Are the severe injuries of cutaneous leishmaniasis caused by an exacerbated Th1 response?

American tegumentary leishmaniasis (ATL) is a disease whose clinical features are strongly related to the type of immune response it induces. Herein we report an atypical presentation of cutaneous leishmaniasis in a woman with a severe and extensive sore located in her leg, and we describe the differences between the usual local immune response in ATL and the local immune response in this patient. We observed an intense inflammatory response characterized by Th1 cells and cytokines with conspicuous expression of Toll-like receptor 3 (TLR-3). Few parasites were present, but there was an extensive tissue damage. We also discuss the immunological factors that could be related to the atypical presentation.

Espressione dell'Enzima ad Azione Immunoregolatoria Indoleamina 2,3-Diossigenasi nella Leucemia Mieloide Acuta dell'Adulto: correlazioni Clinico-Biologiche

L’enzima IDO interviene nella via di degradazione del triptofano, essenziale per la vita cellulare; l’iperespressione di IDO favorisce la creazione di un microambiente immunotollerante. Nelle LAM IDO è funzionalmente attivo nelle cellule blastiche e determina l’acquisizione di un fenotipo regolatorio da parte delle cellule T alloreattive; l’espressione della proteina aumenta in modo consensuale con l’evoluzione clinica della patologia. Scopo della Tesi è indagare l’esistenza di una correlazione tra l’iperespressione di IDO da parte delle cellule leucemiche, le caratteristiche di rischio alla diagnosi e l’outcome dei pazienti. Sono stati esaminati 45 pazienti adulti affetti da LAM afferiti all’Istituto di Ematologia di Bologna. I pazienti sono stati stratificati a seconda di: età di insorgenza della leucemia, secondarietà a Mielodisplasia o radio chemioterapia, iperleucocitosi, citogenetica, biologia molecolare (sono state valutate le alterazioni a carico dei geni FLT3 ed NPM). I pazienti sono stati analizzati per l’espressione del gene IDO mediante RT-PCR, seguita da Western Blot, allo scopo di stabilire la presenza di una proteina attiva; successivamente si è proceduto a verificare l’esistenza di una correlazione tra l’espressione di IDO e le caratteristiche di rischio alla diagnosi per identificare una relazione tra l’espressione del gene ed un subset di pazienti a prognosi favorevole o sfavorevole. Dei 45 pazienti adulti affetti da LAM il 28,9% è risultato negativo per l’espressione di IDO, mentre il rimanente 71,1% è risultato positivo ed è stato suddiviso in tre ulteriori categorie, in base ai livelli di espressione. I dati non sembrano al momento suggerire l’esistenza di una correlazione tra l’espressione di IDO e le caratteristiche di rischio alla diagnosi. Nel gruppo di pazienti ad elevata espressione di IDO si riscontra un rate di resistenza alla chemioterapia di induzione più elevato, con una quota di pazienti resistenti pari al 71,4%, contro il 23,1% nel gruppo di pazienti IDO-negativi.

Die Rolle der Ubiquitin-Ligase Smurf2 in T-Lymphozyten bei der Entwicklung von chronisch entzündlichen Darmerkrankungen

