Дисертації з теми "Autoimmune pancreatiti"

Type 1 diabetes is a disease that results from a disturbed glucose metabolism due to a deficiency in insulin production. This deficiency is the consequence of immune-mediated damage to the insulin-producing p-cells. The cause of type 1 diabetes is presently unknown and probably multifactorial. The initiation and progression of the inflammatory process that destroys the p-cells involves the interplay of environmental factors with an autoimmune-prone genetic background. Abnormal immune regulation explains the autoimmune phenomena observed in diabetic patients and in spontaneous animal models for the disease to a limited extent only. The precise reason for the immune system to target the pancreatic islets of Langerhans is still unclear. The pathogenic process in the pancreas is characterized by the pathology-related intra-islet infiltration of T and B-lymphocytes that mediate islet destruction. This T and B-celUnfiltration is preceded by an accumulation of macrophages and dendritic cells at the islet periphery. The early peri-islet accumulation of these antigen presenting cells probably reflects the first response of the immune system that is progressively heading for islet destruction. Macrophages are involved in every step of the diabetogenic process. In the non-obese diabetic (NOD) mouse that spontaneously develops diabetes, various macrophage-abnormalities like defective maturation, reduced phagocytosis and increased production of IL-12, have been described previously. Moreover, macrophages are present in higher numbers in the pancreas of the NOD mouse from birth onwards, randomly distributed in the connective tissue and exocrine parenchyma. In this thesis we present the results of our studies on the murine pancreatic macrophage compartment. We have questioned in particular the possible underlying causes for the abnormal early peri-islet accumulation of macrophages. Therefore, the studies were performed with an emphasis on the interactions of macrophages with the extracellular matrix of the pancreatic connective tissue. In our first study we show that the depletion of macrophages and dendritic cells from the endocrine pancreas was accompanied by a total disappearance of lymphocytes from the pancreas. Hence, pancreatic macrophages and dendritic cells are critically involved in the local progression of islet inflammation in NOD mice by mediating the retention and possibly the recruitment of lymphocytes to the pancreas. Importantly, this depletion significantly postponed the onset of diabetes, leading to a strongly decreased incidence by 35 weeks of age. In the second study described, we have phenotypically characterized the pancreatic macrophages. The majority of macrophages were characterized by the expression of MMGL and sialoadhesin. Only a minority of these pancreatic macrophages expressed the macrophage marker F4/80 under non-inflammatory conditions. By contrast, macrophages expressing F4/80 were observed massively before the onset of destructive insulitis in the NOD pancreas, and their presence is particularly associated with islet destruction. This suggests that F4/80 identifies a particular subset of inflammatory macrophages in the pancreas that are virtually absent under non- inflammatory conditions. Interestingly, in the fetal pancreas mature macrophages were exclusively identified by their expression of F4/80 and these macrophages lacked the expression of MMGL and sialoadhesin. In addition, we present data that support the conclusion that macrophages develop from pre-existing precursors that are present in the fetal pancreas at E12. 5. Using an in vitro approach, we demonstrated that their numbers significantly increased in fetal pancreas explants cultured with M-CSF. This increase of F4/80-positive cells was paralleled by an increase in the number of insulin-producing cells, suggesting that macrophages support insulin-cell growth in vitro. These results are in line with the results of the third study that is presented in this thesis. Early postnatal pancreases from NOD mice are characterized by an increase in the percentage of endocrine tissue and by enlarged and irregularly-shaped islets, concomitantly with the presence of increased numbers of macrophages. Importantly, the levels of the extracellular matrix protein fibronectin are significantly increased during this pre-weaning period as well. Fibronectin labeling was mostly localized at the islet-ductal pole, islet periphery and in intralobular septa. Interestingly, pancreatic macrophages mainly reside at sites with intense fibronectin-labeling. In a fourth study we demonstrate that, paradoxically, NOD macrophages exhibit impaired fibronectin-mediated adhesion and migration due to the defective expression of the integrin-type fibronectin receptor a-chain, CD49d. In addition, we show that extracellular-regulated kinase-1/2 (ERK-1/2) is a negative regulator of CD49d expression in macrophages. NOD macrophages were characterized by increased levels of the activated form of this kinase when stimulated with the toll-like receptor-4 (TLR-4) ligand, lipopolysaccharide (LPS). LPS-stimulation resulted in the impaired upregulation of CD49d levels in NOD macrophages as compared to macrophages of other mouse strains. We believe that this specific defect in the macrophage compartment of NOD mice might play a role in the observed peri-islet accumulation. At the end of this thesis, we have postulated the working hypothesis that the observed presence of higher levels of the endogenous TLR-4 ligand fibronectin in the NOD pancreas in combination with the inappropriate TLR-4-responsiveness of NOD macrophages contributes to their local retention and activation. This unfortunate activation may damage the neighboring islets, unintentionally provoking an immune response eventually leading to the development of autoimmunity. We have uncovered several abnormalities that may trigger a mechanism that possibly contributes to the development of spontaneous autoimmune diabetes in the NOD mouse. Whether similar abnormalities are present in type 1 diabetic patients remains to be established
De pancreas, ook wel alvleesklier genoemd, is een in de buikholte gelegen orgaan met verschillende functies. Het grootste gedeelte van de pancreas bestaat uit exocrien weefsel dat verteringsenzymen uitscheidt in de dunne darm. Slechts iets meer dan één procent van de pancreas wordt gevormd door de eilandjes van Langerhans. Deze endocriene eilandjes produceren verschillende hormonen die worden uitgescheiden in het bloed. Het belangrijkste hormoon is insuline, gemaakt door de p-cellen die zijn gelegen in het midden van de eilandjes. Insuline verlaagt de glucose-spiegel en reguleert zo de opslag van energie uit ons voedsel. Patiënten met type 1 diabetes hebben een gevaarlijk hoge concentratie van glucose in het bloed door een tekort aan insuline. Dit tekort wordt veroorzaakt door de vernietiging van de p-cellen door het eigen afweersysteem en resulteert in een ernstige verstoring van het metabolisme. Deze immuunreactie tegen deze eiland-cellen ontstaat vaak al op heel jonge leeftijd en is onomkeerbaar. Patiënten met type 1 diabetes zijn daarom genoodzaakt hun verdere leven insuline te gebruiken. Er zijn verschillende afwijkingen gevonden in het immuunsysteem van type 1 diabetes patiënten, die gerelateerd zijn aan het onstaan van autoimmuniteit. Deze autoimmuniteit lijkt echter voornamelijk gericht te zijn tegen de endocriene eilandjes en daarom noemen we type 1 diabetes een orgaan-specifieke autoimmuunziekte. Waarom het immuunsysteem van type 1 diabetes-patiënten zich juist tegen de eigen p-cellen keert is niet duidelijk. Het kan zijn dat de oorzaak van de immuunrespons tegen de eilandjes in de pancreas zelf ligt. Om te bestuderen wat er in de pancreas plaatsvindt voordat de specifieke afweerreactie ontstaat, zijn we genoodzaakt onderzoek te doen in proefdieren. Niet-obese diabetische (NOD)-muizen ontwikkelen spontaan diabetes. Voorafgaand aan de specifieke immuunreactie tegen de p-cellen zien we in de pancreas van deze muizen veel niet-specifieke afweercellen rondom de eilandjes. Deze niet-specifieke immuuncellen worden macrofagen genoemd. Macrofagen zijn in alle organen aanwezig en vormen daar de eerste verdediging tegen pathogene indringers. Macrofagen ruimen bacteriën op en alarmeren andere cellen van het immuunsysteem om een afweerreactie tot stand te brengen. Deze afweerreactie moet specifiek gericht zijn tegen de pathogene indringers en niet tegen ons eigen weefsel. Dit laatste gebeurt helaas wel bij patiënten met type 1 diabetes. Er is echter onvoldoende bewijs dat de afweerreactie tegen de eilandjes samenhangt met een eerdere infectie. Omdat macrofagen mede onze afweerreactie aansturen, kunnen ze een heel belangrijke rol spelen in het ontstaan van autoimmuunziekte. In het onderzoek dat is beschreven in dit proefschrift, hebben we de macrofagen in de pancreas nader bestudeerd en onderzocht wat de oorzaak kan zijn van de vroege accumulatie van macrofagen rondom de eilandjes. We laten zien dat er verschillende typen macrofagen aanwezig zijn in de pancreas. In de volwassen muis bleek een bepaald type macrofaag specifiek geassocieerd met ontsteking en deze is in hoge mate aanwezig in de pancreas van de NOD muis. De pancreas macrofagen bevinden zich in het bindweefsel van de pancreas, tussen het exocriene weefsel maar ook rondom de eilandjes waar veel fibronectine aanwezig is. Fibronectine is een matrix eiwit wat enerzijds een rol speelt bij pancreas¬ontwikkeling en anderzijds bij de ontwikkeling en mobiliteit van macrofagen. In de pancreas van NOD muizen werden allereerst verhoogde concentraties fibronectine gevonden in de periode voorafgaand aan het ontstaan van de afweerreactie tegen de eilandjes. Verder laten wij zien dat de eilandjes in de pancreas van de NOD muis groter en grilliger van vorm zijn dan die in gezonde muizen. Deze afwijkingen kunnen samenhangen met het verhoogde aantal macrofagen dat in de NOD pancreas aanwezig is. In tegenstelling tot wat wij hadden verwacht, vertonen macrofagen van de NOD muis een verzwakte interactie met fibronectine. Ze hebben een verstoorde expressie van een van de eiwitten waarmee macrofagen aan fibronectine binden, het CD49d integrine. Dit leidt tot een defect in de beweeglijkheid van de NOD macrofagen over fibronectine en kan daardoor bijdragen aan de vroege accumulatie van de macrofagen rondom de eilandjes. De oorzaak van de defecte expressie van dit integrine ligt in een verstoorde aansturing van de expressie binnenin de cel. Deze verkeerde aansturing is ook betrokken bij de activatie van macrofagen door moleculen uit de bacteriewand. Stimulatie van de NOD macrofagen door deze moleculen of eiwitten die daar op lijken, leidt tot een verstoorde expressie van CD49d maar ook tot een verstoorde activatie van de macrofaag. Eén van de eiwitten die tot deze verstoorde activatie kan leiden is fibronectine, wat in verhoogde mate aanwezig is in de NOD pancreas. Deze factoren tezamen kunnen bepalend zijn voor de ongewenste beschadiging van de eilandjes door de macrofagen in de NOD muis. De orgaan-specifieke afwijkingen in combinatie met de door ons gevonden defecten in de NOD macrofaag kunnen het lokale evenwicht in de pancreas verstoren, mogelijk gevolgd door schade aan de eilandjes en de initiatie van een specifieke immuunrespons. De beschreven resultaten bieden mogelijk de basis voor een nieuwe hypothese voor het onstaan van lokale activatie van pancreas macrofagen. Ook macrofagen van patiënten met type 1 diabetes vertonen een verstoorde respons op hetzelfde ligand als door ons is getest in de NOD muis. Wij hopen dat met onze bevindingen in de NOD muis een nieuwe weg kan worden geopend in het onderzoek naar het ontstaan van type 1 diabetes in de mens

