Development of a thermostable spray dried outer membrane vesicle pertussis vaccine for pulmonary immunization

Worldwide resurgence of whooping cough calls for improved, next-generation pertussis vaccines that induce broad and long-lasting immunity. A mucosal pertussis vaccine based on outer membrane vesicles (omvPV) is a promising candidate. Further, a vaccine that is stable outside the cold chain would be of substantial advantage for worldwide distribution and application. A vaccine formulated as a powder could both stabilize the vaccine as well as make it suitable for pulmonary vaccination. To that end, we developed a spray dried omvPV with improved stability compared to the liquid omvPV formulation. Spray drying did not affect the structural integrity of the omvPV. The antigenicity of Vag8, a major antigen in omvPV was diminished slightly and an altered tryptophan fluorescence indicated some changes in protein structure. However, when administered via the pulmonary route in mice after reconstitution, spray dried omvPV showed comparable immune responses and protection against challenge with live B. pertussis as liquid omvPV. Mucosal IgA and Th17 responses were established in addition to broad systemic IgG and Th1/Th17 responses, indicating the induction of an effective immunity profile. Overall, a spray dried omvPV was developed that maintained effective immunogenic properties and has an improved storage stability

Measurement of the W mass in e+e−e^+ e^- collisions at 183 GeV

The mass of the W boson is obtained from reconstructed invariant mass distributions in W-pair events. The sample of W pairs is selected from 57 pb$^{-1}$ collected with the ALEPH detector in 1997 at a centre-of-mass energy of 183 GeV. The invariant mass distributions of reweighted Monte Carlo events are fitted separately to the experimental distributions in the $qqbarqqbar$ and all l\nuqqbar channels to give the following W masses: $m_{W}^{hadronic} = 80.461 \pm 0.177(stat.) \pm 0.045(syst.) \pm 0.056(theory) GeV/c^2$, $m_{W}^{semileptonic} = 80.326 \pm 0.184(stat.) \pm 0.040(syst.) GeV/c^2$ where the theory error represents the possible effects of final state interactions. The combination of these two measurements, including the LEP energy calibration uncertainty, gives $m_{W} = 80.393 \pm 0.128(stat.)\pm 0.041(syst.) \pm 0.028(theory)\pm 0.021(LEP) GeV/c^2

Role of plasminogen activator inhibitor type 1 in experimental (post-traumatic) sepsis