TGF-beta ist ein Schlüsselmolekül zellvermittelter Immuntoleranz. So spielt es neben seiner pleiotropen Rolle in Immunzellen auch bei der Tumorentwicklung eine große Rolle. Das TGF-beta hat bei der Tumorentwicklung eine duale Rolle. So dient es in frühen Phasen als Tumorsuppressor, währenddessen es in späten Phasen der Entwicklung als Tumorpromotor wirkt. Eine strikte Regulation des TGF-beta Signalweges ist daher für ein funktionierendes Immunsystem von essentieller Bedeutung. Die Ubiquitin Ligase Smurf2 ist dabei ein wichtiger negativ Regulator des TGF-beta Signalweges.In der vorliegenden Arbeit konnte eine neue Spleißform des Smurf2 (dE2Smurf2) aus murinen CD4+ T-Zellen isoliert werden, deren Funktion in vitro und in vivo in T-Lymphozyten untersucht worden ist. Für diese Spleißform konnte zudem eine humane Relevanz nachgewiesen werden. Mit Hilfe von Überexpressionen in Cos7 Zellen konnte eine veränderte Lokalisation der Smurf2 Spleißformen (WT und dE2) festgestellt werden. Dabei konnten lysosomale und endosomale Kompartimente bei der Kolokalisation mit dem dE2Smurf2 Konstrukt beobachtet werden. Das Spleißen des Exons2 führte dabei zu Änderungen der Topologie der N-terminalen C2-Domäne, wodurch sich eine veränderte Lokalisation in der Zelle beschreiben ließ. Mit der veränderten intrazellulären Verteilung erfuhr auch die Funktion der dE2Smurf2 Ubiquitin Ligase eine Änderung. So konnte überraschenderweise eine positive Signalinduktion des TGF-beta Signalweges beobachtet werden, was im Gegensatz zum beschriebenen WTSmurf2 stand. Durch eine Überexpression des dE2Smurf2 Proteins in T-Lymphozyten wurde der TGF-beta Signalweg in CD4+ und CD8+ Zellen positiv reguliert, dabei wurde der TGFbetaRII vermehrt exprimiert und gleichzeitig fand eine verstärkte Phosphorylierung der Transkriptionsfaktoren Smad2 und Smad3 nach TGF-beta Stimulation statt. Die transgenen T-Lymphozyten waren somit sensitiver gegenüber TGF-beta. Dies führte zur Hypothese, die durch Western Blot Analyse bestätigt werden konnte, daß das dE2Smurf2 nach Überexpression seine WT-Form bindet und dadurch degradiert. Die Degradation der Ubiquitin Ligase war dabei Smad7 abhängig. Zur Analyse des Einflusses der Ubiquitin Ligase dE2Smurf2 auf die Differenzierung von CD4+ T-Zellen, sowie ihre Rolle bei der T-Zell Proliferation, konnte gezeigt werden, daß durch die höhere Sensitivität gegenüber TGF-beta naive T-Zellen unter Einfluß von TGF-beta und IL6 vermehrt in TH17 Zellen differenzierten. Zudem konnte gezeigt werden, daß die Proliferationsrate transgener naiver CD4+ T-Zellen bei geringen Mengen von TGF-beta starkt vermindert war. Weiterhin konnte gezeigt werden, daß bei einer Differenzierung der naiven CD4+ T-Zellen in TH1 Zellen, diese signifkant weniger das proinflammatorische Zytokin INFγ produzierten.So zeigten in vivo Versuche, daß die transgenen Tiere in der Entwicklung von Kolorektalen Karzinomen protektiert waren. Sowohl im kolitisassiziierten Tumor Modell als auch bei der spontanen Entwicklung von Tumoren im APCmin Modell. Dies konnte zum einen auf eine deutlich verminderte Entzündung (geringere Produktion an Zytokinen durch verminderte Proliferation) des Darms und zum anderen durch eine stärkere Produktion an zytotoxischen Genen, wie Perforin, INFγ und Granzym B erklärt werden. Interessanterweise konnte jedoch im Transfer Kolitis Modell eher eine proinflammatorische Wirkung des dE2Smurf2 Proteins nachgewiesen werden. So wiesen die immundefizienten Mäuse, in denen die transgenen T-Zellen injiziert wurden, eine signifikant stärkere Kolitis auf als die Kontrollen. Dies konnte mit einer Überproduktion an IL17 sezernierenden T-Zellen erklärt werden. Klonierungsexperimente führten zudem zur Identifikation einer bisher nicht beschriebenen nicht kodierenden RNA. Diese zeigte in Kombination mit dem dE2Smurf2 Protein in einer Reportergen Analyse eine Hyperaktivierung des Smad3 Promotors. Diese Daten liefern zum einen ein genaueres Modell über die Regulation des TGF-beta Signalweges sowie wichtige Erkenntnisse zur Pathophysiologie chronisch entzündlicher Darmerkrankung und daraus resultierende Tumorerkrankungen. So entwickelt sich das dE2Smurf2, Teil des TGF-beta Signalweges, als attraktives Zielprotein für die Modulation von chronisch entzündlichen Darmerkrankungen und (kolitisassoziierte) Kolonkarzinomen.

