Dispositivi di supporto alla insufficienza epatica

L’organizzazione mondiale della sanità stima che il 10% della popolazione nel mondo presenta una malattia epatica cronica. L’insufficienza epatica fulminante porta alla morte entro novantasei ore in assenza di trapianto. Questa patologia colpisce circa 2500 persone l’anno e il trapianto di fegato è l’unico trattamento efficiente, ma la mancanza di donatori ha causato un elevato tasso di mortalità in pazienti in lista di attesa per l’organo compatibile. Negli ultimi anni è cresciuta sempre più la lista d’attesa per il trapianto, mentre il numero degli innesti disponibili rimane ridotto. Non tutti i pazienti sono candidati al trapianto. Questo tipo di intervento è rischioso e richiede un trattamento d’immunosoppressione per tutta la vita. Si cercano quindi soluzioni sostitutive, ma lo sviluppo di dispositivi efficaci di assistenza epatica rimane uno dei più alti sforzi dell’ingegneria biomedica. Il fegato, infatti, svolge più di 500 funzioni diverse che sono difficili da riprodurre artificialmente. Un dispositivo di assistenza epatica deve sostituire o integrare la funzione epatica quando scende al di sotto della soglia critica del 30% del normale funzionamento. Per garantire il normale funzionamento dell’organismo, la massa di epatociti disponibile in un paziente adulto deve essere approssimativamente di circa 400g. Se questa massa è disponibile si può avere rigenerazione epatica spontanea. Questo spiega la necessità di raggiungere e garantire, comunque, un livello minimo di funzionalità epatica, al di sotto del quale non si ha né rigenerazione di epatociti (impossibilità di ripristinare l’attività metabolica del fegato), né funzionamento di altri organi del corpo, che dipendono dall’attività epatica. La ricerca e lo sviluppo di dispositivi di assistenza epatica è tesa al raggiungimento e al mantenimento della soglia minima di funzionalità; in questo modo si permette al paziente di sopravvivere fino al trapianto, quando questo è l’unica terapia possibile, oppure di poter raggiungere le condizioni di autorigenerazione spontanea nelle patologie meno gravi. La ricerca sui dispositivi di assistenza epatica risale agli anni 50, e sin da allora sono state sviluppate diverse soluzioni, che possiamo distinguere in sistemi meccanici (sistemi non biologici) e sistemi biochimici o biomeccanici (sistemi biologici). I sistemi non biologici possono avere un ruolo nel trattamento di forme specifiche d’insufficienza epatica. L’obiettivo è di fornire purificazione del sangue, ossia rimuovere tutte le sostanze che si accumulano nel sangue durante la disfunzione epatica, causando anomalie neurologiche, lesioni del fegato e di altri organi e inibizione della rigenerazione epatica. I sistemi biologici possono essere utili nel trattamento d’insufficienza epatica, in cui l’obiettivo primario è quello di fornire tutte le funzioni del fegato che sono state compromesse o perse.

Genetic and environmental factors associated with cardiovascular diseases and acute myocardial infarction

Acute myocardial infarction (AMI) is a multifactorial disease with a complex pathogenesis where lifestyle, individual genetic background and environmental risk factors are involved. Altered inflammatory responses seems to be implicated in the pathogenesis of atherosclerosis. To understand which genes may predispose to increased risk of cardiovascular disease gene polymorphism of immune regulatory genes, and clinical events from the Offs of parents with an early AMI were investigated. Genetics data from Offs were compared with those obtained from healthy subjects and an independent cohort of patients with clinical sporadic AMI. Rates of clinical events during a 24 years follow up from Offs and from an independent Italian population survey were also evaluated. This study showed that a genetic signature consisting of the concomitant presence of the CC genotype of VEGF, the A allele of IL-10 and the A allele of IFN-γ was indeed present in the Offs population. During the 24-year follow-up, Offs with a positive familiarity in spite of a relatively young age showed an increased prevalence of diabetes, ischemic heart disease and stroke. In these patients with the genetic signature the EBV and HHV-6 herpes virus were also investigated and founded. These findings reinforce the notion that subjects with a familial history of AMI are at risk of an accelerated aging of cardiovascular system resulting in cardiovascular events. These data suggest that selected genes with immune regulatory functions and envoronmental factors are part of the complex genetic background contributing to familiarity for cardiovascular diseases.N

