Adenylate cyclase toxin (CyaA) produced by the causative agent of whooping cough Bordetella pertussis, is a key virulence factor important for colonization of the host. CyaA targets preferentially myeloid phagocytes expressing CD11b/CD18 integrin. By elevating cytosolic cAMP in the host cells, CyaA interferes with their phagocytic, chemotactic and oxidative burst capacities. Furthermore, CyaA modulates the secretion of cytokines and the maturation state in LPS-stimulated dendritic cells (DC) by affecting the expression of costimulatory molecules. In this study, we investigated the effects of CyaA on the capacity of murine bone-marrow DC to prime CD4+ and CD8+ T cells in response to ovalbumin epitopes delivered by the CyaA-AC- toxoid, as a model antigen. Further, we examined the possible impact of CyaA on the antigen uptake and processing for MHC class I and II-restricted presentation by DC, as we previously observed a decreased T cell stimulatory capacity of CyaA-treated DC in response to soluble ovalbumin. We found out that the high levels of cAMP generated by CyaA in LPS-stimulated DC account for the decreased presentation of ovalbumin epitopes carried by CyaA-AC- toxoid on MHC class I and II molecules, thereby impairing the CD8+ and CD4+ T cell responses. Whereas CyaA did not influence the...

Diss. (sammanfattning) Stockholm : Karolinska institutet, 2010.

<p>Uvod: Karcinom jajnika predstavlja značajan zdravstveni problem.Karakteri&scaron;e ga najveća smrtnost od svih ginekolo&scaron;kih maligniteta. Najveći broj slučajeva karcinoma jajnika dijagostikuje se u uznapredovalim stadijumima bolesti (FIGO st. III i IV), kod kojih petogodi&scaron;nje preživljavanje iznosi ispod 30%, dok se svega 25% slučajeva otkrije u prvom stadijumu gde petogodi&scaron;nje preživljavanje iznosi preko 90%. Do danas nije otkrivena dijagnostička metoda za rano otkrivanje početnog karcinoma jajnika u op&scaron;toj populaciji koja je dovoljno osetljiva i specifična da bi se koristila kao &bdquo;screening&ldquo; metoda. Uspeh u lečenju karcinoma jajnika direktno zavisi od rano postavljene dijagnoze. Cilj istraživanja: Utvrditi značaj tumorskih markera Ca125, HE4, Roma indexa, konvencionalne i dopler transvaginalne sonografije u dijagnostici karcinoma jajnika. Metodologija: Istraživanje je sprovedeno kao prospektivna klinička studija, na Klinici za ginekologiju i aku&scaron;erstvo u Novom Sadu. Ispitivanjem je obuhvaćeno 238 pacijenktinja sa adneksalnim tumorom za operativno lečenje. Preoperativno svim pacijentkinjama je uzeta detaljna anamneza, urađen konvencionalni i dopler transvaginalni ultrazvučni pregled i uzeta krv za određivanje tumorskih markera CA125, HE4, Roma indexa. U zavisnosti od definitivnog patohistolo&scaron;kog nalaza pacijentkinje su podeljene u dve grupe. Grupu A ili ispitivanu grupu su činile ispitanice sa karcinomom i border line tumorima,a grupu B ili kontrolnu grupu,pacijentkinje sa benignim tumorima jajnika. Rezultati: Prosečna starost pacijentkinja je 53 godine. U ukupnom ispitivanom uzorku bilo je statistički značajno vi&scaron;e pacijentkinja u premenopauzi(59,2%) u odnosu na postmenopauzalne pacijentkinje. U ispitivanoj grupi najče&scaron;će zastupljen patohistolo&scaron;ki tip karcinoma je high-grade serozni cistadenokarcinom. Kod najvećeg broja pacijentkinja(49,4%) karcinom je dijagnostikovan u I stadijumu bolesti. U diferencijaciji karcinoma jajnika i benignih tumora jajnika, AUC vrednosti za HE4,Ca125 i Roma index su 0.933, 0.831 i 0.932. Senzitivnost HE4,Ca125,Roma indexa iznosi 0.797/ 0.734 / 0.823. Specifičnost