Individualisierte Krebstherapie : präklinischer „proof of concept“ einer mutationsspezifischen Immuntherapie

Da nicht-synonyme tumorspezifische Punktmutationen nur in malignen Geweben vorkommen und das veränderte Proteinprodukt vom Immunsystem als „fremd“ erkannt werden kann, stellen diese einen bisher ungenutzten Pool von Zielstrukturen für die Immuntherapie dar. Menschliche Tumore können individuell bis zu tausenden nicht-synonymer Punktmutationen in ihrem Genom tragen, welche nicht der zentralen Immuntoleranz unterliegen. Ziel der vorliegenden Arbeit war die Hypothese zu untersuchen, dass das Immunsystem in der Lage sein sollte, mutierte Epitope auf Tumorzellen zu erkennen und zu klären, ob auf dieser Basis eine wirksame mRNA (RNA) basierte anti-tumorale Vakzinierung etabliert werden kann. Hierzu wurde von Ugur Sahin und Kollegen, das gesamte Genom des murinen B16-F10 Melanoms sequenziert und bioinformatisch analysiert. Im Rahmen der NGS Sequenzierung wurden mehr als 500 nicht-synonyme Punktmutationen identifiziert, von welchen 50 Mutationen selektiert und durch Sanger Sequenzierung validiert wurden. rnNach der Etablierung des immunologischen Testsysteme war eine Hauptfragestellung dieser Arbeit, die selektierten nicht-synonyme Punktmutationen in einem in vivo Ansatz systematisch auf Antigenität zu testen. Für diese Studien wurden mutierte Sequenzen in einer Länge von 27 Aminosäuren genutzt, in denen die mutierte Aminosäure zentral positioniert war. Durch die Länge der Peptide können prinzipiell alle möglichen MHC Klasse-I und -II Epitope abgedeckt werden, welche die Mutation enthalten. Eine Grundidee des Projektes Ansatzes ist es, einen auf in vitro transkribierter RNA basierten oligotopen Impfstoff zu entwickeln. Daher wurden die Impfungen naiver Mäuse sowohl mit langen Peptiden, als auch in einem unabhängigen Ansatz mit peptidkodierender RNA durchgeführt. Die Immunphänotypisierung der Impfstoff induzierten T-Zellen zeigte, dass insgesamt 16 der 50 (32%) mutierten Sequenzen eine T-Zellreaktivität induzierten. rnDie Verwendung der vorhergesagten Epitope in therapeutischen Vakzinierungsstudien bestätigten die Hypothese das mutierte Neo-Epitope potente Zielstrukturen einer anti-tumoralen Impftherapie darstellen können. So wurde in therapeutischen Tumorstudien gezeigt, dass auf Basis von RNA 9 von 12 bestätigten Epitopen einen anti-tumoralen Effekt zeigte.rnÜberaschenderweise wurde bei einem MHC Klasse-II restringierten mutiertem Epitop (Mut-30) sowohl in einem subkutanen, als auch in einem unabhängigen therapeutischen Lungenmetastasen Modell ein starker anti-tumoraler Effekt auf B16-F10 beobachtet, der dieses Epitop als neues immundominantes Epitop für das B16-F10 Melanom etabliert. Um den immunologischen Mechanismus hinter diesem Effekt näher zu untersuchen wurde in verschieden Experimenten die Rolle von CD4+, CD8+ sowie NK-Zellen zu verschieden Zeitpunkten der Tumorentwicklung untersucht. Die Analyse des Tumorgewebes ergab, eine signifikante erhöhte Frequenz von NK-Zellen in den mit Mut-30 RNA vakzinierten Tieren. Das NK Zellen in der frühen Phase der Therapie eine entscheidende Rolle spielen wurde anhand von Depletionsstudien bestätigt. Daran anschließend wurde gezeigt, dass im fortgeschrittenen Tumorstadium die NK Zellen keinen weiteren relevanten Beitrag zum anti-tumoralen Effekt der RNA Vakzinierung leisten, sondern die Vakzine induzierte adaptive Immunantwort. Durch die Isolierung von Lymphozyten aus dem Tumorgewebe und deren Einsatz als Effektorzellen im IFN-γ ELISPOT wurde nachgewiesen, dass Mut-30 spezifische T-Zellen das Tumorgewebe infiltrieren und dort u.a. IFN-γ sekretieren. Dass diese spezifische IFN-γ Ausschüttung für den beobachteten antitumoralen Effekt eine zentrale Rolle einnimmt wurde unter der Verwendung von IFN-γ -/- K.O. Mäusen bestätigt.rnDas Konzept der individuellen RNA basierten mutationsspezifischen Vakzine sieht vor, nicht nur mit einem mutations-spezifischen Epitop, sondern mit mehreren RNA-kodierten Mutationen Patienten zu impfen um der Entstehung von „escape“-Mutanten entgegenzuwirken. Da es nur Erfahrung mit der Herstellung und Verabreichung von Monotop-RNA gab, also RNA die für ein Epitop kodiert, war eine wichtige Fragestellungen, inwieweit Oligotope, welche die mutierten Sequenzen sequentiell durch Linker verbunden als Fusionsprotein kodieren, Immunantworten induzieren können. Hierzu wurden Pentatope mit variierender Position des einzelnen Epitopes hinsichtlich ihrer in vivo induzierten T-Zellreaktivitäten charakterisiert. Die Experimente zeigten, dass es möglich ist, unabhängig von der Position im Pentatop eine Immunantwort gegen ein Epitop zu induzieren. Des weiteren wurde beobachtet, dass die induzierten T-Zellfrequenzen nach Pentatop Vakzinierung im Vergleich zur Nutzung von Monotopen signifikant gesteigert werden kann.rnZusammenfassend wurde im Rahmen der vorliegenden Arbeit präklinisch erstmalig nachgewiesen, dass nicht-synonyme Mutationen eine numerisch relevante Quelle von Zielstrukturen für die anti-tumorale Immuntherapie darstellen. Überraschenderweise zeigte sich eine dominante Induktion MHC-II restringierter Immunantworten, welche partiell in der Lage waren massive Tumorabstoßungsreaktionen zu induzieren. Im Sinne einer Translation der gewonnenen Erkenntnisse wurde ein RNA basiertes Oligotop-Format etabliert, welches Eingang in die klinische Testung des Konzeptes fand.rn