Ricerca dell'emissione alle alte energie da parte delle radio galassie fri e frii

Lo scenario di unificazione degli AGN caratterizza le molteplici proprietà di questi oggetti in termini del differente angolo di vista rispetto ad un sistema costituito da un toro oscurante, un disco di accrescimento che alimenta il SMBH e nubi di gas che circondano il buco nero. Circa il 10% degli AGN sono forti sorgenti radio. Questi oggetti, detti AGN Radio-Loud, sono caratterizzati da getti relativistici emessi trasversalmente rispetto al disco di accrescimento e comprendono le radio galassie e i blazar. In accordo con il modello unificato, le radio galassie (MAGN), rappresentano i blazar visti a grandi angoli di inclinazione del getto rispetto alla linea di vista. Nei blazar la radiazione emessa dai getti su scale del pc viene amplificata da effetti relativistici dando origine a spettri piatti con elevata polarizzazione ottica e forte variabilità. Questi oggetti rappresentano le sorgenti più brillanti identificate nel cielo gamma extragalattico. I MAGN, a differenza dei blazar, mostrano spettri ripidi e strutture radio quasi simmetriche. In queste sorgenti, l'effetto del Doppler boosting è meno evidente a causa del grande angolo di inclinazione del getto. In soli 3 mesi di osservazioni scientifiche effettuate con il satellite Fermi è stata rivelata emissione gamma da parte delle radio galassie NGC 1275 e Cen A. I MAGN rappresentano una nuova classe di sorgenti gamma. Tuttavia, il numero di radio galassie rivelate è sorprendentemente piccolo ponendo degli interrogativi sui meccanismi di emissione alle alte energie di questi oggetti. Nel presente lavoro di tesi, si analizzeranno i dati gamma raccolti dal LAT durante i primi 5 anni di osservazioni scientifiche per un campione di 10 radio galassie più brillanti selezionate dai cataloghi B2 e BCS. L'obiettivo principale sarà migliorare la statistica e cercare di comprendere la natura dell'emissione alle alte energie da parte delle radio galassie.

Transkutane Immunisierung - Mechanismen und Optimierungsstrategien

In dieser Arbeit wurde ein etabliertes Immunisierungsmodell auf der Basis eines synthetischen TLR 7-Liganden zur Aufhebung der suppressiven Eigenschaften von UV B-Strahlung verwendet. Es konnte gezeigt werden, dass nach Ermittlung eines geeigneten Immunisierungsprotokolls mit UV B die durch TCI vermittelte ausschließlich primäre Immunantwort verstärkt werden konnte, so dass eine Gedächtnisantwort induziert werden konnte. Durch die Immunisierung verschiedenster knockout sowie transgener Stämme konnte bewiesen werden, dass die Reaktion auch nach zusätzlicher UV B-Bestrahlung abhängig von TLR 7 und dem Adaptormolekül MyD88 ist. Bei der Aufklärung der zellulären Mechanismen, die dieser Immunisierungsmethode zu Grunde liegen, konnten dermale DCs, sowie GR1+-Zellen als wichtige Mediatoren identifiziert werden. Eine wesentliche Funktion von epidermalen Langerhans Zellen konnte in diesem Zusammenhang ausgeschlossen werden. Des Weiteren konnte gezeigt werden, dass auf Zytokinebene Typ I Interferone eine tragende Rolle spielen und die Produktion von IL-12p35 in den Haut-drainierenden Lymphknoten angeregt wird. Supprimiert wird die Immunantwort durch regulatorische T-Zellen, sowie durch die Freisetzung von IL-10, welches nicht ausschließlich von regulatorischen T-Zellen produziert wird. Die Applikation eines blockierenden IL-10-Rezeptor Antikörpers verhinderte die IL-10-vermittelte Suppression und führte zu einer weiteren Verstärkung der Immunantwort. Durch die induzierte Gedächtnisantwort nach UV B/TCI konnte das Immunisierungs-Modell in einem therapeutischen Tumormodell angewandt werden und führt hier zu einer verstärkten Abstoßung von Tumoren, verglichen mit TCI alleine, was die Basis für zukünftige nadelfreie Vakzinierungen für die Behandlung von Krebs oder persistierender Infektionen darstellen könnte.