HE4,Ca125, Roma indexa je 0.881 / 0.838 / 0.774. Senzitivnost konvencionalne i dopler transvaginalne sonografije je 0,937/ 0,750, a specifičnost je 0,736/ 0,931 respektivno.Kod pacijentkinja sa endometriozom, vrednost tumorskog markera HE4 je povi&scaron;ena samo kod 6% pacijentkinja, za razliku od vrednosti Ca125 koje su povi&scaron;ene kod 76% pacijentkinja sa endometriozom. Zaključak: Najsnažniji prediktori u diferencijaciji karcinoma od benignih tumora jajnika su: tumorski marker HE4, Roma index, indeks otpora protoku krvi kroz tumorsko tkivo (RI), neravan unutra&scaron;nji zid tumora i ekrescencije unutar tumora. Najbolju senzitivnost u detekciji karcinoma jajnika pokazala je konvencionalna transvaginalna sonografija u odnosu na druge dve ispitivane metode, dok najbolju specifičnost u odvajanju benignih tumora od karcinoma jajnika pokazuje dopler transvaginalna sonografija.</p> / <p>Background: Ovarian cancer represents very important world health issue. It is characterized by the highest mortality rate of all gynecological malignancies. The majority of ovarian cancer cases are diagnosed in advanced stages (FIGO III and IV) in which 5 year survival rate is less than 30%, and only 25% of cases are diagnosed in stage I with survival rate of 90%. So far no diagnostic method has been discovered that is specific and accurate enough to diagnose ovarian cancer in early stage in general population, so that it can be used as screening method. Success rate of treatment of ovarian cancer is dependent on the stage in which the diagnosis has been made. Objective: to determine the importance of tumor markers CA 125, HE4, Roma index, conventional and Doppler transvaginal ultrasound in diagnosis of ovarian cancer. Method: Research was undertaken as prospective study at Clinic for Gynecology and Obstetrics in Novi Sad. The analysis included 238 women with adnexal tumors indicated for surgery. Preoperatively detailed medical history, blood analysis (CA125,HE4,ROMA index), conventional and Doppler transvaginal ultrasound were done for all patients. Patients were divided into two groups depending on their definite pathohistological finding. Group A included patients with carcinoma and border line tumors. Group B (control group) included patients with benign ovarian tumors. Results: Average age of patient was 53 years. More patients were premenopausal (59.2%). The most frequent pathohistological type of carcinoma was high grade serous cystadenocarcinoma. In most cases diagnosis was made in stage I (49.4%). In differentiation between ovarian carcinoma and benign ovarian tumors AUC for HE4, Ca125and Roma index were 0.933,0.831,0.932. Sensitivity of HE4,Ca125 and Roma index is 0.797,0.734,0.832. Specificity of HE4,Ca125 and Roma index is 0.881,0.838,0.774. Sensitivity of conventional and transvaginal ultrasound is 0.937, 0.750, and specificity is 0.736 and 0.931 respectively. In patients with endometriosis tumor marker HE4 levels were elevated in only 6% of cases, while Ca125 levels were elevated in 76% of cases. Conclusion: The most important predictors in carcinoma/benign tumor differentiation are tumor markers HE4, Roma index, RI, uneven inner walls of tumor and ekrescency inside tumor. The highest sensitivity in ovarian cancer detection showed conventional transvaginal ultrasound when compared to two other used methods. The highest specificity in carcinoma/bening tumor differentiation showed doppler transvaginal ultrasound.</p>

Mémoire numérisé par la Division de la gestion de documents et des archives de l'Université de Montréal.