Acquired factor XIII deficiency: a therapeutic challenge

Less than 60 cases of acquired factor (F)XIII deficiencies have been reported, most having distinct clinical features. To illustrate the therapeutic challenges of acquired FXIII inhibitors, we report a case of a 65-year-old patient with no previous bleeding history who suddenly developed massive haemorrhages associated to a strong and isolated FXIII inhibitor. No underlying disorder has been detected till now after three years of follow-up. Despite aggressive treatment with prednisone, rituximab, cyclophosphamide, immunoglobulin, immunoadsorption and immune tolerance his inhibitor is still present, although at low titre and with a clinical benefit since the patient has no more bleed since more than one year. Moreover the patient had a venous thromboembolic complication. After a review of the management of acquired FXIII deficiency patients and based on the management of acquired haemophilia we discuss a possible strategy for such difficult cases.

Exosomes

Exosomes are small natural membrane vesicles released by a wide variety of cell types into the extracellular compartment by exocytosis. The biological functions of exosomes are only slowly unveiled, but it is clear that they serve to remove unnecessary cellular proteins (e.g., during reticulocyte maturation) and act as intercellular messengers because they fuse easily with the membranes of neighboring cells, delivering membrane and cytoplasmic proteins from one cell to another. Recent findings suggests that cell-derived vesicles (exosomes are also named membranous vesicles or microvesicles) could also induce immune tolerance, suppression of natural killer cell function, T cell apoptosis, or metastasis. For example, by secreting exosomes, tumors may be able to accomplish the loss of those antigens that may be immunogenic and capable of signaling to immune cells as well as inducing dysfunction or death of immune effector cells. On the other hand, dendritic cell-derived exosomes have the potential to be an attractive powerful immunotherapeutic tool combining the antitumor activity of dendritic cells with the advantages of a cell-free vehicle. Although the full understanding of the significance of exosomes requires additional studies, these membrane vesicles could become a new important component in orchestrating responses between cells.

[Implantation: physiology, pathology and therapeutic options in disorders of implantation]

So far, implantation is a poorly understood process, which involves several paradoxical cell-biological mechanisms. First, 50% of the embryo is paternal and immunologically foreign material, and second, both the endometrium and embryo are covered by epithelial tissue to prevent cellular fusion. The adhesion and invasion of the blastocyst require an accurate coordination of embryonic and endometrial physiology and the modulation of maternal immune tolerance. Endometrial function plays an important role in assisted reproduction. Pathologies such as fibroids, hydrosalpinges, endometriosis and the polycystic ovary syndrome have a significant negative impact on implantation but can be treated in most cases. Therapeutic strategies to improve endometrial and embryonic function in recurrent implantation disorders are however still controversially discussed.