Verletzungen und Infektionen lösen einen Entzündungsprozess aus, dessen Ausmaß stark von den individuellen Charakteristiken und der persönlichen Immunantwort des Patienten abhängt. Sepsis wird als systemische Entzündungsantwort auf eine Infektion definiert und ist einer der häufigsten Todesursachen auf Intensivstationen. Ein vorhergegangenes Trauma kann den Verlauf der folgenden Sepsis bedeutend beeinflussen. Das Blutgerinnungssystem bewirkt die Bildung von Fibrin und ist zum einen unentbehrlich für die Eindämmung von Blutungen nach Verletzungen und zum anderen ein wichtiger Bestandteil der angeborenen Immunantwort gegen eindringende Mikroorganismen. Die Gegenspieler der Bluttgerinnung sind natürliche Gerinnungshemmer und die Fibrinolyse. Während einer Sepsis ist die Blutgerinnung im Allgemeinen aktiviert, jedoch gleichzeitig die natürliche Gerinnungshemmung vermindert und die Fibrinolyse stark reduziert. Die Hemmung der Fibrinolyse liegt vor allem an erhöhten Werten von Typ 1 Plasminogen Aktivator Inhibitor (PAI-1). Erhöhte Plasmaspiegel von PAI-1 werden mit einer erhöhten Sterbensrate während der Sepsis assoziiert. Wir haben daher untersucht, ob eine Hemmung von PAI-1 während einer Sepsis die Überlebensrate günstig beeinflusst. Wir verwendeten zuerst das klinisch relevante Model der zäkalen Ligation und Punktion, um eine polymikrobielle, septische Peritonitis in der Maus zu induzieren. Dabei untersuchten wir die Genexpression von PAI 1 in mehreren Organen und zu verschiedenen Zeitpunkten im Verlauf der Sepsis. Im ersten Abschnitt zeigte sich, dass die Genexpression von PAI-1 während der Sepsis sehr organ-spezifisch ist und außerdem vom Zeitpunkt und dem Schweregrad der Sepsis abhängt. Der PAI-1 Plasmaspiegel stieg während der Sepsis stark an, wobei hohe Werte auch höhere Sterbensraten bedeuteten. Im zweiten Abschnitt untersuchten wir, ob Trauma und Blutverlust die eben untersuchte Dynamik von PAI-1 in verschiedenen Gewebetypen während einer folgenden Sepsis beeinflusst. Unsere Daten zeigten, dass vorheriges Trauma mit Blutverlust tatsächlich die PAI-1 Genexpression im vaskulärem Endothel (die Gefäß Innenwand) und in der Leber während der nachfolgenden Sepsis verändert, obwohl der PAI-1 Proteinspiegel im Blut unbeeinflusst bleibt. Zusätzlich zeigten sterbende Mäuse, im Vergleich zu überlebenden Mäusen, höhere PAI-1 Plasmaspiegel (schon zwei Tage bevor Tod) während der posttraumatischen Sepsis. Mittels Bestimmung der Thrombomudulinspiegel in diesen Mäusen konnten wir allerdings zeigen, dass diese Erhöhung der PAI-1 Proteinspiegel unabhängig war von der Stärke des Schadens am vaskulären Endothel. Im dritten Abschnitt dieser Studie wurde bei septischen Mäusen (ohne vorherigem Trauma und Blutverlust) das PAI-1 im Blut mit Hilfe eines spezifischen Antikörpers neutralisiert und die fibrinolytische Aktivität sowie das Überleben der Tiere im Vergleich zur Kontrollantikörperapplikation untersucht. Die Behandlung mit dem spezifischen PAI-1 Antikörper konnte die Hemmung der Fibrinolyse während der Sepsis verhindern, hatte jedoch keinen Einfluss auf das Überleben während der Sepsis. Um möglichst homogene Gruppen zu erreichen, wurden alle septischen Tiere in einem weiteren Durchgang vor der Antikörperapplikation nach ihrem individuellen Entzündungsstatus in zwei Untergruppen eingeteilt: Tiere mit einem niedrigen Risiko zu sterben, beziehungsweise Tiere mit einem hohen Risiko zu sterben. Erst die Unterteilung in zwei homogenere Untergruppen basierend auf dem vorherrschenden Entzündungsstatus (entweder alle in der Gruppe mit hohem oder niedrigem Risiko zu sterben) demonstrierte, dass die Neutralisierung von PAI-1 das Überleben (in beiden Untergruppen) maßgeblich verschlechtert. Auf Gund dieser Ergebnisse können wir folgendes schliessen. Erstens, die organspezifische Genexpression von PAI-1 und dessen Plasmaprotein sind während der Sepsis erhöht. Zweitens, ein vorhergehendes Trauma mit Blutverlust bewirkt wesentliche Veränderungen in der organspezifischen Genexpression von PAI-1 in der anschließenden Sepsis. Drittens, obwohl die Neutralisierung von PAI-1 die Fibrinolyse während Sepsis wieder herstellt, bewirkt dies keinen Überlebensvorteil. Interessanterweise war nur nach Unterteilung der Mäuse in maximal homogene Gruppen (entweder hohes oder niedriges Risiko zu sterben) der nachteilige Effekt der PAI-1 Neutralisierung sichtbar. Diese Ergebnisse bedeuten, dass PAI-1 neben seiner Schlüsselfunktion in der Fibrinolyse, scheinbar eine zusätzliche Funktion im weiteren Entzündungsgeschehen besitzt. Ausserdem erlaubt dieser