La présentation antigénique par le complexe majeur d’histocompatibilité (MHC) I est un processus ubiquitaire permettant la présentation de protéines endogènes qui reflètent l'état de la cellule à la surface cellulaire aux lymphocytes T CD8+ dans le contexte de la surveillance et la réponse immunitaires. Ainsi, l'expression des molécules du MHC I classiques est induite en réponse aux stimuli inflammatoires afin de favoriser la reconnaissance immunitaire et l'élimination des pathogènes. HFE est une molécule du MHC Ib non-classique qui sert de régulateur négatif de l'absorption du fer. HFE est associé au développement de l'hémochromatose héréditaire (HH), maladie associée au métabolisme du fer mais souvent accompagnée de défauts immunitaires. Ainsi, nous avons en premier lieu étudié l'impact de HFE sur la présentation antigénique par MHC I, afin d'expliquer en partie les défauts immunitaires liés à l'HH associée à HFEC282Y. Puis, compte tenu de l'impact de l'inflammation sur l'expression des molécules du MHC I classiques, nous avons étudié la régulation de l'expression de HFE en réponse aux stimuli inflammatoires induits par les cellules du sang périphérique mononucléées (PBMC). Nous avons mis au point un système d’expression antigénique dans lequel nous contrôlons l’expression de MHC I, de HFE et d’un antigène pour lequel nous avons généré des lymphocytes T CD8+ spécifiques. Nos résultats démontrent que la forme sauvage de HFE (HFEWT), contrairement à sa forme mutée (HFEC282Y), inhibe la reconnaissance de complexes MHC I/peptide (pMHC). Nous avons également démontré que l'inhibition de la reconnaissance est maintenue, indépendamment des niveaux d'expression de MHC I à la surface, d'une compétition pour la β2-microglobuline, de la capacité de HFE d'interagir avec le récepteur de la transferrine, de l'origine de l'antigène ou de l'affinité de celui-ci. Par ailleurs, nous avons identifié les domaines α1-2 de HFEWT comme étant responsables de l'inhibition de la reconnaissance antigénique. Par contre, la reconnaissance de peptides chargés de manière externe sur les molécules du MHC I présentes à la surface n'a démontré aucune inhibition en présence de HFEWT, suggérant que HFEWT pourrait affecter la reconnaissance en interférant avec le processus d'apprêtement antigénique intracellulaire. À l’inverse, nous avons souhaité déterminer si les lymphocytes T activés pouvaient influencer les niveaux d'expression de HFE. En termes de régulation de l'expression de HFE, nous avons établi que HFE est exprimé dans les tissus sains chez l'humain et induit chez les lignées de cancers du colon, du sein, du poumon, du rein et du mélanome. Par ailleurs, en co-cultivant des lymphocytes T activés avec ces lignées tumorales, nous avons démontré que l'expression de HFE est fortement inhibée dans toutes ces lignées tumorales lorsqu'exposées à des lymphocytes T activés. Finalement, la modulation de l'expression de HFE est indépendante du contact cellulaire et semble médiée en partie par le GM-CSF, l'IFN-γ et le TNF. En somme, ces résultats suggèrent que les lymphocytes T de l'hôte modulent l'expression de HFE dans le microenvironnement inflammatoire, ce qui pourrait promouvoir la reconnaissance des antigènes présentés sur les molécules du MHC I présentées aux lymphocytes T CD8+ antigène-spécifiques. De plus, ces études soulèvent la possibilité d'un nouveau rôle physiologique de HFEWT dans la voie de présentation antigénique par MHC I, qui pourrait moduler l'immunogénicité des antigènes et la réponse immunitaire cellulaire chez l'hôte. / MHC class I antigen presentation is an ubiquitous process by which cells present endogenous proteins to CD8+ T lymphocytes during immune surveillance and response. Accordingly, classical MHC I molecules are up-regulated in response to inflammatory stimuli to favor immune recognition and pathogen clearance. HFE is a non-classical, MHC Ib molecule which acts as a negative regulator of iron absorption. HFE has been linked to the development of hereditary hemochromatosis (HH), an iron overload disease often associated to immune defects. Firstly, we studied the impact of HFE expression on MHC I antigen presentation, as a hypothesis for HH-associated immunological defects observed in HFEC282Y-mutated HH patients. Secondly, we evaluated whether, like its classical MHC I counterparts, HFE expression could be modulated in response to peripheral blood mononuclear cell (PBMC) inflammation. We developed an antigen presentation system in which we control MHC I expression, HFE expression, and expression of a model antigen for which we have generated antigen-specific CD8+ T lymphocytes. Our results demonstrate that wild-type HFE (HFEWT), but not C282Y-mutated HFE (HFEC282Y), inhibits recognition of MHC I antigens. We further demonstrate that inhibition of antigen recognition is maintained regardless of MHC I surface levels, β2-microglobulin competition, HFE ability to interact with transferrin receptor, antigen origin, or epitope affinity. We identified the α1-2 domains of HFEWT as being responsible for inhibiting antigen recognition. However, recognition of externally peptide-pulsed 293-A2 remained uninhibited in presence of HFEWT, indicating that HFE may affect T cell recognition by interfering with intracellular antigen processing. We also questioned whether activated T lymphocytes may influence HFE expression. We established that HFE is widely expressed in healthy human tissues and induced in colon cancer, breast cancer, lung cancer, kidney cancer and melanoma cell lines. Furthermore, HFE mRNA expression was drastically inhibited in all tumor cell lines when exposed to activated T lymphocytes. Down-regulation of HFE mRNA expression was independent of cell contact and appears to be partially mediated by GM-CSF, IFN-γ, and TNF. Overall, these data suggest that host T lymphocytes may alter HFE expression levels in the inflammatory microenvironment, which could, in turn, promote recognition of MHC I antigens presented to antigen-specific CD8+ T lymphocytes. Accordingly, this could suggest a new physiological role for HFEWT in the MHC I antigen presentation pathway, which could modulate antigen immunogenicity and the cellular immune response.