Self-antigen does not accelerate immature B cell apoptosis, but stimulates receptor editing as a consequence of developmental arrest

In pre-B lymphocytes, productive rearrangement of Ig light chain genes allows assembly of the B cell receptor (BCR), which selectively promotes further developmental maturation through poorly defined transmembrane signaling events. Using a novel in vitro system to study immune tolerance during development, we find that BCR reactivity to auto-antigen blocks this positive selection, preventing down-regulation of light chain gene recombination and promoting secondary light chain gene rearrangements that often alter BCR specificity, a process called receptor editing. Under these experimental conditions, self-antigen induces secondary light chain gene rearrangements in at least two-thirds of autoreactive immature B cells, but fails to accelerate cell death at this stage. These data suggest that in these cells the mechanism of immune tolerance is receptor selection rather than clonal selection.

Altered peptide ligand vaccination with Flt3 ligand expanded dendritic cells for tumor immunotherapy

Most tumor-associated antigens represent self-proteins and as a result are poorly immunogenic due to immune tolerance. Here we show that tolerance to carcinoembryonic antigen (CEA), which is overexpressed by the majority of lethal malignancies, can be reversed by immunization with a CEA-derived peptide. This peptide was altered to make it a more potent T cell antigen and loaded onto dendritic cells (DCs) for delivery as a cellular vaccine. Although DCs are rare in the blood, we found that treatment of advanced cancer patients with Flt3 ligand, a hematopoietic growth factor, expanded DCs 20-fold in vivo. Immunization with these antigen-loaded DCs induced CD8 cytotoxic T lymphocytes that recognized tumor cells expressing endogenous CEA. Staining with peptide-MHC tetramers demonstrated the expansion of CD8 T cells that recognize both the native and altered epitopes and possess an effector cytotoxic T lymphocyte phenotype (CD45RA+CD27−CCR7−). After vaccination, two of 12 patients experienced dramatic tumor regression, one patient had a mixed response, and two had stable disease. Clinical response correlated with the expansion of CD8 tetramer+ T cells, confirming the role of CD8 T cells in this treatment strategy.

Tolerância ao leite processado em altas temperaturas em pacientes com alergia ao leite de vaca mediada pela imunoglobulina E

INTRODUÇÃO: A incidência de pacientes apresentando alergia à proteína do leite de vaca (APLV) após os 5 anos de idade vem crescendo. Definir se estes pacientes tolerariam a ingestão de alimento produzido com leite processado a altas temperaturas (LPAT) proporcionaria melhor qualidade de vida, definiria melhor prognóstico e possibilitaria avaliar a indicação de dessensibilização com muffin. OBJETIVO: (1) identificar quais pacientes com APLV persistente aos quatro anos poderiam tolerar a ingestão de LPAT, (2) descrever as características clínicas e laboratoriais dos grupos reativo e não reativo ao LPAT, e (3) compara-las entre os dois grupos. MÉTODOS: Estudo transversal, utilizando amostra de conveniência, incluindo todos os pacientes acompanhados no ambulatório de alergia alimentar do Instituto da Criança HCFMUSP que preenchiam os critérios de inclusão e que concordaram em realizar o TPO, entre janeiro/2013 e novembro/2014. Os pacientes foram admitidos em hospital-dia sob supervisão médica e submetidos à ingestão de um muffin contendo 2,8 gramas de proteína do leite de vaca. Foram definidos como tolerantes se não apresentassem nenhuma reação alérgica. Estes pacientes foram submetidos na sequência a novo TPO com leite de vaca in natura para excluir a tolerância ao leite de vaca. RESULTADOS: Foram realizados 38 TPO com LPAT, sendo que 30 pacientes (15 masculinos) preencheram todos os critérios de inclusão. A mediana da idade foi de 7 anos e 7 meses (4a10m -14a2m). 14 pacientes (46%) não apresentaram reação após a ingestão do muffin, sendo considerados como não reativos. A análise comparativa entre os grupos reativos e não reativos ao LPAT, não mostrou diferença estatisticamente significante quanto às características clínicas: idade (p=0,8), sexo (p=0,4), história pessoal de rinite (p=0,7), história pessoal de asma (p=0,7), história pessoal de outras alergias (p=0,6), história familiar de rinite (p=0,7), história familiar de asma (p=0,3), história familiar de outras alergias (p=0,1), relato de anafilaxia prévia (p=0,07), relato de ingestão de traços de leite previamente ao TPO (p=0,4), relato de reação alérgica no último ano antes da provocação (p=0,6), relato de anafilaxia no último ano antes do TPO (p=0,6). Não se observou diferença estatisticamente significante entre os dois grupos para IgE total (p=0,1) e eosinófilos (p=0,6). O teste de puntura para leite de vaca e frações mostrou diferença estatisticamente significante para ?-lactoalbumina (p= 0,01) e para a caseína (p = 0,004); em relação ao ImmunoCAP® apenas para a caseína (p= 0,05) essa diferença foi significante. Ao avaliar estes pacientes 1 ano após o TPO, nenhum dos 16 pacientes que foram reativos ao LPAT estava ingerindo leite de vaca, enquanto 28% dos pacientes que foram tolerantes ao LPAT estavam consumindo leite de vaca in natura sem reação (p=0,037). CONCLUSÃO: O estudo mostrou que os pacientes com APLV desta amostra brasileira apresentaram 2 diferentes fenótipos, sendo que aproximadamente metade tolerou o LPAT. Sendo assim, o TPO para LPAT deve ser considerado para pacientes com APLV, sempre sob supervisão médica e estrutura segura e adequada, pois pode contribuir para uma mudança no paradigma do seguimento destes pacientes. Teste de puntura e ImmunoCAP® para caseína podem sugerir quais pacientes estariam tolerantes ao TPO com LPAT, reforçando dados da literatura internacional