The role of B cells during acute and latent infections with murine cytomegalovirus and Leishmania major

This thesis focuses on the role of B cells in mCMV and Leishmania major infection. B cells are an essential component of the adaptive immune system and play a key role in the humoral immune response. In mCMV infection we analyzed the influence of B cells on the virus-specific CD8 T cell response, in detail the role of B cells as IL-10 secreting cells, as source of immunoglobulin (Ig) and as antigen presenting cells. In Leishmania major infection we investigated the role of Ig in Th1 and Th2 directed disease.rnWe found in mCMV infection that the B cell secreted IL-10 suppresses effectively the acute virus-specific CD8 T cell response, while the IL-10 secreted by dendritic cell has no obvious effect. Ig has no effect in the acute virus-specific CD8 T cell response, but in memory response Ig is essential. If Ig is missing the CD8 T cell population remains high in memory response 135 days post infection. The complete absence of B cells dramatically reduces the acute virus-specific CD8 T cell response, while B cell reconstitution just partially rescues this dramatic reduction. A comparison of this reduction in a B cell free organism to an organism with depleted dendritic cells gave a similar result. To exclude a malfunction of the CD8 T cells in the B cell deficient mice, the decreased virus-specific CD8 T cell population was confirmed in a B cell depletion model. Further, bone marrow chimeras with a B cell compartment deficient for CD40-/- showed a decrease of the virus-specific response and an involvement of CD40 on B cells. Taken together these results suggest a role for B cells in antigen presentation during mCMV infection.rnFurther we took advantage of the altered mCMV specific CD8 T cell memory response in mice without Ig to investigate the memory inflation of CD8 T cells specific for distinct mCMV specifc peptides. Using a SIINFEKL-presenting virus system, we were able to shorten the time until the memory inflation occurs and show that direct presentation stimulates the memory inflation. rnIn Leishmania major infection, Ig of Th2 balanced BALB/c mice has a central role in preventing a systemic infection, although the ear lesions are smaller in IgMi mice without specific Ig. Here the parasite loads of ears and spleen are elevated, and an IMS-reconstitution does not affect the parasite load. In contrast in Th1 balanced C57BL/6 mice, reconstitution of IgMi mice with serum of either untreated or immunized mice decreased the parasite load of spleen and ear, further IMS treatment reduces the size of the spleen and the cytokine-levels of IL-10, IL-4, IL-2 and IFN-γ to a level comparable to wt mice. rn

Flagellin:allergen fusion proteins as novel vaccines for the treatment of severe type I allergies