Antigen presentation during which antigen fragments in complex with MHC glycoproteins are recognized by T cell antigen-specific receptors is necessary for the initiation of adaptive immune response. During this process, immunological synapse is assembled at the site of contact between the T cell and the antigen-presenting cell (APC). This leads to the activation of receptors on the surface of both cells followed by triggering of multiple signaling pathways. However, our knowledge about the signaling occurring at the APC-side of the IS is limited in comparison to the T cell side. Here, we analyze role of Src family kinases in the APC signaling pathways. For this purpose, constructs targeting Csk kinase to the plasma membrane of APCs were prepared to inhibit SFKs there. We show that expression of these constructs inhibits activation of SFKs, calcium mobilization and cell activation of K46 B cell line. Further, expression of these constructs in hematopoietic progenitors attenuates their differentiation into dendritic cells which then results in their decreased ability to stimulate T cells.

Von TRAIL, FasL und APRIL, drei Mitgliedern der TNF-Liganden-Familie, ist bekannt, dass Trimerstabilität und Oligomerisierungsstatus maßgeblich das Rezeptoraktivierungspotential dieser Liganden beeinflussen. Für die immunstimulatorischen TNF-Liganden CD27L, CD40L, OX40L, 41BBL und GITRL war hingegen vor der Durchführung dieser Arbeit praktisch nicht bekannt, inwieweit Trimerbildung, Stabilisierung und Oligomerisierung wichtig für deren Aktitvität sind. Dies wurde in dieser Arbeit systematisch untersucht. CD40L besaß bereits als trimeres Molekül eine hohe Aktivität, die durch sekundäre Oligomerisierung nur wenig gesteigert wurde. Die spezifische Aktivität konnte durch Stabilisierung mit Hilfe der Tenascin-C (TNC)-Trimerisierungsdomäne nur geringfügig gesteigert werden. CD27L war als lösliches Flag-markiertes sowie als hexameres Fc-Protein selbst nach Quervernetzen nicht in der Lage, seinen Rezeptor CD27 zu binden und zu aktivieren. Die TNC-stabilisierte trimere Form des CD27L hingegen induzierte nach Oligomerisierung mit einem anti-Flag-Antikörper ein starkes Signal. Trimerer OX40L und trimerer 41BBL konnten nur in oligomerisierter Form ihre Rezeptoren aktivieren, wobei die Aktivität der TNC-stabilisierten Form signifikant stärker ausgeprägt war. GITRL aktivierte seinen Rezeptor bereits als stabilisiertes Trimer und Hexamer, die Aktivität konnte durch Quervernetzen nur gering gesteigert werden. Zusammenfassend kann man sagen, dass CD27L, OX40L und 41BBL zu der Untergruppe der TNF-Ligandenfamilie gehört, für die eine Stabilisierung des trimeren Moleküls und dessen Oligomerisierung nötig sind, um eine starke Rezeptoraktivierung zu ermöglichen. Im Gegensatz dazu zeigten CD40L und GITRL bereits oligomerisierungsunabhängig eine hohe Aktivität. GITRL benötigte allerdings die Stabilisierung des trimeren Moleküls durch die TNC-Domäne, um gute Aktivität zu zeigen. Im Weiteren wurden Antikörperfragment (scFv-)-TNF-Ligand-Fusionsproteine konstruiert und untersucht, die ein Zelloberflächenantigen binden. Eine starke Zelloberflächenantigen-spezifische Aktivierung des jeweiligen Rezeptors konnte für scFv-41BBL und für scFv-OX40L gezeigt werden, wohingegen scFv-CD40L und scFv-GITRL bereits auf antigennegativen Zellen stark aktiv waren. scFv-CD27L war selbst auf antigenpositiven Zellen inaktiv. Verwendet man an Stelle des Antikörperfragments eine extrazelluläre Proteinbindedomäne, z.B. die eines TNF-Rezeptors, erhält man Fusionsproteine, die zum einen eine selektive Aktivierung der TNF-Ligandendomäne und somit die Aktivierung des korrespondierenden Rezeptors auf der Zielzelle ermöglichen, zum