Type 1 diabetes (T1D) is a chronic autoimmune disease characterized by the immune-mediated destruction of insulin-producing beta-cells of pancreatic islets, culminating in critical insulin deficiency. Both genetic and environmental factors likely orchestrate an immune-mediated functional loss of beta cell mass, leading to the clinical manifestation of disease and lifelong dependence on insulin therapy. Additional evidence suggests the role of innate and adaptive immune mechanisms leading to inflammation in beta cells mediated by proinflammatory cytokines and chemokines, activation of beta-cell-reactive T cells,and failure of immune tolerance. Viral infections have been proposed as causal determinants or initiating triggers for T1D but remain unproven. Understanding the relationship between viral infections and the development of T1D is essential for T1D prevention. Importantly, virus-induced innate immune responses, particularly type I interferon (IFN-I, IFN-a/b), have been implicated in the initiation of islet autoimmunity and development of T1D. The goal of my thesis project is to investigate how the IFN-I signaling pathway affects the development of T1D using the LEW.1WR1 rat model of autoimmune diabetes. My hypothesis is that disrupting IFN-Isignaling via functional deficiency of the IFN-I interferon receptor (IFNAR) prevents or delays the development of virus-induced diabetes.For this purpose, I generated IFNAR subunit 1(IFNAR1)-deficient LEW.1WR1 rats using CRISPR-Cas9 genome editing and confirmed the functional disruption of IFNAR1. The absence of IFNAR1 results in a significant delay in onset and frequency of diabetes following poly I:C challenge and reduces the incidence of insulitis after poly I:C treatment. The frequency of diabetes induced by Kilham rat virus (KRV) is also reduced in IFNAR1-deficient LEW.1WR1 rats. Furthermore, I observe a decrease in CD8+T cells in spleens from KRV-infected IFNAR1-deficient rats relative to that in KRV-infected wild-type rats. While splenic regulatory T cells are depleted in WT rats during KRV-infection, no such decrease is observed in KRV-infected IFNAR1-deficient rats. A comprehensive bulk RNA-seq analysis reveals a decrease of interferon-stimulated genes and inflammatory gene expression in IFNAR1-deficient rats relative to wild-type rats following KRV challenge. Collectively, the results from these studies provided mechanistic insights into the essential role of virus-induced, IFN-I-initiated innate immune responses in the early phase of autoimmune diabetes pathogenesis.

Cyclin D3 and CDK11 are downregulated in pancreatic islet endocrine cells during the autoimmune attack progression in autoimmune-prone NOD (Non-obese diabetic) mouse strain. D-type cyclins are crucial in order to connect mitogenic signals with the Rb/E2F pathway, which regulates transcription of factors involved in further cell cycle progression. CDK11, protein-kinase PITSLRE, exhibits two gene products: p58 and p110 (p130 in mouse) in humans. CDK11p110 regulates transcription and RNA splicing. CDK11p110 is expressed in all cell cycle phases, while CDK11p58 is only expressed during mitosis (G2/M) and is essential in apoptosis. The interaction between CDK11p58 and cyclin D3 has been reported and it represses certain nuclear receptors action. This observation may suggest that in pancreatic beta cells simultaneous downregulation of cyclin D3 and CDK11 may obey to a coordinated regulation of both molecules. In this thesis we have studied whether there is a causal relationship between coordinated cyclin D3 and CDK11 downregulation and type 1 diabetes in vivo and in vitro.The outcome of our research will allow us to establish whether cyclin D3 and CDK11 are molecular targets in type1 diabetes
La ciclina D3 i CDK11 estan regulades a la baixa en les cèl•lules endocrines de l'illot pancreàtic durant la progressió atac autoimmune en la soca de ratolins NOD propensos a la diabetis autoimmune. Les ciclines de tipus D són crucials per connectar senyals mitogèniques amb la via Rb/E2F , que regula la transcripció de factors implicats en la progressió del cicle cel • lular . El gen que codifica per a la CDK11 , proteïna-quinasa PITSLRE, té dos productes gènics en humans: p58 i p110 ( p130 al ratolí ). LaCDK11p110 regula la transcripció i processament del 'ARN. La CDK11p110 s'expressa en totes les fases del cicle cel•lular , mentre que la CDK11p58 només s'expressa durant la mitosi (G2 / M) i participa en processos apoptòtics . La interacció entre CDK11p58 i ciclina D3 reprimeix certa acció d'alguns receptors nuclears, per exemple afecta negativament l'activitat transcripcional del receptor d'andrògens . Aquesta observació pot suggerir que en les cèl•lules beta del pàncrees la regulació a la baixa simultània de ciclina D3 i CDK11 pot obeir a una regulació coordinada de les dues molècules . En aquesta tesi s'ha estudiat si hi ha una relació causal entre la regulació a la baixa coordinada de ciclina D3 i CDK11 i l'aparició de la diabetis tipus 1 , in vivo i in vitro .El resultat de la nostra recerca ens permetrà establir si la ciclina D3 i la CDK11 podran ser blancs moleculars en la diabetis tipus 1.
Ciclina D3 y CDK11 están regulados a la baja en las células endocrinas del islote pancreático durante la progresión ataque autoinmune en la cepa de ratones NOD predispuesta genéticamente a la autoinmunidad. Los ciclinas de tipo D son cruciales para conectar las señales mitogénicas con la vía Rb/E2F, la cual regula la transcripción de factores implicados en la progresión del ciclo celular. El gen que codifica para la CDK11, la proteína-quinasa PITSLRE, tiene dos productos génicos en humanos: p58 y p110 ( p130 en el ratón). CDK11p110 regula la transcripción y procesameinto del ARN. CDK11p110 se expresa en todas las fases del ciclo celular, mientras que CDK11p58 sólo se expresa durante la mitosis (G2 / M) y está implicada en procesos apoptóticos. La interacción entre CDK11p58 y ciclina D3 reprime cierta acción de algunos receptores nucleare, por ejemplo, afecta negativamente a la actividad transcripcional del receptor de andrógenos. Esta observación puede sugerir que en las células beta del páncreas la regulación a la baja simultánea de ciclina D3 y CDK11 puede obedecer a una regulación coordinada de ambas moléculas. En esta tesis se ha estudiado in vivo e in vitro si existe una relación causal entre la regulación a la baja coordinada de ciclina D3 y CDK11 y la aparición de la diabetes tipo 1.El resultado de nuestra investigación nos permitirá establecer ciclina D3 y CDK11 como blancos moleculares en la diabetes tipo 1.

