<p>Uvod: Nespecifičnost kliničke slike sepse, velike individualne razlike u odgovoru organizma na infekciju kao i neophodnost adekvatne inicijalne procene težine kliničke slike, toka i ishoda bolesti, čine istraživanja biomarkera koji bi doprineli pravovremenom postavljanju dijagnoze i adekvatnoj prognozi bolesti izuzetno značajnim. Do sada je ispitivano preko 200 biomarkera od kojih ni jedan nije pokazao zadovoljavajuću senzitivnost i specifičnost. Uloga B limfocita u patogenezi sepse pri tome je nedovoljno istražena. Članovi superfamilije tumor-nekrozis faktora alfa: A proliferation inducing ligand (APRIL), Bcell activating factor (BAFF) i solubilni transmembrane activator and calcium modulator cyclophilin ligand interactor (sTACI) su citokini koji imaju ključnu ulogu u homeostazi B limfocita. Cilj istraživanja bio je da se ispita dijagnostički i prognostički značaj citokina članova superfamilije tumor nekrozis faktora alfa (APRIL, BAFF, sTACI) za procenu težine kliničke slike, razvoja multiorganske disfunkcije (MODS) u prvih 48h hospitalizacije i letalnog ishoda sepse. Ispitanici i metode: Istraživanjem je obuhvaćeno 150 obolelih od sepse lečenih na Klinici za infektivne bolesti i Odeljenju anestezije i reanimacije Kliničkog centra Vojvodine i 30 zdravih dobrovljnih davalaca krvi. Kod svih bolesnika evidentirani su demografski i ostali podaci iz istorije bolesti kao i laboratorijske analize u okviru rutinske dijagnostike sepse. Iz dodatnih 5ml venske krvi svim ispitanicima određene su koncentracije APRIL-a, BAFF-a, sTACI-ja ELISA metodom komercijalnim testovima proizvođača R&amp;D Systems. Rezultati pokazuju da su koncentracije sva tri citokina (APRIL, BAFF i sTACI) statistički značajno povi&scaron;ene kod obolelih od sepse u odnosu na zdravu populaciju (p&lt;0.001), pri čemu APRIL pokazuje najveću senzitinvost (99%) i specifičnost (97%). Najveći dijagnostički značaj BAFF-a ogleda se u sposobnosti distinkcije između sepsi uzrokovanih Gram pozitivnim i Gram negativnim bakterijama (p=0,03). U predikciji razvoja MODS-a i letalnog ishoda sepse multivarijantnom regresionom analizom kao nezavisni prediktori pokazali su se jedino antiinflamatorni biomarker sTACI receptor i klinička procena pacijenta iskazana kroz APACHE II i SOFA skor. Senzitivnost i specifilnost sTACI receptora u predikciji razvoja MODS-a i letalnog ishoda daleko nadma&scaron;uje do sada rutinski kori&scaron;ćen prokalcitonin. Zaključak: Dobijeni rezultati ukazuju na to da su citokini koji učestvuju u regulaciji funkcije B limfocita značajni dijagnostički i prognostički parametri u sepsi. Predominacija antiinflamatornog odgovora na koju ukazuju povi&scaron;ene koncentracije sTACI receptora pokazala se pored APACHE II i SOFA skora kao jedini nezavisni prediktor razvoja MODS-a i letalnog ishoda septičnih bolesnika. Neophodna su dalja istraživanja u pravcu određivanja momenta kada u imunskom odgovoru organizam prelazi iz stanja dominacije proniflamatornog u dominaciju antiinflamatornog odgovora radi pravovremenog reagovanja imunomodulatornom terapijom.</p> / <p>Introduction: The nonspecific clinical presentation of sepsis and great individual response variations, as well as huge significance of adequate early prognosis of its clinical course and outcome made sepsis biomarkers research extremely significant. The properties of more than 200 biomarkers have been evaluated for prognostic value, but none have adequate specificity and sensitivity. The role of the B cells in sepsis pathogenesis also remains unclear. Tumor necrosis factor alpha (TNF-&alpha;) superfamily members: A proliferation inducing ligand (APRIL), Bcell activating factor (BAFF) and soluble transmembrane activator and calcium modulator cyclophilin ligand interactor (sTACI) are key factors in B cell biology. The aim of the study was to evaluate the diagnostic and prognostic significance of determining the concentrations of tumor necrosis factor alpha superfamily members for the prediction of MODS development in the first 48h of hospitalization as well as outcome prediction.<br />Subjects and methods: The study included 150 patients suffering from sepsis treated at the Clinic for infectious diseases and Department for anesthesiology and reanimatology of the Clinical center of Vojvodina, and 30 healthy volunteer blood donors. The demographic and other data regarding routine blood analysis performed during sepsis treatment of the patients has been acquired from their hospitalization documentation. Additional 5 ml of venous blood was taken from the patients and the concentrations of APRIL, BAFF and sTACI have been determined using the ELISA method by using R&amp;D Systems commercial kit&rsquo;s. Results: There is a statistically significant difference in concentrations of APRIL, BAFF and sTACI between healthy blood donors and septic patients (p&lt;0.001). APRIL showed the highest sensitivity (99%) and specificity (97%) in distinguishing sepsis from healthy subjects. BAFF showed statistically significant higher concentrations in Gram positive than in Gram negative sepsis (p=0,03). In the multivariate logistic regression analysis, only anti-inflammatory cytokine sTACI and APACHE II or SOFA score remained significant predictors of MODS and lethal outcome. sTACI showed greater sensitivity and specificity for MODS and outcome prediction then the widely used procalcitonin.&nbsp; Conclusions: The concentrations of TNF superfamily members, the main regulators of B cell function, have a significant diagnostic and prognostic value in predicting sepsis course and outcome. The predomination of the anti-inflammatory response, as being pointed out by elevated concentrations of sTACI receptors, has proved to be the only independent predictor, besides APACHE II and SOFA score, in MODS and lethal outcome development in sepsis. Further research is needed in order to accurately determine the exact moment when the immunological response shifts from the predominance of the pro-inflammatory response to the predominance of the anti-inflammatory response, so as to ensure the timely application of therapy that modulates the immunological response.</p>

