schlussfolgernd kann zusammengefasst werden, dass sich das Model der equinen Vollblutzellkultur hervorragend eignet, um nach erfolgreicher Mitogenstimulation den zeitabhängigen Freisetzungsverlauf von Zytokinen evaluieren zu können. Somit bietet das Model der equinen Vollblutzellkultur durch die Vorteile einer einfachen, günstigen Durchführung im in vivo-nahen, physiologischen Milieu, die Möglichkeit den Zytokinstatus gesunder sowie kranker Pferde zu beurteilen und stellt seinen Nutzen und die Verlässlichkeit unter Beweis potentielle Arzneimittel und immunologische Zusammenhänge des Pferdes untersuchen zu können.:INHALTSVERZEICHNIS I ABBILDUNGSVERZEICHNIS III TABELLENVERZEICHNIS III ABKÜRZUNGSVERZEICHNIS IV 1 EINLEITUNG 1 2 LITERATURÜBERSICHT 3 2.1 Allgemeine wissenschaftliche Hintergründe 3 2.1.1 Das Blut und das Immunsystem des Pferdes 3 2.1.1.1 Zusammensetzung des equinen Blutes 3 2.1.1.2 Allgemeiner Aufbau des Immunsystems 4 2.1.2 Zytokine und Entzündungsreaktionen - Mediation der Immunantwort durch Zytokine und Chemokine 14 2.1.2.1 Pro-inflammatorische Zytokine: Tumornekrosefaktor-α und Interferon-γ 17 2.1.2.2 Anti-inflammatorische Zytokine: Interleukin-1 Rezeptor-Antagonist 19 2.2 Therapeutische Beeinflussung der Zytokin- und Mediator-Freisetzung 21 2.2.1 Inhibition der Zytokinfreisetzung durch Glukokortikoide 21 2.2.2 Inhibition der Zytokinfreisetzung durch weitere Pharmaka und Substanzen 23 2.2.2.1 NSAID 23 2.2.2.2 Small molecules und Anti-Zytokinantikörper 24 2.3 Equine Zellkulturmodelle zur Zytokindetektion 25 2.3.1 Stimulation der Zytokinfreisetzung 26 2.3.2 Vollblutzellkultursysteme und Systeme mit isolierten Zellen 27 2.4 Fragestellung der Dissertation 30 3 PUBLIKATIONEN 31 3.1 Freisetzungskinetik von TNF-α und IL-1Ra im equinen Vollblut 32 3.2 Modulation der Freisetzung von TNF-α, IL-1Ra und IFN-γ in der equinen Vollblutzellkultur durch Glukokortikoide 40 4 DISKUSSION 45 4.1 Zytokinfreisetzung im equinen Vollblut 46 4.2 Der Einfluss von Glukokortikoiden auf die Zytokinfreisetzung 52 4.3 Schlussfolgerungen 56 4.4 Ausblick 56 5 ZUSAMMENFASSUNG 58 6 SUMMARY 60 7 LITERATURVERZEICHNIS 62 8 ANHANG 72 8.1 Freisetzungskinetik von IFN-γ 72 8.2 Konzentration von TNF-α, IL-1Ra und IFN-γ in der equinen Vollblutzellkultur 72 9 DANKSAGUNG 74
Introduction The communication and coordination between the cellular and humoral effector compartments of the innate and adaptive immunity were mainly accomplished by cytokines. Immunocompetent cells themselves represent the main source for cytokines. Pro- and anti-inflammatory cytokines not only play a pivotal role within the cell signaling of expiring immune- and inflammatory reactions but also take part in the pathogenesis of several equine diseases. Despite various studies based on different experimental setups no uniform availability of data about validated, comparable in vivo cell culture systems exists which enables the description of the kinetically time course of cytokines as foundation of further investigations of cytokine interactions as well as the testing of potential drugs. These days especially glucocorticoids are still frequently used for treatment of equine diseases because of their anti-inflammatory and immunomodulatory, but with respect to cytokines unspecific properties. Objectives of the investigations The aim of the study was to develop an easy, quick, cheap and reproducible ex vivo method for measuring cytokines (tumor necrosis factor alpha [TNF-α], interleukin-1 receptor antagonist [IL-1Ra] and interferon gamma [IFN-γ]) and their time- and concentration-dependent release in the equine whole blood cell culture. Whereby the impact of the glucocorticoids dexamethasone (DEX) and hydrocortisone (HC) on production of TNF-α, IL-1Ra and IFN-γ should be investigated subsequently. Currently, cytokines, their release and eventuation of their mediated effects are objects of actual