Background: Intestinal inflammation elicited by environmental determinants including dietary proteins and microbes is implicated in type 1 diabetes (T1D) pathogenesis. Also, intrinsic pancreatic abnormalities could precede classic insulitis, contributing to T1D. Materials and Methods: Spontaneous rat T1D models were used for in situ analyses of gut and pancreas to explore novel disease pathways using immunohistochemistry and detailed morphometry, gene expression studies, and molecular screening analyses. Results: In BBdp rats, feeding a cereal diet stimulated T1D under germ-free or specific pathogen-free (SPF) conditions compared with a protective hydrolyzed casein (HC) diet. Cereal-induced T1D was paralleled by increased gut T cell infiltration and TH1-associated pro-inflammatory transcription. HC-fed rats displayed an increased number of anti-inflammatory CD163+ M2 macrophages compared with cereal-fed rats. Cereal-associated promotion of T1D in Lewis diabetes-prone (LEW-DP) rats, a different rat model, similarly featured gut T cell infiltration in conjunction with decreased immunoregulation. The Camp gene was induced in diet-protected HC-fed BBdp rats. Camp encodes the cathelicidin antimicrobial peptide (CAMP), a pleiotropic immunomodulatory host defence factor. Intestinal CAMP was enriched in CD163+ M2 macrophages and could represent a novel marker of these tolerogenic innate immune cells. CAMP expression was also discovered in pancreatic lymph nodes (PLN) and islets, indicating a novel role for this factor in target tissue homeostasis. There was a positive correlation between pancreatic CAMP and total islet number. Also, islet-associated CAMP+ cells were increased in rats with islet inflammation, suggesting upregulation in parallel with insulitis. Exogenous CAMP/LL-37 injections increased the abundance of T1D-protective probiotic bacteria and promoted islet neogenesis in BBdp rats. A prospective partial pancreatectomy (PPx) study was performed to obtain pre-diabetic pancreas biopsies from iii pre-insulitic BBdp rats. The number of endothelium-associated CD68+ macrophages was increased in pre-diabetic pancreata, indicating that perivascular inflammation was an early lesion in the animals. In addition, pre-diabetic pancreata featured enhanced regenerative Reg3a and Reg3b gene expression, indicating abnormal islet expansion preceding insulitis. Conclusions: Small intestinal inflammation paired with deficits in local immunoregulation parallels T1D development. CAMP represents a novel factor in T1D that could have several pleiotropic functions including regulation of commensal microbes, intestinal homeostasis, and pancreatic homeostasis. In addition, target tissue abnormalities precede insulitis and T1D. This research focused on the integrative biology of T1D pathogenesis in spontaneous rat models. This work provides a novel working model that incorporates key roles for gut lumen antigens, intestinal immunity, and the role of islets and altered regenerative capacity in T1D. This research could lead to new therapeutic opportunities for T1D treatment.