Over the last decades the prevalence of food allergies has continually increased on a world wide scale. While there are effective treatments available for bee and wasp venom allergic patients, there is currently no established therapy for the treatment of severe food allergies. Aim of the project was to genetically fuse different food allergens with the immune modulating Toll-like receptor 5 (TLR5)-ligand flagellin and to test these constructs for their immune modulatory capacities both in vitro and in vivo. Chicken ovalbumin (Ova) as model antigen, Pru p 3, and Ara h 2 the respective major allergens from peach and peanut were used as allergens. The potential vaccine candidates were characterized by protein biochemical methods (purity, folding, endotoxin contaminations). Moreover, their immune modulating effects on cell culture lines (TLR5-receptor activation) and primary mouse immune cells (myeloid and plasmacytoid dendritic cells) were investigated. Additionally, the prophylactic and therapeutic use of the flagellin Ova fusion protein (rflaA:Ova) were investigated in a mouse model of intestinal allergy. In myeloid dendritic cells (mDC) stimulation with the fusion proteins led to a strong cell activation and cytokine secretion. Here, the fusion proteins proved to be a much stronger stimulus than the equimolar amount of both proteins provided alone or as a mixture. Noteworthy, stimulation with rflaA:Ova induced the secretion of the anti-inflammatory cytokine IL-10 from mDC. In co-culture experiments this IL-10 secretion suppressed the Ova-induced secretion of Th1 and Th2 cytokines from Ova-specific CD4 T cells. Using MyD88-deficient mDC this repression of cytokine secretion was shown to be TLR-dependent. Finally, the potency of the rflaA:Ova construct was investigated in a mouse model of Ova-induced intestinal allergy. In a prophylactic vaccination approach rflaA:Ova was shown to prevent the establishment of the intestinal allergy and all associated symptoms (weight loss, temperature drop, soft faeces). This fusion protein-mediated protection was accompanied by a reduced T cell activation, and reduced Th2 cytokines in intestinal homogenates. These effects were paralleled by a strong induction of Ova-specific IgG2a antibodies in rflaA:Ova-vaccinated sera, while Ova-specific IgE antibody production was significantly reduced. Therapeutic vaccination with rflaA:Ova reduced allergic symptoms and T cell activation but did not influence weight loss and antibody production. In all in vivo experiments vaccination with both proteins either provided alone or as a mixture did not have comparable effects. Future experiments aim at elucidating the mechanism and further optimization of the therapeutic vaccination approach. The results presented in this thesis demonstrate, that fusion proteins containing flagellin have strong immune modulatory capacities both in vitro and in vivo. Therefore, such constructs are promising vaccine candidates for the therapy of type I allergies.

Etablierung eines Triple-Kultur-Modells der oberen Atemwege unter Einbeziehung immunkompetenter Zellen

Organophosphate können chronische Lungenerkrankungen und Vergiftungen hervorrufen. Bei der Vergiftung erfolgt eine Immunreaktion, welche noch nicht erforscht ist. In dieser Arbeit wurden Toxizitätsstudien an dendritischen Zellen und einem bronchialen Triple-Kultur-Modell durchgeführt. Dendritische Zellen spielen bei der ersten Immunabwehr in der Lunge eine große Rolle. Aus der Zell-Linie THP-1 und primären Monozyten wurden reife dendritische Zellen differenziert und mittels Durchflusszytometrie und Immunfluoreszenz auf spezifische Zellmarker, wie zum Beispiel CD11c, CD83 oder auch CD209, charakterisiert und etabliert. Durch die Vergiftung der Zellen mit Dimethoat und Chlorpyrifos konnte eine Erhöhung des Zelltodes, die Sekretion von proinflammatorischen Mediatoren, Veränderungen in der Morphologie der Zellen und ein Effekt auf den Proteinkinase-Signalweg festgestellt werden. Spezifische dendritische Zellmarker (CD83, CD209) wurden inhibiert und die Dendriten der Dendritischen Zellen kürzer und beschädigt. Die Schädigung von Chlorpyrifos war erheblich größer, als die bei Dimethoat.rnDie weiteren Toxizitätsstudien wurden an einem bronchialen Triple-Kultur-Modell durchgeführt. Hierzu wurden auf Transwell-Filtermembranen bronchiale Epithelzellen, Fibroblastenzellen und Dendritische Zellen verwendet. Die bronchialen Epithelzellen und Fibroblastenzellen waren hier physiologisch voneinander getrennt, konnten aber durch Poren in der Membran miteinander interagieren. Die Etablierung des Triple-Kultur-Modells erfolgte durch die Untersuchung von Entzündungsprozessen, durch Stimulation mit LPS, TNF-alpha und Interferon-gamma. In der Ko-Kultur konnten Zell-Zell-Kontakt Schädigungen und Erhöhung von proinflammatorischen Markern, wie zum Beispiel IL-1ß, IL-6 oder auch IL-8 gemessen werden. Versuche in der Triple-Kultur zeigten den positiven Effekt von Dendritischen Zellen. Bei höheren Konzentrationsbereichen von Dimethoat und Chlorpyrifos konnte ein Wandern der Zellen zu den geschädigten Zell-Zell-Kontakten nachgewiesen werden. Die Ausschüttung der proinflammatorischen Mediatoren wurde inhibiert, vor allem bei IL-10 war eine deutliche Reduktion, um mehr als 70% messbar. Ebenso konnten Veränderungen in dem Apoptose-Signalblick festgestellt werden. Vor allem anti-apoptotische Proteine wurden nach einer Vergiftung der Triple-Kultur induziert. Interventionsstudien mit Vitamin C zeigten allerdings keinen positiven Effekt.