Survival strategy of Echinococcus multilocularis in the human host

As exemplified by aborted calcified liver lesions commonly found in patients from endemic areas, Echinococcus multilocularis metacestodes develop only in a minority of individuals exposed to infection with the papasite. Clinical research has disclosed some aspects of the survival strategy of E. multilocularis in human hosts. Clinical observations in liver transplantation and AIDS suggest that suppression of cellular/Th1related immunity increases disease severity. Most of the studies have stressed a role for CD8+ T cells and for Interleukin-10 in the development of tolerance. A spontaneous secretion of IL-10 by the PBMC seems to be the immunological hallmark of patients with progressive forms of alveolar echinococcosis (AE). IL-10-induced inhibition of effector macrophages, but also of antigen-presenting dendritic cells, may be operating and allowing parasite growth and survival. The genetic correlates of susceptibility to infection with E. multilocularis are clearer in humans than in the mouse model. A significant link between MHC polymorphism and clinical presentation of AE has been shown, and the spontaneous secretion of IL-10 in patients with a progressive AE is higher in patients with the HLA DR3+, DQ2+ haplotype. Clustering of cases in certain families, in communities otherwise exposed to similar risk factors, also points to immuno-genetic predisposition factors that may allow the larva to escape host immunity more easily. The first stage of larval development may be crucial in producing danger signals stimulating the initial production of cytokines. Therapeutic use of Interferon alpha is an attempt to foil the survival strategy of E. multilocularis. (C) 2005 Elsevier Ireland Ltd. All rights reserved.

Linfócitos T : uma análise do perfil diferencial da expressão gênica na imunorregulação

Tese (doutorado)—Universidade de Brasília, Faculdade de Medicina, Pós-Graduação em Patologia Molecular, 2016.

Nitrosative stress, hypernitrosylation, and autoimmune responses to nitrosylated proteins: new pathways in neuroprogressive disorders Including depression and chronic fatigue syndrome.

Nitric oxide plays an indispensable role in modulating cellular signaling and redox pathways. This role is mainly effected by the readily reversible nitrosylation of selective protein cysteine thiols. The reversibility and sophistication of this signaling system is enabled and regulated by a number of enzymes which form part of the thioredoxin, glutathione, and pyridoxine antioxidant systems. Increases in nitric oxide levels initially lead to a defensive increase in the number of nitrosylated proteins in an effort to preserve their function. However, in an environment of chronic oxidative and nitrosative stress (O&NS), nitrosylation of crucial cysteine groups within key enzymes of the thioredoxin, glutathione, and pyridoxine systems leads to their inactivation thereby disabling denitrosylation and transnitrosylation and subsequently a state described as "hypernitrosylation." This state leads to the development of pathology in multiple domains such as the inhibition of enzymes of the electron transport chain, decreased mitochondrial function, and altered conformation of proteins and amino acids leading to loss of immune tolerance and development of autoimmunity. Hypernitrosylation also leads to altered function or inactivation of proteins involved in the regulation of apoptosis, autophagy, proteomic degradation, transcription factor activity, immune-inflammatory pathways, energy production, and neural function and survival. Hypernitrosylation, as a consequence of chronically elevated O&NS and activated immune-inflammatory pathways, can explain many characteristic abnormalities observed in neuroprogressive disease including major depression and chronic fatigue syndrome/myalgic encephalomyelitis. In those disorders, increased bacterial translocation may drive hypernitrosylation and autoimmune responses against nitrosylated proteins.