La diabetis tipus 1 (DM1) és una malaltia autoimmune causada per la destrucció de cèl·lules productores d'insulina β en els illots pancreàtics, aquesta destrucció és mediada per limfòcits. En aquesta tesi es pretén donar a conèixer les dianes moleculars de les cèl·lules β responsables de la mort de les cèl·lules β causada per l'atac limfocític. Es van realitzar assajos de microarrays per tal d’identificar els gens expressats diferencialment en les cèl·lules endocrines d’illots, com a conseqüència de la infiltració insulítica, mitjançant la comparació de cèl·lules endocrines d’ illots provinents de ratolins NOD (Non Obese Diabetic)- model de ratolí predisposat genèticament a la diabetis autoimmune-, amb les provinents del seu homòleg lliure de limfòcits, NOD / SCID. Curiosament, vam descobrir que la ciclina D3 (CcnD3) es regula a la baixa a conseqüència de la inflamació d'una manera dosi-dependent, mentre que l'activitat proliferativa de les cèl·lules β no es veu alterada per aquesta disminució en l’expressió de ciclina D3. A més, les cèl·lules NIT-1 que sobreexpressen Ciclina D3 resten protegides de l'apoptosi espontània i de l'apoptosi induïda per un entorn proinflamatori proporcionat per IL-β. Per demostrar la relació de causalitat entre la repressió de CcnD3 repressió i la mort de les cèl·lules β in vivo, hem estudiat l’aparició de diabetis espontània en ratolins NOD deficients en CcnD3 (NODCcnD3KO). Els ratolins NODCcnD3KO desenvolupen diabetis exacerbada en comparació amb companys de llodrigada de tipus salvatge (WT) (88% enfront de 61% respectivament), i aquest fet deu únicament a la deficiència de CcnD3 a les cèl·lules β i no a un augment de diabetogenicitat del repertori limfocític dels ratolins NODCcnD3KO. No obstant això, l'exacerbació de la diabetis requereix de la complicitat de tots dos, la deficiència CcnD3 i la inflamació, ja que els ratolins NOD/SCIDCcnD3KO, els quals no tenen limfòcits, no desenvolupen diabetis espontània. CcnD3 té també un paper essencial en la fisiologia les de cèl·lules β del pàncrees, ja que illots pancreàtics deficients en CcnD3 no experimenten canvis adients en les concentracions intracel·lulars de Ca2+ en resposta a concentracions canviants de glucosa en el medi, el qual denota una sensibilitat deficient a la glucosa en aquests illots. Aquest deteriorament d'acoblament concentració de glucosa en el medi extracel·lular i l’augment a la concentració intracel·lular de Ca2+ no és a causa dels canvis en els nivells d’expressió del transportador de glucosa GLUT-2 entre illots NODCcnD3 KO i NODWT. D'altra banda, hem desenvolupat ratolins transgènics que sobreexpressen ciclina D3 en cèl·lules β pancreàtiques (NOD/RIPCcnD3). Aquests ratolins mostren protecció contra la diabetis autoimmune i rescaten ratolins NOD CcnD3KO de desenvolupar diabetis exacerbada.
Type 1 Diabetes (T1D) is an autoimmune condition caused by the lymphocytemediated destruction of insulin-producing β cells in pancreatic islets. This thesis aims to unveil the final targets in the β cells responsible for the β cell loss caused by the lymphocytic attack. Microarray studies were performed to assess differentially expressed genes in islet endocrine cells as a consequence of insulitic infiltration by comparing the autoimmune-prone Non-Obese Diabetic (NOD) mouse model with its congenic, lymphocyte-free, NOD/SCID strain. Interestingly, we discovered that cyclin D3 (CcnD3) underwent downregulation in beta cells upon inflammatory insult in a dose-dependent manner, while the proliferative activity of beta cells downregulating CcnD3 was not altered. Moreover, NIT-1 cells stably overexpressing CcnD3 were protected from spontaneous apoptosis and from apoptosis induced by a proinflammatory environment provided by IL-1β. To demonstrate the causal link between CcnD3 repression and β cell death in vivo, we studied spontaneous diabetes onset in CcnD3 deficient NOD mice (NODCcnD3KO). NODCcnD3KO mice developed exacerbated diabetes compared to the wild type (WT) littermates (88% versus 61% respectively), and this fact was solely due to the CcnD3 deficiency in the β cells and not to an increased diabetogenicity of the NODCcnD3KO lymphocytic repertoire. However, diabetes exacerbation required the complicity of both CcnD3 deficiency and inflammation, since plain NOD/SCID CcnD3KO mice did not develop spontaneous diabetes. CcnD3 also plays an essential role in pancreatic β-cell fitness, since pancreatic islets deficient in CcnD3 do not experience proper intracellular Ca2+ influx changes in response to different concentrations of glucose. This impairment, coupling glucose concentration in the extracellular milieu and intracellular increase in Ca2+ concentration, is not due to changes in Glut-2 expression levels between the CcnD3 KO and WT islets. Moreover, we developed transgenic mice overexpressing cyclin D3 in pancreatic β cells (NOD/RIPCcnD3). These mice exhibit protection from autoimmune diabetes and NOD CcnD3KO mice are prevented from developing exacerbated diabetes.
La diabetes tipo 1 (T1D) es una enfermedad autoinmune causada por la destrucción de las células β productoras de insulina en los islotes pancreáticos por parte de los linfocitos. En esta tesis pretendemos dar a conocer dianas moleculares de las células β responsables de pérdida de la célula β causadas por el ataque linfocitico. Desarrollamos un estudio de microarreglos en el cual encontramos genes expresados diferencialmente en células endócrinas del islote como consecuencia de la infiltración insulítica mediante la comparación de un modelo de ratón autoinmune susceptible No Obeso Diabético (NOD) con una cepa congénica libre de linfocitos NOD/SCID. Interesantemente, descubrimos que, la ciclina D3 (CcnD3) experimentó una disminución como consecuencia de un asalto inflamatorio de forma dosis dependiente, mientras que la actividad de proliferación de las células β no fue alterada. Además, células NIT-1 que sobreexpresan CcnD3 fueron protegidas de apoptosis espontánea y apoptosis inducida por un entorno proinflamatorio proporcionado por la IL-1β. Para demostrar la relación causal entre la represión de la CcnD3 y la muerte de las células β in vivo, hemos estudiado la aparición de la diabetes espontánea en ratones NOD deficientes en CcnD3 (NODCcnD3KO). Los ratones NODCcnD3KO desarrollan una diabetes exacerbada en comparación con sus compañeros de camada de tipo salvaje (WT) (88% contra el 61% respectivamente) y este hecho debido únicamente a la deficiencia de CcnD3 de las células β y no a un aumento de diabetogenicidad del repertorio linfocítico de los ratones NODCcnD3KO. Sin embargo, la exacerbación de la diabetes requiere la complicidad de ambos, la deficiencia de CcnD3 e inflamación, ya que los ratones NOD/SCIDCcnD3KO, los cuales carecen de linfocitos, no desarrollan diabetes espontánea. CcnD3 tiene también un papel escencial en la fisiología de las células β del páncreas, ya que los islotes deficientes de CcnD3 no experimentan cambios apropiados en las concentraciones intracelulares de Ca2+ en respuesta a diferentes concentraciones de glucosa en el medio. Este deterioro en el acoplamiento de la concentración de glucosa en el medio extracelular y aumento de la concentración intracelular de Ca2 + no es debido al cambio del nivel de expresión del transportador de GLUT-2 entre islotes NODCcnD3 KO y NODWT. Por otro lado, hemos desarrollado ratones transgénicos que sobreexpresan la ciclina D3 en células β pancreáticas (NOD/RIPCcnD3). Estos ratones muestran protección contra la diabetes autoinmune y rescatan a ratones NOD CcnD3KO de desarrollar diabetes exacerbada.

In type 1 diabetes (T1D) a combination of genetic predisposition and environmental factors triggers islet inflammation (insulitis) leading to a selective and gradual destruction of the pancreatic beta cells. Beta cells mainly die through apoptosis, triggered at least in part by pro-inflammatory cytokines such as IL-1β, TNF-α and IFN-γ. Recent findings suggest that the mitochondrial pathway of cell death is involved in this death cascade. Array analysis indicated that TNF-α+IFN-γ induces transcription factors such as NF-ĸB, STAT1, and AP-1 in beta cells. We presently aimed to examine the pathway(s) of apoptosis triggered by TNF-α+IFN-γ in beta cells.

TNF-α+IFN-γ induces beta cell apoptosis through the intrinsic pathway of cell death. This involved activation of the BH3 only proteins DP5, PUMA and Bim. Knockdown (KD) of either DP5 or PUMA or both led to a partial protection of INS-1E cells (12-20%), while silencing Bim led to about 60% protection against cytokine-induced apoptosis. Bim is transcriptionally induced by activated STAT1. TNF-α+IFN-γ also induces downregulation of Bcl-XL, an anti-apoptotic Bcl-2 gene which inhibits Bim. Knocking down Bcl-XL alone led to increase in apoptosis, but this was prevented by the parallel KD of Bim.

The ultimate goal of our research is to protect beta cells from the autoimmune assault. Previous data revealed that JunB inhibits ER stress and apoptosis in beta cells treated with IL-β+IFN-γ. Here, TNF-α+IFN-γ up-regulated the expression of JunB which was downstream of activated NF-ĸB. JunB KD exacerbated TNF-α+IFN-γ induced beta cell death in primary rat beta cells and INS-1E cells. The gene networks affected by JunB were studied by microarray analysis. JunB regulates 20-25% of the cytokine-modified beta cell genes, including the transcription factor ATF3 and Bcl-XL. ATF3 expression was increased in cytokine-treated human islets and in vitro silencing of JunB led to >60% reduction in ATF3 overexpression. We confirmed direct JunB regulation of the ATF3 promoter by its binding to an ATF/CRE site. Silencing of ATF3 aggravated TNF-α+IFN-γ induced cell death in beta cells and led to the downregulation of Bcl-XL expression in INS-1E cells. Pharmacological upregulation of JunB using forskolin led to upregulation of ATF3 and consistent protection of these cells against cytokine-induced cell death, while genetic overexpression of JunB in mice increased ATF3 expression in the pancreatic islets and reversed the pro-apoptotic effects of cytokines on beta cells (±40 % protection).

As a whole, our findings indicate that TNF-α+IFN-γ triggers beta cell apoptosis by the upregulation of the pro-apoptotic protein Bim and downregulation of the Bcl-XL protein. These deleterious effects are at least in part antagonized by JunB via activation of ATF3.

Dans le diabète de type 1 (DT1), la combinaison de facteurs génétiques de prédisposition et de l'environnement déclenche l'inflammation des îlots de Langerhans (insulite) conduisant à une destruction sélective et progressive des cellules bêta du pancréas. Les cellules bêta meurent principalement d’apoptose, déclenchée au moins en partie par les cytokines pro-inflammatoires sécrétées par les cellules immunitaires comme l’IL-β, le TNF-α l’IFN-γ. De récentes découvertes suggèrent que la voie mitochondriale de la mort cellulaire jouerait un rôle dans la mort de ces cellules. L'analyse de réseaux de gène utilisant les biopuces d’ADN indique que l’association TNF-α+IFN-γ induit l’activation de facteurs de transcription tels que NF-ĸB, STAT1 et AP-1 dans la cellule bêta. Dans ce contexte, nous avons cherché à examiner les voies de l'apoptose déclenchées par le TNF-α+IFN-γ dans la cellule bêta.