Das Ubiquitin-Proteasom-System ist an der Degradation cytosolischer Proteine und der Generierung von Antigenen beteiligt, die über MHC Klasse I Moleküle CD8+ T Zellen präsentiert werden. Die Antigenprozessierung wird durch Typ I und II Interferone beeinflusst, welche die Formierung des Immunoproteasoms und des Proteasomen-Aktivators PA28 induzieren und so die katalytische Aktivität des Ubiquitin-Proteasom-Systems qualitativ verändern. In der vorliegenden Arbeit wurde im Zellkulturmodell unter dem Einfluss von IFN gamma die zunehmende Inkorporation der Immunountereinheiten in de novo assemblierende 20S Proteasomen und die daraus resultierende Veränderung der proteolytische Aktivität untersucht. Die Inkorporation der Immunountereinheiten wurde mittels 2D Gelelektrophorese und Western Blots von 20S Proteasomen untersucht, die nach unterschiedlicher Stimulationsdauer mit IFN gamma aus HeLa Zellen isoliert wurden. Es konnte gezeigt werden, dass innerhalb der ersten 24h einer IFN gamma Stimulation die strukturelle Heterogenität des zellulären Proteasomenpools zunimmt, indem sowohl intermediäre als auch Immunoproteasomen assemblieren. In der Nativ-PAGE von Lysaten IFN gamma stimulierter Zellen wurde eine Zunahme des 20S Proteasoms als freier Komplex und in Assoziation mit PA28 beobachtet, während die Menge des zum ATP-abhängigen Abbau von polyubiquitinierten Proteinen notwendigen 26S Proteasoms unverändert blieb. Die Stimulation mit IFN gamma hatte eine Steigerung der gesamtproteasomalen Aktivität zur Folge, die unter Inhibition der Interaktion zwischen 20S Proteasom und PA28 verzögert erfolgte. Die katalytischen Eigenschaften isolierter Proteasomen wurden anhand der Generierung eines immunrelevanten Hepatitis C CTL Epitops des viralen Core Proteins in vitro untersucht. Im Verlauf der IFN gamma Stimulation de novo assemblierte Proteasomen wiesen jeweils unterschiedliche Präferenzen für die Generierung des untersuchten CTL Epitops auf. Eine weitere, proteasomen-spezifische Änderung der katalytischen Aktivität bewirkte die Assoziation des Proteasomen-Aktivators. Innerhalb der ersten zwölf Stunden einer IFN gamma Stimulation wurde das Epitop vermehrt mit der Unterstützung des Proteasomen-Aktivators generiert, nach 24 Stunden zunehmend durch freies 20S Proteasom. Die Ergebnisse der vorgestellten Arbeit zeigen, dass Strukturvarianten des Proteasoms zusammen mit PA28 redundant funktionieren und eine hohe proteolytische Plastizität des UPS gewährleisten. / The ubiquitin proteasome system is responsible for the degradation of cytosolic proteins and the processing of MHC class I restricted antigens. The generation of these antigens is influenced by type I and II interferons which induce the expression of immunoproteasomes and the proteasome activator PA28; and thereby impact the quality of peptides processed by the proteasome system. The adoption of the proteasome system to a proinflammatory environment has been investigated in a cell culture model by isolating proteasomes after different stages of IFN gamma stimulation. The composition of isolated proteasomes was analysed by 2D PAGE and western blot approach. The presented work shows that within 24h of IFN gamma stimulation an increasing heterogeneity of the cellular proteasome pool is observed, resulting from the assembly of both intermediate type proteasomes and immunoproteasomes at the early stage of IFN gamma stimulation. It could be shown by native PAGE of HeLa cell lysates that IFN gamma induces increasing amounts of 20S proteasomes and PA28 associated proteasomes without decreasing the amount of 26S proteasomes that are necessary for the ATP dependent degradation of ubiquitinated proteins; and resulting in an enhanced total proteasomal activity in vitro. This increase in activity was delayed when the interaction of 20S proteasomes and PA28 was inhibited. A comparative analysis of the ability of isolated 20S proteasomes to generate a known hepatitis C virus derived CTL epitope in vitro proved that during early IFN gamma stimulation de novo assembled proteasomes exhibited a structure specific preference to generate the HCV CTL epitope either alone or in combination with the proteasome activator PA28. Within the first 12h of IFN gamma stimulation the epitope was generated with higher efficiency by 20S proteasomes in association with PA28, whereas after 24h the impact of PA28 on the proteasome pool was less pronounced. The presented work shows that IFN gamma induces a heterogeneity of 20S proteasomes in the early stage of stimulation, acting in combination with the proteasome activator in a redundant manner; and provides a high proteolytic placticity of the proteasome system.