research with the aim to reveal specific interactions and thus be able to establish purposeful therapeutic agents. This could be beneficial especially for horses which display a susceptibility to septic diseases or equine asthma. Material and Methods Therefore horse whole blood diluted to 10%, 20% and 50% was stimulated with lipopolysaccharide (LPS), a combination of phytohemagglutinin, concanavalin A, pokeweed mitogen and LPS (PCPwL) or equine recombinant TNF-α (erTNF-α). To generate cytokine kinetics TNF-α, IL-1Ra and IFN-γ were analyzed in culture supernatants, which were collected at different time points using specific enzyme-linked immunosorbent assays (ELISA). In further investigations within the equine whole blood cell culture DEX and HC were applied with concentrations between 10-12 and 10-5 M to modulate LPS- or PCPwL-induced cytokine release. Statistics were performed by calculation of means with associated standard errors. Statistical significances were assessed by one- and two-way analysis of variance. Results The evaluations revealed that cytokines could be detected optimal in whole blood cell cultures with 20% blood volume. TNF-α, IL-1Ra and IFN-γ were released time-dependently and differing kinetics were displayed. PCPwL-induced TNF-α and IL-1Ra release was enhanced continuously over 24 - 48 hours, respectively. Similarly, LPS-stimulated TNF-α was at maximum at time points between 8 - 12 hours and started to decrease thereafter, whereas IL-1Ra peaked 24 hours later and rather continued to accumulate beyond 48 hours. ErTNF-α could induce also the IL-1Ra release. PCPwL- induced IFN-γ release started time displaced and showed a continuously enhanced course over 48 - 72 hours. In subsequent investigations within equine whole blood cell culture, LPS-induced TNF-α and IL-1Ra as well as PCPwL-induced IFN-γ production were more potently suppressed concentration-dependently by DEX than by HC. DEX inhibited cytokine release with the inhibition concentration (IC50) 0.09 μM (TNF-α), 0.453 μM (IL-1Ra) and 0.001 μM (IFN-γ), whereas HC with IC50 values of 1.45 μM (TNF-α), 2.96 μM (IL-1Ra) and 0.09 μM (IFN-γ). Conclusion In conclusion our results could suggest the eminent suitability of equine whole blood cell culture to assess the release of a variety of cytokines following successful mitogen stimulation. Therefore the model of the equine whole blood cell culture provides, because of its advantages including simple and cheap performance in an in vivo close physiological ambient, the opportunity to evaluate the cytokine status of healthy and diseased horses. Furthermore it could give the proof of its benefit and reliability to evaluate potential equine drugs and immunological coherences of the horse.:INHALTSVERZEICHNIS I ABBILDUNGSVERZEICHNIS III TABELLENVERZEICHNIS III ABKÜRZUNGSVERZEICHNIS IV 1 EINLEITUNG 1 2 LITERATURÜBERSICHT 3 2.1 Allgemeine wissenschaftliche Hintergründe 3 2.1.1 Das Blut und das Immunsystem des Pferdes 3 2.1.1.1 Zusammensetzung des equinen Blutes 3 2.1.1.2 Allgemeiner Aufbau des Immunsystems 4 2.1.2 Zytokine und Entzündungsreaktionen - Mediation der Immunantwort durch Zytokine und Chemokine 14 2.1.2.1 Pro-inflammatorische Zytokine: Tumornekrosefaktor-α und Interferon-γ 17 2.1.2.2 Anti-inflammatorische Zytokine: Interleukin-1 Rezeptor-Antagonist 19 2.2 Therapeutische Beeinflussung der Zytokin- und Mediator-Freisetzung 21 2.2.1 Inhibition der Zytokinfreisetzung durch Glukokortikoide 21 2.2.2 Inhibition der Zytokinfreisetzung durch weitere Pharmaka und Substanzen 23 2.2.2.1 NSAID 23 2.2.2.2 Small molecules und Anti-Zytokinantikörper 24 2.3 Equine Zellkulturmodelle zur Zytokindetektion 25 2.3.1 Stimulation der Zytokinfreisetzung 26 2.3.2 Vollblutzellkultursysteme und Systeme mit isolierten Zellen 27 2.4 Fragestellung der Dissertation 30 3 PUBLIKATIONEN 31 3.1 Freisetzungskinetik von TNF-α und IL-1Ra im equinen Vollblut 32 3.2 Modulation der Freisetzung von TNF-α, IL-1Ra und IFN-γ in der equinen Vollblutzellkultur durch Glukokortikoide 40 4 DISKUSSION 45 4.1 Zytokinfreisetzung im equinen Vollblut 46 4.2 Der Einfluss von Glukokortikoiden auf die Zytokinfreisetzung 52 4.3 Schlussfolgerungen 56 4.4 Ausblick 56 5 ZUSAMMENFASSUNG 58 6 SUMMARY 60 7 LITERATURVERZEICHNIS 62 8 ANHANG 72 8.1 Freisetzungskinetik von IFN-γ 72 8.2 Konzentration von TNF-α, IL-1Ra und IFN-γ in der equinen Vollblutzellkultur 72 9 DANKSAGUNG 74