En présence de TNF-α+IFN-γ les cellules bêta meurent par apoptose via la voie intrinsèque. L’activation des protéines pro-apoptotiques « BH3-seulement » dont DP5, PUMA et Bim étaient en cause de cette apoptose. Le « knockdown »1 (KD), de DP5 ou de PUMA, ou des deux en même temps conduit à une protection partielle des cellules INS-1E (12-20%), tandis que le KD de Bim conduit à environ 60% de protection contre l’apoptose induite par cette combinaison de cytokines. La transcription de Bim est induite par STAT1 activé. Parallèlement à la régulation positive de Bim, TNF-α+IFN-γ conduit à la régulation négative de la protéine Bcl-XL. Bcl-XL est une protèine anti-apoptotique de la famille de protèines Bcl-2 qui en general inhibe Bim. Réduire l’expression de Bcl-XL seul induit une augmention de l'apoptose, alors que le KD de Bim et Bcl-XL en parallèle empêche l'apoptose.

Le but ultime de notre recherche est de protéger les cellules bêta des agressions autoimmunitaires. Les données antérieures ont révélé que JunB inhibe le stress du réticulum endoplasmique et l'apoptose dans les cellules bêta traitées avec IL-β+IFN-γ. Nous avons observé que TNF-α+IFN-γ induit l'expression de JunB qui se produit en aval de NF-ĸB activé. Il est important de noter que l’inactivation de JunB par des agents interférants de l’ARN (siRNA) exacerbe la mort des cellules primaires bêta de rat et de cellules INS-1E induite par les cytokines. Les réseaux de gènes touchés par JunB ont été étudiés grâce a l'analyse en microréseaux. JunB règule 20-25% des gènes modifiés par des cytokines dans les cellules bêta, y compris le facteur de transcription ATF3 et Bcl-XL. L’expression d’ATF3 est augmenté dans les îlots humains traités avec les cytokines et la répression in vitro de JunB conduit à une réduction de >60% de l’expression d’ATF3. Nous avons confirmé la régulation d’ATF3 par JunB en montrant que JunB est directement lié au promoteur d’ATF3 via le site ATF/CRE. La diminution d’expression d’ATF3 en presence de TNF-α+IFN-γ a aggravé la mort cellulaire induite dans les cellules bêta et a conduit à la régulation négative de l'expression de Bcl-XL dans les cellules INS-1E. L’augmentation pharmacologique de JunB dans les cellules INS-1E par l’utilisation de forskolin a conduit à la régulation positive en aval d’ATF3 et par conséquente à la protection de cellules bêta vis-a-vis de effets indésirables des cytokines. Dans cette optique, la surexpression génétique de JunB dans le modèle Ubi-JunB de souris transgénique a conduit à une surexpression d’ATF3 dans les îlots pancréatiques et a permir d’inverser les effets pro-apoptotiques de cytokines sur la cellule bêta (protection ± 40%).

Globalement, ces résultats indiquent que TNF-α+IFN-γ déclenche l'apoptose des cellules bêta par la régulation positive du gène pro-apoptotique Bim et la régulation négative du gène anti-apoptotique Bcl-XL. Ces effets indésirables sont inhibé en partie par JunB via l’activation de ATF3.

1Pas d’équivalent en français. Signifie la réduction de l’expression d’un gène via utilisation d’un siRNA (agent interférant de l’ARN).

Doctorat en Sciences biomédicales et pharmaceutiques
info:eu-repo/semantics/nonPublished

La pancreatite autoimmune rappresenta a tuttoggi una patologia di difficile inquadramento diagnostico ed approccio terapeutico. Infatti, il trattamento steroideo si è dimostrato efficace nella terapia a breve termine, ma non è in grado di prevenire le recidive di malattia. Esiste quindi una sottopopolazione di pazienti che necessita di un trattamento con immunosoppressori. Le caratteristiche cliniche, bioumorali e strumentali di questa sottopopolazione di pazienti a rischio di recidiva sembrano essere rappresentate dall’età avanzata, dall’ittero all’esordio, dall’aumento delle IgG4 sieriche e dalla presenza di una malattia sistemica coinvolgente altri organi, che sembrano quindi rappresentare dei fattori prognostici di recidiva. In questi pazienti, un trattamento con immunosoppressori è indicato. L’azatioprina sembra essere il trattamento di scelta, sia dai risultati del nostro studio, sia da quelli riportati in letteratura. L’uso dell’azatioprina sembra non esporre a maggior rischio di pancreatite, almeno nella casistica da noi riportata e da quelle presenti in letteratura. La revisione metanalitica degli studi sul rischio di pancreatite nei pazienti con IBD trattati con azatioprina, ci permette di non affermare in maniera definitiva che la pancreatite acuta sia un evento avverso in questa popolazione di pazienti. E’ al contrario, più verosimile che possa trattarsi di iperenzimemie pancreatiche o, forse, espressione di un coinvolgimento autoimmunitario del pancreas. Infine il profilo clinico della pancreatite autoimmune associata a colite ulcerosa è differente da quello dei pazienti senza tale patologia. Le recidive di malattia sembrano essere non differenti in maniera significativa in questi due gruppi di 58 pazienti. Pertanto, la presenza di colite ulcerosa non è un criterio per non trattare questi pazienti. Dal momento che la pancreatite autoimmune associata a colite ulcerosa è nella maggior parte dei pazienti di tipo 2, anche questa forma di malattia può giovarsi di un trattamento con immunosoppressori. Studi prospettici multicentrici sono tuttavia necessari al fine di rendere le conclusioni della nostra ricerca definitive.
Autoimmune pancreatitis (AIP) is a disease with difficult diagnostical and therapeuthical approach. Indeed, steroid theraphy is effective in the short term treatment, but ii do not prevent the disease relapse. A part of AIP patients need therefore a long term immunosuppressive therapy. Clinical, biochemical and instrumental characteristic of patients at risk for relapse seems to be the presence of jaundice at clinical onset, increase of serum levels of IgG4, and the extrapancreatic involvement, that seems therefore to be prognostic factor for relapse. In these patients, immunosuppressant is indicated. Azathioprin has been suggested the treatment of choice on the basis of the results of our study. Furthermore, our systematic review and our results on AIP patients treated whit this drug seems not to increase the risk of pancreatitis during the treatment of azathioprin. The clinical profile of AIP patients suffering from ulcerative colitis is not different from the other AIP patients. Since ulcerative colitis may be treated with immunosuppressant, its presence do not exclude a possible treatment with azathioprin. Prospective multicenter studies are suggested to confirm our conclusions.

Chronic pancreatitis is considered a risk factor for pancreatic cancer. An exact mechanism how chronic inflammation of the pancreas leads to pancreatic cancer is not yet understood; the possibility of a shared genetic predisposition for both diseases is also assumed. A similar association in patients with AIP has not yet been demonstrated. The aim of our work was to expand the knowledge about relationship between chronic pancreatitis and pancreatic cancer. We studied the association of the diseases in two synchronous projects. In the first one, we examined the occurrence of pancreatic cancer in patients with autoimmune pancreatitis. In the second project, we investigated the presence of genetics variants associated with chronic pancreatitis in patients with pancreatic cancer. In the retrospective study of our cohort of patients, we were one of the very first in the world to show occurrence of pancreatic cancer in patients with autoimmune pancreatitis, and as the only one, we have defined the characteristics of such patients. To assess the association of the diseases, we performed a systematic review where we identified all reported cases of coincidence of pancreatic cancer and autoimmune pancreatitis; the incidence of cancer in patients with autoimmune pancreatitis was similar to that of patients...