Influenza della tipologia di cereale e di fermentazione sulle possibili proprietà salutistiche del pane

Il rinnovato interesse da parte dei consumatori per le caratteristiche nutrizionali degli alimenti incentiva la ricerca in campo agroalimentare a sviluppare nuovi prodotti per il mercato attuale sempre più attento a salute e benessere. È in questo contesto che nasce a livello europeo il progetto Bake4Fun mirato allo sviluppo di soluzioni biotecnologiche innovative per la messa a punto di nuovi prodotti da forno funzionali. Nell’ambito di questo progetto europeo, lo scopo dello studio oggetto di questo elaborato di laurea è stato quello di valutare le proprietà antiossidanti e antinfiammatorie di diverse tipologie di pani creati ad hoc con farina di frumento o di farro e sottoposti a fermentazione convenzionale o con pasta madre (sourdough). Il farro appartiene alla famiglia dei cosiddetti “cereali antichi” e grazie al suo elevato profilo nutrizionale si presenta come un promettente candidato per la produzione di alimenti dalle proprietà benefiche. Similmente anche diverse tipologie di fermentazione sembrano in grado di possedere caratteristiche salutistiche. Pertanto, nell’ambito di B4F sono stati formulati e prodotti diversi tipi di pane e se ne sono voluti studiare i possibili effetti antiossidanti ed antiinfiammatori in vivo sul modello sperimentale del suino. Dopo 30 giorni di dieta arricchita con i diversi prodotti sperimentali miscelate in un rapporto di 1:1 con una dieta standard è stato valutato lo stato redox plasmatico mediante l’analisi di tre indicatori del danno ossidativo, quali TAC, GSH e TBARS. Il grado d’infiammazione è stato invece valutato attraverso l’analisi di otto citochine (IFNα, IFNγ, IL-1β, IL-4, IL-6, IL-8, IL-10 e TNFα) utilizzando un saggio multiparametrico di tipo ELISA. Tutti gli animali impiegati godevano di buona salute ed i loro plasmi sono stati analizzati sia all’inizio (T0) sia alla fine (T30) dell’esperimento. Sebbene i risultati conseguiti in relazione allo status ossidativo ed infiammatorio nei suini impiegati non abbiano evidenziato particolari differenze tra i diversi tipi di diete sperimentali, non si esclude che eventuali variazioni delle condizioni sperimentali possano portare a conclusioni diverse o che gli effetti siano maggiormente visibili a livello di altri parametri quali, ad esempio, la variazione della microflora intestinale o l’indice glicemico dei prodotti. Questa possibilità unitamente all’importanza degli effetti salutistici dei cereali antichi e delle diverse tipologie di fermentazione, giustificate da altre ricerche in letteratura, sottolineano la necessità di ulteriori studi in questo ambito.