博士
國立中央大學
生命科學系
107
Macrophages and dendritic cells (DCs) are important in innate immune system, where they recognize pathogens and initiate inflammatory responses. Elevation of cAMP by inhibition of phosphodieasterases 4 (PDE4), enzymes that degrade cAMP with high affinity, in these cells is known to suppress various inflammatory responses. Among the four PDE4 isoforms (PDE4A-4D), PDE4B has been shown to play a major role in some of these responses. In this study, using PDE4 inhibitors and PDE4-deficient mice and cells, we demonstrate that PDE4B is involved in modulating additional immune responses in these cells under endotoxin lipopolysaccharide (LPS) stimulation. Activation of TLR4 by LPS is known to induce both pro-inflammatory and anti-inflammatory cytokine production in macrophages. Here in the first part of the study, we show that PDE4 inhibitors, such as rolipram, enhance the anti-inflammatory cytokine interleukin-1 receptor antagonist (IL-1Ra) secretion in LPS-stimulated mouse peritoneal macrophages, and this response was regulated at the transcriptional level rather than an increased IL-1Ra mRNA stability. Studies with PDE4-deficient macrophages revealed that the IL-1Ra upregulation elicited by LPS alone is PKA-independent, whereas the rolipram-enhanced response was mediated by inhibition of only PDE4B, one of the three PDE4 isoforms expressed in macrophages, and it requires PKA but not Epac activity. However, both pathways activate CREB to induce IL-1Ra expression. PDE4B ablation also promoted STAT3 phosphorylation (Tyr705) to LPS stimulation, but this STAT3 activation is not entirely responsible for the IL-1Ra upregulation in PDE4B-deficient macrophages. In a model of LPS-induced sepsis, only PDE4B-deficient mice displayed an increased circulating IL-1Ra, suggesting a protective role of PDE4B inactivation in vivo. These findings demonstrate that PDE4B negatively modulates anti-inflammatory cytokine expression in macrophages. In the second part of the study, we demonstrate that during in vitro differentiation of mouse bone marrow (BM) cells to immature DCs (imDCs), rolipram or ablation of PDE4B, but PDE4A or PDE4D, significantly reduced CD11c+ imDC population and the cell surface level of CD11c. The CD11c+ population was further decreased by rolipram and PDE4B ablation following LPS induction of DC maturation. Quantitative PCR analysis revealed that the mRNA expression of Toll-like receptor (TLR) 1, 2, 3, 6, 7, and 9 in BMDCs was markedly upregulated at 4 h of LPS stimulation and then declined to near or lower than basal levels at 24 or 36 h. Among these TLRs, the induction of TLR1, 6, 7, and 9 mRNA was greatly inhibited by rolipram and this inhibition was shown to be mediated mainly by inhibition of PDE4B, indicating an essential role of PDE4B in the expression of these innate immune receptors. In a murine model of TLR7 agonist-induced psoriasis, we further showed that PDE4 inhibitor significantly attenuated the severity of psoriatic symptoms in mice treated with imiquimod. These data demonstrate an important role of PDE4, particularly PDE4B, in regulation of DC development and functions under inflammatory conditions. Taken together, our findings provide further evidence that the development of PDE4B-selective inhibitor should retain the therapeutic benefits but devoid the side effects of non-selective PDE4 inhibitors.