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2015
Background: A doença relacionada com a imunoglobulina G4 (DR-IgG4) é uma entidade sistémica caracterizada por elevação da IgG4 sérica, hipertrofia dos orgãos afectados com infiltração linfoplasmocítica maciça com plasmócitos IgG4-positivos e fibrose tecidular. A pancreatite autoimune (PAI) é uma forma de pancreatite com provável etiologia autoimune que pode apresentar envolvimento difuso ou focal do pâncreas. A PAI tipo 1 é a manifestação pancreática da DR-IgG4 e a Tipo 2 uma doença específica do pâncreas. Na forma focal a PAI partilha características com o adenocarcinoma do pâncreas. Objectivos: Realizar uma revisão do conhecimento actual da PAI quanto à patogénese, epidemiologia, características clínicas, histológicas, imagiológicas, manifestações extrapancreáticas da DR-IgG4 e tratamento. Focam-se as descobertas recentes quanto ao diagnóstico e diagnóstico diferencial com adenocarcinoma do pâncreas. Métodos: Pesquisa na base de dados da Pubmed usando as palavras DR-IgG4 e PAI. Seleccionei apenas os artigos entre 2006-2015. Conclusões: Apesar da intensa investigação, o diagnóstico diferencial entre PAI e adenocarcinoma do pâncreas ainda é desafiador para os clínicos. A sua correcta diferenciação pode evitar cirúrgia desnecessária em doentes com PAI, pois estes podem ser tratados com sucesso com corticoesteroides e rituximab. Se não for possível excluir com segurança o adenocarcinoma do pâncreas a cirurgia é mandatória.
Background: IgG4-Related Disease (IgG4-RD) is a systemic disease characterized by elevation of serum IgG4, enlargement of the affected organs with lymphoplasmacitic infiltration with IgG4-positive plasmocytes and tissue fibrosis. Autoimmune pancreatitis (AIP) is a form of pancreatitis with probable autoimmune etiology that can present as focal or diffuse form of involvement. Type 1 AIP is the pancreatic manifestation of IgG4-RD whereas Type 2 is a pancreas-specific disorder. The AIP focal form shares features with pancreatic cancer. Objectives: We intend to make a brief review on the current knowledge about AIP regarding patogenesis, epidemiology, histologic, clinical and imagiological features, laboratory findings, extrapancreatic involvement and treatment. We will focus the scientific breakthroughs in the last years concerning the diagnosis and differential diagnosis with pancreatic adenocarcinoma. Methods: Selective literature research in Pubmed database regarding IgG4-RD and AIP. Only those articles between 2006 and 2015 were selected. Conclusion: Although the intensive investigation in the last years, the differential diagnosis between AIP and pancreatic adenocarcinoma is still a challenge for clinicians. The correct differentiation between them can prevent unnecessary surgery in AIP patients since these can be successfully treated conservatively with steroids and rituximab. If pancreatic adenocarcinoma cannot be excluded with safety surgery is mandatory.

OBIETTIVI: La pancreatite autoimmune (AIP) di tipo 1 rappresenta una possibile manifestazione della malattia IgG4-relata (IgG4-RD), la cui diagnosi è a volte ancora difficile da formulare. I plasmablasti circolanti sono stati proposti come possibile marcatore sierico sensibile e specifico, anche se non è chiaro se possano differenziare tra AIP di tipo 1, di tipo 2 e altre condizioni pancreatiche. Obiettivo dello studio è stato valutare il valore diagnostico dei livelli di plasmablasti totali e di IgG4+ circolanti nell'AIP e in altre malattie del pancreas. METODI: Sono stati arruolati prospetticamente da Gennaio 2018 a Maggio 2020 pazienti con diagnosi di AIP di tipo 1 attiva (Gruppo AIP-1, n = 19) secondo i criteri diagnostici internazionali, insieme a pazienti affetti da AIP attiva di tipo 2 (AIP 2) o non altrimenti specificata (AIP NOS) (Gruppo AIP 2/NOS, n=10) adenocarcinoma pancreatico (Gruppo PDAC, n=17), pancreatite cronica (Gruppo CP, n=20), neoplasia mucinosa papillare intraduttale (IPMN) o iperenzimemia pancreatica cronica asintomatica (CAPH) (Gruppo IPMN-CAPH, n=21) come gruppi di controllo. La citometria a flusso è stata utilizzata per misurare la conta dei plasmablasti totali e dei plasmablasti IgG4+ nel sangue periferico utilizzando i seguenti anticorpi per le cellule CD45+CD19+CD38hiCD20-CD24-CD27+ e CD45+CD19+CD38hiCD20-CD24-CD27+ IgG4+. Inoltre, queste cellule sono state misurate nei pazienti con AIP dopo un mese di terapia, dopo 2-4 mesi dalla fine del trattamento e dopo un anno dall'arruolamento. RISULTATI: Il gruppo AIP-1 ha mostrato livelli significativamente più alti di plasmablasti totali (media 6365, SD 5522 cellule/mL) rispetto sia al gruppo PDAC (media 3216, SD 1228 cellule/mL) (p=0.0067) che al gruppo IPMN-CAPH (media 1065, SD 781 cellule/mL)(p<0.0001). Tuttavia, la conta dei plasmablasti totali non era significativamente differente nel gruppo AIP 1 rispetto a AIP 2/NOS. I plasmablasti IgG4+ hanno invece distinto il tipo 1 da tutti gli altri gruppi, incluso l'AIP di tipo 2, con una sensibilità dell'80% e una specificità del 97% utilizzando un cut-off di 210 cellule IgG4+/mL. Inoltre, i livelli di plasmablasti IgG4+ diminuiscono significativamente dopo la terapia. CONCLUSIONI: Solo i plasmablasti IgG4+ sembrano essere un biomarcatore potenzialmente utile per differenziare l'AIP di tipo 1 dall'AIP di tipo 2/NOS e da altre condizioni pancreatiche, in particolare il PDAC.
OBJECTIVES: Type 1 autoimmune pancreatitis (AIP) is a manifestation of IgG4-related disease (IgG4-RD) whose diagnosis is still challenging. Circulating plasmablasts seem to be a useful tool in this setting. Whether they may differentiate type 1, type 2 AIP, and other pancreatic conditions is still unknown. The aim of the study was to investigate the diagnostic value of circulating total and IgG4+ plasmablasts levels in AIP and other pancreatic diseases. METHODS: Patients diagnosed with active type 1 AIP (Group AIP-1, n=19) according to International Consensus Diagnostic Criteria from January 2018 to May 2020, were prospectively enrolled together with patients suffering from active type 2 or not otherwise specified AIP (Group AIP 2/NOS, n=10) pancreatic adenocarcinoma (Group PDAC, n=17), chronic pancreatitis (Group CP, n=20), intraductal papillary mucinous neoplasia (IPMN) or chronic asymptomatic pancreatic hyperenzymemia (CAPH) (Group IPMN-CAPH, n=21) as control groups. Flow cytometry was used to measure total plasmablasts’ and IgG4+ plasmablasts’ counts by gating peripheral blood for CD45+CD19+CD38hiCD20-CD24-CD27+ cells and CD45+CD19+CD38hi CD20-CD24-CD27+IgG4+ cells. Moreover, these cells were measured after one month of therapy, after 2-4 months from the end of treatment, and after one year from the enrollment in AIP patients groups only. RESULTS: Group AIP-1 showed significantly higher levels of total plasmablasts (mean 6365, SD 5522 cells/mL) compared to both Group PDAC (mean 3216, SD 1228 cells/mL)(p=0.0067) and Group IPMN-CAPH (mean 1065, SD 781 cells/mL)(p<0.0001). However, plasmablasts count was not significantly higher in Group AIP 1 compared to AIP 2/NOS. IgG4+ plasmablasts distinguished type 1 AIP from all other pancreatic disorders with a sensitivity of 80% and a specificity of 97% using a cut-off of 210 IgG4+ cells/mL. IgG4+ plasmablasts significantly decrease after steroids. CONCLUSIONS: Only IgG4+ plasmablasts may be a potentially useful biomarker to differentiate type 1 AIP from type 2 or NOS AIP and other pancreatic conditions, especially PDAC.