anderen aber durch die Bindung an den membranständigen Liganden dessen Aktitvät neutralisieren können. Für CD40-, RANK- und B7-2-FasL konnte der immobilisationabhängige Aktivierungseffekt auf entsprechenden Zelloberflächenmolekül-exprimierenden Zellen gezeigt werden. Anhand von T47D-Zellen, die durch eine autokrine CD40L-CD40-Signalschleife vor Apoptose geschützt sind, konnte gezeigt werden, dass durch die Bindung von CD40-FasL an membranständigen CD40L die CD40L-CD40-Interaktion gestört und gleichzeitig Apoptose verstärkt induziert werden kann. Das Prinzip der antigenabhängigen Aktivierung von TNF-Liganden könnte Anwendung in der Tumortherapie finden, da bei Verwendung entsprechender selektiv exprimierter Marker eine lokale Rezeptoraktivierung erreicht und so Nebenwirkungen minimiert werden können. / Trimer stability and oligomerization status of TRAIL, FasL and APRIL, three members of the TNF ligand family, critically determine their receptor activating potential. However, detailed information for the immunostimmulatory ligands CD27L, CD40L, OX40L, 41BBL and GITRL regarding the importance of trimer formation, stabilization and oligomerization for ligand activity was lacking. These aspects were investigated systematically in this work. CD40L was highly active as a trimeric molecule. Secondary oligomerization and/or stabilization via the tenascin-C (TNC) trimerization domain slightly enhanced its specific activity. As soluble Flag-tagged and as hexameric Fc protein CD27L failed to bind and activate its cognate receptor CD27, even after crosslinking. However, the TNC stabilized form of CD27L induced a strong signal after oligomerization with anti-Flag antibody. Receptor signaling was only activated by oligomerized molecules of trimeric OX40L and 41BBL whereas the respective TNC fusion protein showed significant stronger activity. Stabilized GITRL trimers and hexamers already activated their receptor whereas oligomerization of GITRL just slightly enhanced the specific activity. Taken together, CD27L, OX40L and 41BBL belong to a TNF ligand family subgroup which requires oligomerization and stabilization of the trimeric molecule to ensure strong receptor activation. In contrast, CD40L and GITRL already display high oligomerization-independent activity, though the latter needs stabilization by the TNC domain. Furthermore, antibody fragment (scFv)-ligand fusion proteins targeting specific cell surface antigens were designed and analyzed. Strong cell surface antigen-selective TNF receptor activation was achieved for scFv-41BBL and scFv-OX40L whereas scFv-CD40L and scFv-GITRL already induced signaling in the absence of antigen-positive cells. scFv-CD27L lacked activity even on antigen-positive cells. Using an extracellular protein binding domain for example the ligand binding domain of a TNF receptor instead of an antibody fragment resulted in fusion proteins that on the one hand activate the TNF ligand domain and thus the corresponding receptor on target cells and on the other hand neutralize membrane ligand activity by binding. The effect of cell surface immobilization-mediated activation of these fusion proteins on cells expressing the corresponding target molecule was shown here for CD40-, RANK- and B7-2-FasL. The CD40-FasL fusion protein simultaneously blocked CD40L-CD40 interaction and induced strong apoptosis in T47D cells displaying an antiapoptotic autocrine CD40L-CD40 signaling loop. The principle of antigen-dependent activation of TNF ligands could be of use in tumor treatment due to the fact that tumor specific marker targeting leads to locally restricted receptor activation on antigen positive cells, promising a reduction in potential off target effects.