Imaging Diagnostico utilizzato in neuroradiologia per lo studio degli Aneurismi Cerebrali

Durante la mia tesi mi sono occupata di una grave patologia, l’Aneurisma Cerebrale, che rappresenta una delle principali cause di ospedalizzazione provocando il 10/12% della mortalità globale annua nei paesi industrializzati. In Italia , l’incidenza di aneurismi cerebrali è di 100.000 nuovi casi all’anno. In generale , nei paesi industrializzati, la prevalenza è stimata a circa 600 per 100.000 abitanti . Tuttavia la prevalenza degli aneurismi cerebrali nelle popolazioni in generale , è molto più elevato stimato intorno al 2,3%,il che suggerisce indirettamente che la maggior parte degli aneurismi non va mai incontro a rottura. Negli ultimi vent’anni lo sviluppo della tecnologia ha visto significativi progressi che ci hanno permesso di agire sul problema in modo sempre migliore. Nei primi capitoli ho descritto alcune apparecchiature biomediche che sono di fondamentale importanza nello studio degli aneurismi cerebrali, mettendo in evidenza le varie peculiarità che le contraddistinguono. A seguire, ho parlato del trattamento endovascolare, che consiste nell’esclusione della cavità aneurismatica dal flusso di sangue, il quale viene incanalato in una protesi posizionata all’interno del lume vasale eliminando il rischio di rottura o di embolizzazione di materiale trombotico proveniente dalla sacca aneurismatica. I vantaggi della tecnica endovascolare consistono nella minore invasività rispetto alla tecnica chirurgica standard, la craniotomia. A fronte di ciò, si deduce quanto l’ingegneria biomedica sia una disciplina in evoluzione. Le condizioni di vita delle persone che subiscono questi tipi di interventi sono notevolmente migliorate ottenendo risultati di completa o semi-completa guarigione.

Contributo dei coralliti sub-fossili di Cladocora caespitosa (L.,1767)(Cnidaria, Scleractinia) ai sedimenti dei fondali circostanti le isole del Golfo di La Spezia.

I sedimenti superficiali dei fondali circostanti l’arcipelago del Golfo di La Spezia sono stati analizzati dal punto di vista granulometrico e composizionale al fine di ottenere la mappatura delle concentrazioni di coralliti sub-fossili di Cladocora caespitosa nel sedimento. Mediante lo studio del sedimento campionato in trentacinque stazioni, sono state individuate tre zone di accumulo di coralliti: (i) in corrispondenza del capo occidentale dell’Isola Palmaria con le concentrazioni più elevate comprese tra il 25 e 55% (ii) sul lato sud-orientale della stessa isola con concentrazioni tra il 10 e 12% e (iii) una fascia contornante l’Isola del Tinetto con quantità inferiori al 3%. La concentrazione anomala di coralliti è il risultato dello scarico di materiali di dragaggio provenienti dal porto di La Spezia, scaricati al largo delle coste occidentali dell’arcipelago tra gli anni ‘50 e ’70 e progressivamente ridistribuiti verso sud-est dalla deriva litorale.

Effects and uptake of gold nanoparticles deposited at the air–liquid interface of a human epithelial airway model

The impact of nanoparticles (NPs) in medicine and biology has increased rapidly in recent years. Gold NPs have advantageous properties such as chemical stability, high electron density and affinity to biomolecules, making them very promising candidates as drug carriers and diagnostic tools. However, diverse studies on the toxicity of gold NPs have reported contradictory results. To address this issue, a triple cell co-culture model simulating the alveolar lung epithelium was used and exposed at the air-liquid interface. The cell cultures were exposed to characterized aerosols with 15 nm gold particles (61 ng Au/cm2 and 561 ng Au/cm2 deposition) and incubated for 4 h and 24 h. Experiments were repeated six times. The mRNA induction of pro-inflammatory (TNFalpha, IL-8, iNOS) and oxidative stress markers (HO-1, SOD2) was measured, as well as protein induction of pro- and anti-inflammatory cytokines (IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, GM-CSF, TNFalpha, INFgamma). A pre-stimulation with lipopolysaccharide (LPS) was performed to further study the effects of particles under inflammatory conditions. Particle deposition and particle uptake by cells were analyzed by transmission electron microscopy and design-based stereology. A homogeneous deposition was revealed, and particles were found to enter all cell types. No mRNA induction due to particles was observed for all markers. The cell culture system was sensitive to LPS but gold particles did not cause any synergistic or suppressive effects. With this experimental setup, reflecting the physiological conditions more precisely, no adverse effects from gold NPs were observed. However, chronic studies under in vivo conditions are needed to entirely exclude adverse effects.