Introduzione: La ricerca svolta nel corso di questo dottorato è stata orientata verso la valutazione della pancreatite associata a mutazioni geniche e della pancreatite autoimmune. Nell’ambito dello studio della pancreatite associata a mutazioni geniche, l’indirizzo di ricerca si è orientato (1) al confronto tra l’evoluzione clinico-morfologica di queste forme rispetto alle pancreatiti ad altra eziologia e (2) alla valutazione delle caratteristiche radiologiche delle calcificazioni pancreatiche che si osservano nelle fasi più avanzate di malattia. Nell’ambito dello studio della pancreatite autoimmune, l’indirizzo di ricerca si è orientato (3) alle caratteristiche cliniche e all’evoluzione nel tempo della malattia, differenziando la forma diffusa da quella formante massa e (4) alla ricerca di un marcatore sierico. Risultati e conclusioni: (1) Studio delle mutazioni dei geni CFTR, SPINK1 e PRSS1 con valutazione clinica radiologica e funzionale di 34 pazienti portatori di mutazioni geniche confrontati con 164 pazienti affetti da pancreatite cronica e negativi ai tests genetici. Conclusioni: a.L’outcome clinico dei pazienti affetti da pancreatite cronica associata a mutazioni geniche sembra essere differente da quanto osservato nei pazienti affetti da pancreatite cronica ad altra eziologia. b.Lo sviluppo di insufficienza pancreatica esocrina ed endocrina sembra essere più tardivo nel gruppo di pazienti affetti da positivi ai tests genetici. c.L’alcol, anche in piccole quantità, ed il fumo di sigaretta influenzano in modo considerevole lo sviluppo di calcificazioni pancreatiche nei pazienti con mutazioni geniche. (2) Valutazione del ruolo della TC Addome nell’individuazione di calcoli con aspetto “bull’s eye” per distinguere i pazienti affetti da pancreatite cronica associata a mutazioni geniche rispetto a quelli con tests genetici negativi. Conclusioni: il riscontro alla TC di calcoli di grandi dimensioni (>15 mm) e struttura a “bull’s eye” è fortemente suggestivo della presenza di una mutazione genica associata alla pancreatite cronica, in particolare in assenza di altri fattori eziopatogenetici. (3) Analisi delle caratteristiche cliniche e radiologiche di una serie prospettica di pazienti affetti da AIP (87 tot., di cui 55 affetti da forma focale di malattia e 32 da forma diffusa), seguiti per un lungo periodo di tempo, ponendo attenzione tra le due forme di malattia. Conclusioni: a.La forma focale e quella diffusa presentano differenze cliniche significative. b.La colite ulcerosa è la patologia autoimmune più frequentemente associata alla pancreatite autoimmune. c.Le ricorrenze di malattia sono più frequenti nei pazienti anziani, affetti dalla forma focale, in particolare se fumatori ed in presenza di elevati livelli di IgG4 sieriche. d.L’insorgenza d’insufficienza pancreatica esocrina ed endocrina non è correlata alla chirurgia pancreatica e sembra essere progressiva, suggerendo la natura cronica della malattia, anche in assenza di segni e sintomi clinici. (4) Identificazione di un marcatore sierico in grado di discriminare la pancreatite autoimmune focale dall’adenocarcinoma pancreatico. A tale scopo è stato utilizzato un approccio di biologia molecolare già applicato con risultati soddisfacenti nello studio di altre malattie autoimmuni. Conclusioni: abbiamo identificato un peptide di 7 amminoacidi, che presenta una analogia con una proteina dell’H.pylori, riconosciuto da quasi tutti i sieri dei pazienti affetti da pancreatite autoimmune. Tale peptide è in grado di discriminare questi pazienti da quelli affetti da altre patologie infiammatorie e neoplastiche pancreatiche, in particolare da quelli affetti da adenocarcinoma del pancreas, e da altre patologie autoimmuni.
Introduction: the aim of the studies was to evaluate chronic pancreatitis associated to gene mutations and autoimmune pancreatitis. In chronic pancreatitis associated to gene mutations, the search was addressed to evaluate (1) clinical-morphological evolution and (2) radiological characteristic of pancreatic calcifications compared with chronic pancreatitis negative to genetic tests. In autoimmune pancreatitis, the search was addressed (3) to evaluate clinical aspects and evolution of diffuse and focal forms and (4) to find a serological marker of the disease. Results and conclusions: (1) Radiological, clinical and functional investigation of 34 patients suffering from chronic pancreatitis associated with CFTR, SPINK1 and PRSS1 genes mutations compared with 164 patients with chronic pancreatitis and negative genetic tests. Conclusions: a.The clinical outcome of patients suffering from chronic pancreatitis associated with genes mutations seems to be different from those with negative genetic tests. b.The onset of pancreatic exocrine and endocrine insufficiency seems to be delayed in patients with chronic pancreatitis and gene mutations. c.Alcohol, even in small quantities, and cigarette smoking consumption enhance the onset of pancreatic calcifications in patients with chronic pancreatitis associated with gene mutations. (2)Role of CT in the evaluations of the presence of pancreatic calcifications to distinguish 16 patients suffering from chronic pancreatitis associated with gene mutations from 32 with negative genetic tests. Conclusions: diameter of pancreatic calcifications (>15 mm) and “bull’s eye” aspects are strongly correlated with positive genetic tests. (3) Clinical and radiological characteristic of patients suffering from 87 patients suffering from autoimmune pancreatitis (55 of focal and 32 of diffuse type), followed for a long period of time. Conclusions: a.Focal and diffuse type of the disease are clinically different. b.Ulcerative colitis is the most common autoimmune disease associated with autoimmune pancreatitis. c.Recurrences of the disease are more commonly observed in aged patients, with focal form, in smokers and in patients with elevated serum level of IgG4. d.The onset of exocrine and endocrine pancreatic insufficiency is not related to surgery and seems to be progressive, suggesting that the process is chronic even in the absence of clinical signs. (4) Identification of a serological marker able to discriminate between autoimmune pancreatitis and pancreatic adenocarcinoma. Conclusions: a 7 amminoacids-peptide, that present a homology with a H.pylori protein, was recognized by serum of patients suffering from autoimmune pancreatitis. This peptide is able to discriminate these patients from those suffering from inflammatory and neoplastic pancreatic diseases, particularly from pancreatic adenocarcinoma, and from other autoimmune diseases.

La pancreatite autoimmune (AIP) è una forma particolare di malattia infiammatoria del pancreas che può coinvolgere diffusamente o focalmente il parenchima pancreatico. Sul piano anatomopatologico AIP è stata classificata in AIP tipo 1 e AIP tipo 2. La pancreatite autoimmune può essere associata con la colite ulcerosa (UC) (20-30%) e questa associazione si correla con AIP tipo 2. Circa il 30 % dei pazienti affetti da AIP va incontro ad una recidiva di malattia e le recidive sono state osservate nei pazienti con AIP tipo 1. L’utilizzo di immunosopressori è stato suggerito come terapia della pancreatite autoimmune recidivante ma solo pochi studi che riportano l’uso della azatioprina , con limitato numero di pazienti sono stati pubblicati fino ad oggi. L’azatioprina è stata proposta come arma terapeutica per il trattamento della AIP anche se l’AZA è stata indentificata come farmaco inducente la pancreatite. Lo scopo primario di questo studio è stato di valutare molti aspetti di questa problematica. In particolare è stata valutata 1. l’efficacia della azatioprina nella pancreatite recidivante; 2. la frequenza di associazione tra AIP e UC e le diverse caratteristiche in ambito clinico, strumentale e del outcome della AIP nei pazienti con e senza UC; 3.le caratteristiche istologiche della mucosa colica dei pazienti affetti con AIP nel sospetto di una infiammazione cronica intestinale (IBD) e le carrateristiche distintive tra pazienti con AIP- IBD e pazienti IBD; 4. il rischio di pancreatite in pazienti con IBD trattati con azatioprina. I risultati principali di questi studi permettono di formulare le seguenti conclusioni: 1. il profilo clinico e strumentale dei pazienti con AIP trattati con AZA è diverso rispetto a quello dei pazienti non trattati. Le caratteristiche distintive sono sovrapponibili con i fattori prognostici riportati in letteratura. Tuttavia è stato identificato un sottogruppo di pazienti che potrebbe trarre beneficio dalla terapia immunosoppressiva. 2. la colite ulcerosa si associa con AIP e pazienti AIP con IBD recidivano ugualmente come i pazienti senza IBD e comunque possono essere trattati con AZA 3. le caratteristiche anatomopatologiche compresa l’immunoistochimica per plasmacellule IgG4+ sulla mucosa colica dei pazienti AIP-IBD sono simili a quelle dei pazienti IBD. 4. I risultati di una revisione sistematica indicano un aumentato rischio di pancreatite nei pazienti IBD trattati con AZA. Riassumendo la definizione della pancreatite in questo studio è insufficiente e non permette nessuna conlusione definitiva.
Autoimmune pancreatitis (AIP) is a particular form of inflammatory pancreatic disease that may involve diffusely or focally the pancreatic parenchyma. Pathologically, AIP has been classified in type 1 and type 2 disease. AIP may be associated with ulcerative colitis (UC) (20-30%) and this association is related to AIP type 2. About 30% of patients suffering from AIP experienced a clinical relapse of the disease, and relapses have been observed in AIP type 1. Non-steroidal immunosuppressant drugs has been suggested as treatment of relapsing AIP, but only reports with limited number of patients have been published up to now using azathioprine (AZA). AZA has been proposed in the treatment of AIP, despite AZA has been identified as a drug-inducing pancreatitis. The aim of this study has been to evaluate many aspects of this issue,particularly: 1. the efficacy of azathioprine in relapsing AIP; 2. the frequency of association between AIP and UC and differences in clinical, instrumental and outcome of AIP patients with and without UC; 3. histological findings of colon mucosa of AIP patients with suspected colon inflammation (inflammatory bowel disease – IBD) and distinctive features between AIP-IBD and IBD; 4. risk of pancreatitis in patients suffering from IBD treated with azathioprine. The main results of these investigations seems to allow the following conclusions: 1. the clinical and instrumental profile of AIP patients treated with AZA is different compared to those not-treated. The distinctive features found are the same of prognostic factors suggested in the literature. A subgroup of patients who may benefit from immunosuppressive treatment has been identify. 2. Ulcerative colitis is associated with AIP and AIP patients with IBD relapses as well as AIP patients without IBD and therefore can be treated with AZA. 3. The pathological features, including immunohistochemistry for IgG4+ plasmacells, of colon mucosa of AIP-IBD patients is similar to those suffering from IBD. 4. The results of systematic review indicate an increased risk of pancreatitis in IBD patients treated with AZA. However, the definition of pancreatitis in this study is far too low and does not allow any definitive conclusion.