O presente estudo avaliou a ocorrência de imunoparalisia e sua associação com pior prognóstico em pacientes pediátricos internados em uma UTI de hospital terciário. Para determinar a presença de imunoparalisia procedeu-se a dosagem da expressão de mHLA-DR usando o QuantiBRITE TM Anti HLA-DR/ Anti- Monocyte, um novo reagente que padroniza os valores da citometria de fluxo para o mHLA-DR. Determinamos a expressão de mHLA-DR em 30 pacientes com sepse grave ou choque sépticos admitidos na UTI Pediátrica no período do estudo, mHLA-DR foi quantificado por duas vezes: entre os dias 3 a 5 (mHLA-DR1) e 5 a 7 (mHLA-DR2) após o inicio do quadro séptico. Também foi calculado o deltamHLA-DR (mHLA-DR2 - mHLA-DR1). Dosamos, ainda, o mHLA-DR em vinte e um controles hígidos. O objetivo do estudo foi determinar se a expressão de mHLA-DR correlaciona-se com a mortalidade em pacientes sépticos pediátricos. Os resultados mostram que o mHLA-DR foi significativamente menor nos pacientes sépticos do que nos controles (p = 0.0001). A mortalidade foi de 46% nos pacientes com valores negativos ou < 1000 mAb/cell de deltaHLA-DR, e 7% em pacientes com valores positivos ou > 1000 mAb/cell de deltaHLADR. O deltamHLA-DR médio foi significativamente diferente entre sobreviventes e pacientes que foram a óbito (p = 0.023). Dessa forma, após a análise estatística dos resultados concluímos que o deltaHLA-DR correlaciona-se com a mortalidade em pacientes pediátricos com sepse grave e choque séptico / This study analysis the presence of Immunoparalysis and its association with prognosis in pediatric septic patients of a Tertiary Intensive Care Unit. To determine the presence of immunoparalysis we performed the mHLA-DR dosage using the QuantiBRITE TM Anti HLA-DR/ Anti- Monocyte, a novel reagent that standardizes flow cytometry values. We determined mHLA-DR expression in 30 patients with severe sepsis or septic shock admitted to PICU, mHLA-DR expression was quantified between days 3-5 and 5-7 after the onset of sepsis and calculated the deltamHLA-DR (mHLA-DR2 - mHLADR1). We also measured mHLA-DR levels in twenty-one healthy patients. The objective of this study was to determine if mHLA-DR values correlate with mortality in pediatric septic patients. The results showed that the mean mHLA-DR expression was significantly lower in septic patients compared with controls (p = 0.0001). Mortality was 46% in patients with negative deltaHLA-DR or < 1000 mAb/cell and 7% in patients with positive deltaHLA-DR or > 1000 mAb/cell. Mean deltamHLA-DR levels were significantly different between survivors and non-survivors (p = 0.023). After statistical analysis we concluded that deltaHLA-DR correlates with mortality in pediatric patients with septic shock or severe sepsis

O presente estudo avaliou a ocorrência de imunoparalisia e sua associação com pior prognóstico em pacientes pediátricos internados em uma UTI de hospital terciário. Para determinar a presença de imunoparalisia procedeu-se a dosagem da expressão de mHLA-DR usando o QuantiBRITE TM Anti HLA-DR/ Anti- Monocyte, um novo reagente que padroniza os valores da citometria de fluxo para o mHLA-DR. Determinamos a expressão de mHLA-DR em 30 pacientes com sepse grave ou choque sépticos admitidos na UTI Pediátrica no período do estudo, mHLA-DR foi quantificado por duas vezes: entre os dias 3 a 5 (mHLA-DR1) e 5 a 7 (mHLA-DR2) após o inicio do quadro séptico. Também foi calculado o deltamHLA-DR (mHLA-DR2 - mHLA-DR1). Dosamos, ainda, o mHLA-DR em vinte e um controles hígidos. O objetivo do estudo foi determinar se a expressão de mHLA-DR correlaciona-se com a mortalidade em pacientes sépticos pediátricos. Os resultados mostram que o mHLA-DR foi significativamente menor nos pacientes sépticos do que nos controles (p = 0.0001). A mortalidade foi de 46% nos pacientes com valores negativos ou < 1000 mAb/cell de deltaHLA-DR, e 7% em pacientes com valores positivos ou > 1000 mAb/cell de deltaHLADR. O deltamHLA-DR médio foi significativamente diferente entre sobreviventes e