Attenuation of cerebrospinal fluid inflammation by the nonbacteriolytic antibiotic daptomycin versus that by ceftriaxone in experimental pneumococcal meningitis

Antibiotic-induced bacteriolysis exacerbates inflammation and brain damage in bacterial meningitis. Here the quality and temporal kinetics of cerebrospinal fluid (CSF) inflammation were assessed in an infant rat pneumococcal meningitis model for the nonbacteriolytic antibiotic daptomycin versus ceftriaxone. Daptomycin led to lower CSF concentrations of interleukin 1beta (IL-1beta), IL-10, IL-18, monocyte chemoattractant protein 1 (MCP-1), and macrophage inflammatory protein 1 alpha (MIP-1alpha) (P < 0.05). In experimental pneumococcal meningitis, daptomycin treatment resulted in more rapid bacterial killing, lower CSF inflammation, and less brain damage than ceftriaxone treatment.

Ablation of the tumor suppressor histidine triad nucleotide binding protein 1 is protective against hepatic ischemia/reperfusion injury

The identification of cellular pathways capable of limiting ischemia/reperfusion (I/R) injury remains a frontier in medicine, and its clinical relevance is urgent. Histidine triad nucleotide binding protein 1 (HINT1) is a tumor suppressor that influences apoptosis. Because apoptotic pathways are a feature of I/R injury, we asked whether Hint1 influences hepatic I/R injury. Hint1(-/-) and C57BL/6 mice were subjected to 70% liver ischemia followed by reperfusion for 3 or 24 hours or to a sham operation. The serum aminotransferase levels, histological lesions, apoptosis, reactive oxygen species, and expression of B cell lymphoma 2-associated X protein (Bax), heme oxygenase 1 (HO-1), interleukin-6 (IL-6), IL-10, tumor necrosis factor-a, Src, nuclear factor kappa B (p65/RelA), and c-Jun were quantified. The responses to toll-like receptor ligands and nicotinamide adenine dinucleotide phosphate oxidase activity in Kupffer cells were compared in Hint1(-/-) mice and C57BL/6 mice. After I/R, the levels of serum aminotransferases, parenchymal necrosis, and hepatocellular apoptosis were significantly lower in Hint1(-/-) mice versus control mice. Furthermore, Bax expression decreased more than 2-fold in Hint1(-/-) mice, and the increases in reactive oxygen species and HO-1 expression that were evident in wild-type mice after I/R were absent in Hint1(-/-) mice. The phosphorylation of Src and the nuclear translocation of p65 were increased in Hint1(-/-) mice, whereas the nuclear expression of phosphorylated c-Jun was decreased. The levels of the protective cytokines IL-6 and IL-10 were increased in Hint1(-/-) mice. These effects increased survival after I/R in mice lacking Hint1. Hint1(-/-) Kupffer cells were less activated than control cells after stimulation with lipopolysaccharides. CONCLUSION: The Hint1 protein influences the course of I/R injury, and its ablation in Kupffer cells may limit the extent of the injury.