Trabalho de Projeto do Mestrado Integrado em Medicina apresentado à Faculdade de Medicina
Introdução: A Pancreatite Autoimune (PAI) representa, na prática clínica, uma patologia de relevância crescente, embora persistam algumas reservas quanto à sua definição e caracterização. Inicialmente, a elevação sérica da fração IgG4, destacar-se-ia como possível marcador específico. Contudo, foi comprovado que a especificidade inerente a este e outros marcadores séricos era limitada. Dada a complexidade e ausência de um consenso diagnóstico,foram inicialmente criados os critérios HISORt. Posteriormente, os International Consensus Diagnostic Criteria for Autoimmune Pancreatitis (ICDC) foram apresentados e definidos como a guideline internacional para o diagnóstico, classificação e tratamento da PAI. Contudo, o diagnóstico de PAI continua a ser complexo, exigindo um elevado grau de suspeição clínica e dependendo, frequentemente, da colheita de material para estudo histológico. Objetivos: O objetivo primordial deste projeto consistiu em estabelecer uma relação entre o diagnóstico de PAI e a presença ou ausência de níveis elevados de IgG4. Foi ainda elaborado um estudo comparativo entre os critérios HISORt e ICDC, para averiguar as principais diferenças subjacentes a estes. Estabeleceu-se a respetiva acuidade diagnóstica, utilizando como gold standard a conjugação de dados clínicos, analíticos, imagiológicos, histológicos e a resposta à terapêutica. Materiais e Métodos: Foram analisados todos os doentes com ≥18 anos no momento do diagnóstico, submetidos a doseamentos de IgG4, de 2012 a 2015. Os dados incluíram variáveis demográficas, clínicas, analíticas, imagiológicas, histológicas e terapêuticas. Resultados: Foram identificados 1110 doentes com doseamentos de IgG4, 117 (10,5%) dos quais apresentavam níveis >135mg/dL. O diagnóstico definitivo de PAI foi estabelecido em 13 doentes, dos quais 6 (46,2%) apresentavam níveis normais de IgG4. Os critérios HISORt demonstraram uma sensibilidade de 30,8%, na identificação de doentes com PAI, face à sensibilidade de 46,2% apresentada pelos critérios ICDC. 28,5% dos doentes classificados pelos critérios ICDC como diagnósticos prováveis/definitivos de PAI Tipo I apresentaram doseamentos de IgG4 positivos. Conclusão: A IgG4 revelou pouca utilidade no diagnóstico de PAI. A aplicação dos dois grupos de critérios revelou uma inferioridade diagnóstica dos critérios HISORt face aos ICDC. Uma maior independência face à serologia da IgG4 e a capacidade de diferenciação dos dois subtipos de PAI foram as duas principais características que puderam justificar a discrepância existente entre os critérios HISORt e os ICDC.
Introduction: Autoimmune Pancreatitis (AIP) remains a growing relevant pathology in the clinical practice, albeit the ambiguity regarding its definition and characterization still prevails. Initially, the increase of the IgG4 fraction was thought to be its main specific marker. Nevertheless, it was proved that the specificity inherent to this particular and other serologic markers was limited. Due to the complexity and lack of a diagnostic consensus, the HISORt criteria were designed. Thereafter, the ICDC criteria were delineated and presented as the International Guideline for the diagnosis, classification and treatment of AIP. Notwithstanding, the diagnosis of AIP remains complex, demanding a high level of clinical suspicion and frequently requiring histological analysis of gathered samples. Objectives: The primordial objective of this project consisted in establishing a relation between the diagnosis of AIP and the presence or absence of increased IgG4 levels. A comparative study between the HISORt and ICDC criteria was also performed, in order to investigate the main differences regarding these two classifications. The diagnostic accuracy was assessed by a gold standard made up of clinical, analytical, imagiological, histological and therapeutic response data. Materials and Methods: All the patients, aged ≥18 at the time of the diagnosis and to whom the levels of IgG4 were determined between 2012 and 2015, were included. Demographic, clinical, analytical, imagiological, histological and therapeutic variables were considered. Results: 1110 patients with IgG4 determinations were identified, of which 117 (10.5%) presented levels >135mg/dL. 6 (46.2%) within the 13 patients established with a definitive diagnosis of AIP featured normal IgG4 levels. The HISORt criteria revealed a 30.8% sensitivity in identifying patients with AIP, whilst the ICDC criteria presented a 46.2% sensitivity. 28.5% of those considered, by the ICDC criteria, as probable/definitive Type I AIP had positive IgG4 determinations. Conclusion: IgG4 revealed to be of limited value on the diagnosis of AIP. The application of the two criteria demonstrated a diagnostic inferiority when comparing the HISORt to the ICDC. A major independence regarding the IgG4 serology and the ability to discriminate the two distinct AIP subtypes were the two main features that could justify the discrepancy between the HISORt and ICDC criteria.

博士
國防醫學院
醫學科學研究所
99
Heme oxygenase 1 (HO-1) has strong antiapoptotic, anti-inflammatory and antioxidative effects that help protect cells against various forms of immune attack. We investigated whether the transgenic expression of HO-1 in pancreatic  cells would protect nonobese diabetic (NOD) mice from autoimmune damage and prolong graft survival following islet transplantation. We generated an insulin promoter-driven murine HO-1 (pINS-mHO-1) transgenic NOD mouse model to evaluate the protective effect of  cell-specific HO-1 in autoimmune diabetes. Transgene expression, insulitis and the incidence of diabetes in mice were characterized. Lymphocyte composition, the development of Th1, Th2 and Treg cells, T cell proliferation and lymphocyte-mediated disease transfer were analyzed. The potential effects of transgenic islets and islet transplantation on apoptosis, inflammation and the generation of reactive oxygen species (ROS) were evaluated. Transgenic mice showed less severe insulitis and a lower incidence of diabetes than nontransgenic control littermates. Lymphocyte compositions and functions were not affected. Islets from transgenic mice expressed lower levels of proinflammatory cytokines/chemokines, proapoptotic gene expression and amounts of ROS, and were more resistant to TNF- and IFN--induced apoptosis. Moreover, islet grafts from transgenic mice survived longer in diabetic recipients than control islets. Transgenic overexpression of HO-1 in  cells protected NOD mice from diabetes and delayed the autoimmune destruction of islet grafts, providing valuable insight toward the development of better strategies for clinical islet transplantation in patients with type 1 diabetes.