pacientes que foram a óbito (p = 0.023). Dessa forma, após a análise estatística dos resultados concluímos que o deltaHLA-DR correlaciona-se com a mortalidade em pacientes pediátricos com sepse grave e choque séptico / This study analysis the presence of Immunoparalysis and its association with prognosis in pediatric septic patients of a Tertiary Intensive Care Unit. To determine the presence of immunoparalysis we performed the mHLA-DR dosage using the QuantiBRITE TM Anti HLA-DR/ Anti- Monocyte, a novel reagent that standardizes flow cytometry values. We determined mHLA-DR expression in 30 patients with severe sepsis or septic shock admitted to PICU, mHLA-DR expression was quantified between days 3-5 and 5-7 after the onset of sepsis and calculated the deltamHLA-DR (mHLA-DR2 - mHLADR1). We also measured mHLA-DR levels in twenty-one healthy patients. The objective of this study was to determine if mHLA-DR values correlate with mortality in pediatric septic patients. The results showed that the mean mHLA-DR expression was significantly lower in septic patients compared with controls (p = 0.0001). Mortality was 46% in patients with negative deltaHLA-DR or < 1000 mAb/cell and 7% in patients with positive deltaHLA-DR or > 1000 mAb/cell. Mean deltamHLA-DR levels were significantly different between survivors and non-survivors (p = 0.023). After statistical analysis we concluded that deltaHLA-DR correlates with mortality in pediatric patients with septic shock or severe sepsis

Chlamydia trachomatis serovars D-K are the leading cause of bacterially-acquired sexually transmitted infections in the United States. As an obligate intracellular pathogen, C. trachomatis infects columnar epithelial cells of the genital mucosae and can cause deleterious sequelae such as pelvic inflammatory disease, infertility, and ectopic pregnancy. Several chlamydial antigens reach the host cell cytosol prior to the natural release of chlamydiae at the end of the developmental cycle. While some of these extra-inclusion antigens traffic to the host cell surface, others remain intracellular where they are proposed to influence vital host cell functions and antigen trafficking and presentation. The research herein examines the escape and trafficking of the immunodominant chlamydial antigens MOMP, LPS, and cHsp60 within C. trachomatis serovar E-infected polarized human endometrial epithelial cells. Studies using high-resolution transmission electron microscopy (TEM) and immuno-TEM report the novel escape mechanism of chlamydial antigens via vesicles everted/pinched off from the inclusion membrane, an occurrence observed both in the presence and absence of the antibiotic azithromycin. These extra-inclusion vesicles were differentiated from Golgi vesicles and were shown to deliver chlamydial heat shock protein 60 (cHsp60)-homologs 2 and 3, but not homolog 1, to the infected cell surface. Examination of the iron-responsiveness of the three cHsp60 homologs by immuno-TEM revealed a significant increase in cHsp60-2 following iron deprivation. Further investigation of the trafficking of chlamydial MOMP and LPS antigens enveloped within the protective everted inclusion membrane vesicles within host cells involved density gradient centrifugation for the separation of epithelial secretory pathway components followed by SDS-PAGE and Western blot to determine whether the chlamydial antigen-containing vesicles could fuse with and deliver the antigens to host cell organelles. Coupled with immuno-TEM, these data confirmed the presence of major chlamydial antigens within the endoplasmic reticulum of infected host cells. Additionally, chlamydial lipopolysaccharide (LPS) was co-localized with CD1d, a lipid antigen-presenting molecule. Collectively, these studies (i) establish a novel escape mechanism for chlamydial antigens, (ii) identify cHsp60-2 as a marker of iron stress response in C. trachomatis, and (iii) define for the first time the host cell ER as a destination for selected chlamydial antigens during infection.