La schizophrénie est une maladie mentale grave qui présente une comorbidité fréquente avec la toxicomanie et avec divers troubles immunitaires. Une méta-analyse réalisée récemment dans notre laboratoire a montré une augmentation d’IL-6 (une cytokine pro-inflammatoire), du récepteur soluble d’IL-2 (un marqueur d’activation du système immunitaire), et d’IL-1RA (une cytokine anti-inflammatoire) dans la schizophrénie, suggérant l’existence d’un syndrome inflammatoire dans cette maladie. La toxicomanie aussi est associée au dérèglement du réseau des cytokines inflammatoires, mais les effets dépendent du type de drogues et ils sont parfois diamétralement opposés. On dispose encore de peu d’informations sur le statut immunitaire et inflammatoire des patients qui ont un double diagnostic de schizophrénie et de toxicomanie. Le but de ce travail était d’explorer l’existence d’un état inflammatoire systémique chez les patients schizophrènes et toxicomanes, et l’influence du traitement avec un médicament antipsychotique atypique, la quétiapine. Les objectifs spécifiques étaient : 1) Mesurer les concentrations plasmatiques des cytokines inflammatoires chez les schizophrènes et toxicomanes avant, pendant et après traitement avec la quétiapine ; et 2) Faire des études de corrélations entre les taux de cytokines, les symptômes cliniques, et la consommation de drogues. Les résultats montrent que comparativement aux contrôles normaux, les patients avec un double diagnostic présentent une augmentation d’IL-6, d’IL-1RA, du sIL-2R et d’IL-8 avant traitement à la quétiapine. Les augmentations des concentrations plasmatiques d’IL-1RA sont particulièrement importantes chez les patients avec double diagnostic, si on les compare à celles publiées chez les schizophrènes sans toxicomanie. Le traitement à la quétiapine n’influence pas les concentrations plasmatiques de ces cytokines, sauf sIL-2R qui augmente davantage au cours du traitement. Des corrélations positives de puissance modérée sont retrouvées entre IL-6 et dépression, IL-6 et alcool, IL-1RA et cognition, IL-8 et dépression, IL-8 et alcool, sIL-2R et cannabis. Notre étude révèle que la réponse inflammatoire est activée chez les schizophrènes et toxicomanes. De plus, la toxicomanie semble jouer un rôle facilitant ou potentialisateur dans les augmentations des taux circulants d’IL-1RA. Les études en cours sur différentes populations de schizophrènes avec ou sans toxicomanie, et chez des toxicomanes non schizophrènes permettront de préciser le rôle des différentes drogues d’abus dans le syndrome inflammatoire chez les schizophrènes, ainsi que les implications de ce syndrome sur le plan clinique et thérapeutique.
Schizophrenia is a psychosis which presents a frequent comorbidity with substance use disorders (SUD) and with various immune alterations. Using meta-analysis, we have demonstrated previously establishment of an inflammatory syndrome in schizophrenia patients, illustrated by elevated circulating levels of IL-6 (a pro-inflammatory cytokine), sIL-2R (marker of immune activation) and IL-1RA (an anti-inflammatory cytokine). SUD is also associated with dysregulation of inflammatory cytokines, but the effects may depend on the type of substance of abuse. The goal of this project was: 1) To measure plasma concentrations of inflammatory cytokines in schizophrenia patients with comorbid SUD, before, during and after treatment with an atypical antipsychotic, quetiapine; and 2) To perform correlation studies between plasma concentrations of inflammatory cytokines and clinical symptoms, including positive and negative symptoms, cognition, depression and substance use. Relative to normal controls, patients with a dual diagnosis showed increased plasma concentrations of IL-6, IL-1RA, sIL-2R, and IL-8 at baseline, IL-1RA increases being the most important. Quetiapine treatment did not influence plasma cytokine concentrations, except sIL-2R which increased further. Moderate positive correlations were found between IL-6 and depression, IL-6 and alcohol, IL-1RA and cognition, IL-8 and depression, IL-8 and alcohol and between sIL-2R and cannabis. This study demonstrates that the immune and inflammatory response is activated in schizophrenia patients with comorbid SUD. Furthermore, SUD may play a facilitating or potentiating role in the increases in peripheral levels of IL-1RA. Ongoing studies in different patient populations with schizophrenia with or without SUD, and patients with SUD alone will help elucidate the role of different substances of abuse in the inflammatory syndrome in schizophrenia, as well as the clinical and therapeutic relevance of this syndrome.