Evolution of cytokine receptor signaling.

Cytokines represent essential mediators of cell-cell communication with particularly important roles within the immune system. These secreted factors are produced in response to developmental and/or environmental cues and act via cognate cytokine receptors on target cells, stimulating specific intracellular signaling pathways to facilitate appropriate cellular responses. This review describes the evolution of cytokine receptor signaling, focusing on the class I and class II receptor families and the downstream JAK-STAT pathway along with its key negative regulators. Individual components generated over a long evolutionary time frame coalesced to form an archetypal signaling pathway in bilateria that was expanded extensively during early vertebrate evolution to establish a substantial "core" signaling network, which has subsequently undergone limited diversification within discrete lineages. The evolution of cytokine receptor signaling parallels that of the immune system, particularly the emergence of adaptive immunity, which has likely been a major evolutionary driver.

Immune response to insulin and changes in the gut immune system in children with or at risk for type 1 diabetes

Type 1 diabetes (T1D) is considered to be an autoimmune disease. The cause of T1D is the destruction of insulin-producing β-cells in the pancreatic islets. The autoimmune nature of T1D is characterized by the presence of autoreactive T-cells and autoantibodies against β-cell molecules. Insulin is the only β-cell-specific autoantigen associated with T1D but the insulin autoantibodies (IAAs) are difficult to measure with proper sensitivity. T-cell assays for detection of autoreactive T-cells, such as insulin-specific T-cells, have also proven to be difficult to perform. The genetic risk of T1D is associated with the HLA gene region but the environmental factors also play an important role. The most studied environmental risk factors of T1D are enteroviruses and cow's milk which both affect the immune system through the gut. One hypothesis is that the insulin-specific immune response develops against bovine insulin in cow's milk during early infancy and later spreads to include human insulin. The aims of this study were to determine whether the separation of immunoglobulin (Ig)G from plasma would improve the sensitivity of the IAA assay and how insulin treatment affects the cellular immune response to insulin in newly diagnosed patients. Furthermore, the effect of insulin concentration in mother's breast milk on the development of antibodies to dietary insulin in the child was examined. Small intestinal biopsies were also obtained from children with T1D to characterize any immunological changes associated with T1D in the gut. The isolation of the IgG fraction from the plasma of T1D patients negative for plasma IAA led to detectable IAA levels that exceeded those in the control children. Thus the isolation of IgG may improve the sensitivity of the IAA assay. The effect of insulin treatment on insulin-specific T-cells was studied by culturing peripheral blood mononuclear cells with insulin. The insulin stimulation induced increased expression of regulatory T-cell markers, such as Foxp3, in those patients treated with insulin than in patients examined before initiating insulin treatment. This finding suggests that insulin treatment in patients with T1D stimulates regulatory T-cells in vivo and this may partly explain the difficulties in measuring autoantigen-specific T-cell responses in recently diagnosed patients. The stimulation of regulatory T-cells by insulin treatment may also explain the remission period often seen after initiating insulin treatment. In the third study we showed that insulin concentration in mother's breast milk correlates inversely with the levels of bovine insulin-specific antibodies in those infants who were exposed to cow's milk proteins in their diet, suggesting that human insulin in breast milk induces tolerance to dietary bovine insulin. However, in infants who later developed T1D-associated autoantibodies, the insulin concentration in their mother's breast milk was increased. This finding may indicate that in those children prone to β-cell autoimmunity, breast milk insulin does not promote tolerance to insulin. In the small intestinal biopsies the presence of several immunological markers were quantified with the RT-PCR. From these markers the expression of the interleukin (IL)-18 cytokine was significantly increased in the gut in patients with T1D compared with children with celiac disease or control children. The increased IL-18 expression lends further support for the hypothesis that the gut immune system is involved in the pathogenesis of T1D.