Assessment of endothelium and inflammatory response at the onset of reperfusion injury in hand surgery

Background Activation of the endothelium, complement activation and generation of cytokines are known events during ischemia-reperfusion (I/R) that mediate tissue injury. Our aim was to elucidate their respective participation at the onset of the reperfusion phase. Tourniquet application in hand surgery causes short-term ischemia, followed by reperfusion and was therefore used as the model in this study. Methods Ten patients were included in the study after obtaining informed consent. A tourniquet was placed on the upper arm and inflated to 250 mmHg for 116 ± 16 min, during which the surgery was performed. Venous blood and tissue samples from the surgical area were taken at baseline as well as 0, 2, and 10 min after reperfusion and analyzed for the following parameters: Endothelial integrity and/or activation were analyzed by measuring heparan sulfate and syndecan-1 in serum, and vWF, heparan sulfate proteoglycan as well as CD31on tissue. Complement activation was determined by C3a and C4d levels in plasma, levels of C1-inhibitor in serum, and IgG, IgM, C3b/c, and C4b/c deposition on tissue. Cytokines and growth factors IL-5, IL-6, IL-7, IL-8, IL-10, IL-17, G-CSF, GM-CSF, MCP-1, TNFα, VEGF, and PDGF bb were measured in the serum. Finally, CK-MM levels were determined in plasma as a measure for muscle necrosis. Results Markers for endothelial activation and/or integrity as well as complement activation showed no significant changes until 10 min reperfusion. Among the measured cytokines, IL-6, IL-7, IL-17, TNFα, GM-CSF, VEGF, and PDGF bb were significantly increased at 10 min reperfusion with respect to baseline. CK-MM showed a rise from baseline at the onset of reperfusion (p < 0.001) and dropped again at 2 min (p < 0.01) reperfusion, suggesting ischemic muscle damage. Conclusions In this clinical model of I/R injury no damage to the endothelium, antibody deposition or complement activation were observed during early reperfusion. However, an increase of pro-inflammatory cytokines and growth factors was shown, suggesting a contribution of these molecules in the early stages of I/R injury.

Effects of Echinaforce® treatment on ex vivo-stimulated blood cells

The herb Echinacea purpurea, also called purple coneflower, is regarded as an immune modulator. This study examined changes in cytokine production in blood samples from 30 volunteers before and during 8-day oral administration with an ethanolic extract of fresh Echinacea purpurea (Echinaforce(®)). Daily blood samples were ex vivo stimulated by LPS/SEB or Zymosan and analysed for a series of cytokines and haematological and metabolic parameters. Treatment reduced the proinflammatory mediators TNF-α and IL-1β by up to 24% (p<0.05) and increased anti-inflammatory IL-10 levels by 13% (p<0.05) in comparison to baseline. This demonstrated a substantial overall anti-inflammatory effect of Echinaforce(®) for the whole group (n=28). Chemokines MCP-1 and IL-8 were upregulated by 15% in samples from subjects treated with Echinaforce(®) (p<0.05). An analysis of a subgroup of volunteers who showed low pre-treatment levels of the cytokines MCP-1, IL-8, IL-10 or IFN-γ (n=8) showed significant stimulation of these factors upon Echinaforce(®) treatment (30-49% increases; p<0.05), whereas the levels in subjects with higher pre-treatment levels remained unaffected. We chose the term "adapted immune-modulation" to describe this observation. Volunteers who reported high stress levels (n=7) and more than 2 colds per year experienced a significant transient increase in IFN-γ upon Echinaforce(®) treatment (>50%). Subjects with low cortisol levels (n=11) showed significant down-regulation of the acute-phase proteins IL1-β, IL-6, IL-12 and TNF-α by Echinaforce(®) (range, 13-25%), while subjects with higher cortisol levels showed no such down-regulation. This is the first ex vivo study to demonstrate adapted immune-modulation by an Echinacea preparation. While Echinaforce(®) did not affect leukocyte counts, we speculate that the underlying therapeutic mechanism is based on differential multi-level modulation of the responses of the different types of leukocytes. Echinaforce(®) thus regulates the production of chemokines and cytokines according to current immune status, such as responsiveness to exogenous stimuli, susceptibility to viral infection and exposure to stress.