La thérapie génique représente l'un des défis de la médecine des prochaines décennies dont la réussite dépend de la capacité d'acheminer l'ADN thérapeutique jusqu'à sa cible. Des structures non virales ont été envisagées, dont le chitosane, polymère cationique qui se combine facilement à l’ADN. Une fois le complexe formé, l’ADN est protégé des nucléases qui le dégradent. Le premier objectif de l'étude est de synthétiser et ensuite évaluer différentes nanoparticules de chitosane et choisir la mieux adaptée pour une efficacité de transfection sélective in vitro dans les cellules carcinomes épidermoïdes (KB). Le deuxième objectif de l'étude est d'examiner in vivo les effets protecteurs du gène de l'IL-1Ra (bloqueur naturel de la cytokine inflammatoire, l’Interleukine-1β) complexé aux nanoparticules de chitosane sélectionnées dans un modèle d'arthrite induite par un adjuvant (AIA) chez le rat. Les nanoparticules varient par le poids moléculaire du chitosane (5, 25 et 50 kDa), et la présence ou l’absence de l’acide folique (FA). Des mesures macroscopiques de l’inflammation seront évaluées ainsi que des mesures de concentrations de l’Interleukine-1β, Prostaglandine E2 et IL-1Ra humaine secrétés dans le sérum. Les nanoparticules Chitosane-ADN en présence de l’acide folique et avec du chitosane de poids moléculaire de 25 kDa, permettent une meilleure transfection in vitro. Les effets protecteurs des nanoparticules contenant le gène thérapeutique étaient évidents suite à la détection de l’IL-1Ra dans le sérum, la baisse d'expressions des facteurs inflammatoires, l’Interleukine-1 et la Prostaglandine-E2 ainsi que la diminution macroscopique de l’inflammation. Le but de cette étude est de développer notre méthode de thérapie génique non virale pour des applications cliniques pour traiter l’arthrite rhumatoïde et d’autres maladies humaines.
Considered to be one of the medical challenges of the coming decade, the success of gene therapy depends on the ability to deliver therapeutic DNA to target cells. Non-viral polymers, such as chitosan (Ch), a cationic polymer, can be easily combined with DNA. Once a complex is formed, DNA is protected from degradation by nucleases. The first objective of this study was to define the characteristics of the best-suited Ch nanoparticle for maximum selective transfection in human epidermoid carcinoma (KB) cells in vitro. Nanoparticles varied by the presence or absence of folic acid (FA) and Ch’s molecular weight (MW 5, 25 and 50 kDa). They were then selected and combined with interleukin-1 receptor antagonist (IL-1Ra) gene, a natural blocker of the inflammatory cytokine interleukin-1beta (IL-1β). The second objective was to inject these carriers by the hydrodynamic method in a rat model of adjuvant-induced arthritis and to evaluate the inhibitory effects of IL-1Ra against inflammation in vivo. Ch-DNA nanoparticles with FA and Ch25 demonstrated selective transfection and significantly increased it in KB cells in vitro. The inhibitory effects of IL-1Ra gene therapy in vivo were evident from lower expression levels of inflammatory factors (IL-1 and prostaglandin E2) and decreased macroscopic limb inflammation. The results also revealed the presence of human recombinant IL-1Ra protein in rat sera. Non-viral gene therapy with Ch-PEG-FA-DNA nanoparticles containing the IL-1Ra gene appears to significantly decrease inflammation in this experimental model of arthritis.