Effekt die Mutmaßung, dass eine Unterteilung von septischen Patienten in klar definierte, homogene Gruppen, für die Erkennung negativer Effekte von neuartigen Therapien gegen Sepsis durchaus entscheidend sein kann.Injury and infection induce a host response with a magnitude that strongly depends on the individual characteristics of the patient. Sepsis, defined as a systemic inflammation in the presence of an infection, is one of the most common causes of death on the intensive care unit. Posttraumatic sepsis is the septic syndrome that develops after severe injury (trauma), in which the preceding trauma may influence the host response during the subsequent sepsis. The blood clotting system is crucial for the restriction of bleeding (hemorrhage) upon trauma and, moreover, an important part of the innate defense against invading microorganisms. Activation of blood clotting (coagulation) results in the formation of fibrin, which is regulated by natural anticoagulants and the process called fibrinolysis. During sepsis, coagulation is generally activated, natural anticoagulation is attenuated and fibrinolysis is strongly impaired. Impairment of fibrinolysis is mainly due to up-regulation and release of plasminogen activator inhibitor type 1 (PAI-1). Fibrin deposition and high levels of PAI-1 are associated with the development of multi organ dysfunction and mortality during sepsis. Therefore, we investigated whether inhibition of PAI-1 could prove a beneficial therapeutic option during sepsis. In this thesis, we first used the mouse clinically relevant model of cecal ligation and puncture-induced polymicrobial sepsis to study the gene expression of PAI-1 at various time points during the development of sepsis, which was highly specific on the organ-level studied, the time point and sepsis severity. Levels of circulating PAI-1 increased during sepsis and we were able to link high PAI-1 levels to increased mortality. Next, we studied whether combined trauma and hemorrhage influences the dynamics of PAI-1 in the various compartments and over time. Our data suggested that preceding trauma and hemorrhage altered PAI-1 gene expression in the vascular endothelium (inner lining of blood vessels) and in the liver, whereas PAI-1 levels in the plasma were not changed by prior trauma-hemorrhage. Furthermore, dying subjects displayed higher levels of plasma PAI-1 in post-traumatic sepsis compared to survivors, which was irrespective of the level of damage to the vascular endothelium. PAI-1 levels during posttraumatic sepsis can herald death up to two days prior. Finally, we neutralized the circulating PAI-1 in the blood of septic subjects with a specific antibody and compared their fibrinolytic capacity and survival with animals receiving treatment with a control antibody. To establish more homogeneous treatment groups, some animals were divided into two groups prior to treatment: those who where predicted to die and those predicted to survive based on their inflammatory status. We observed that treatment with the antibody was able to prevent impairment of fibrinolysis during sepsis. In the heterogeneous group, in which animals were treated irrespective of their inflammatory status, treatment with the PAI 1 neutralizing antibody did not alter overall sepsis survival. However, in animals with more homogeneous inflammatory response (either high or low), neutralization of PAI-1 by the antibody significantly impaired survival. From our data, it can be concluded that PAI-1 is an essential component of the host response against sepsis, a function that may be independent of its role in fibrinolysis. PAI-1 organ-specific gene expression and its plasma levels are increased after trauma-hemorrhage and sepsis, and preceding trauma modulates the PAI-1 gene expression during sepsis in a tissue-specific fashion. Although neutralizing PAI-1 restored fibrinolysis during sepsis, it did not provide a survival benefit. Remarkably, only after a maximal homogenous separation of septic subjects into high and low risk-of-death cohorts, the detrimental effect of PAI-1 neutralization became apparent. This implies that a separation of septic patients into various well-defined, homogenous cohorts is critical in revealing negative effects of tested anti-sepsis therapeutics.submitted by Pierre RaevenAbweichender Titel laut Übersetzung der Verfasserin/des VerfassersZsfassung in dt. SpracheThromb Res. 2012 May;129(5):e238-45.Wien, Med. Univ., Diss., 2012OeBB(VLID)188353

A Non-Lethal Traumatic/Hemorrhagic Insult Strongly Modulates the Compartment-Specific PAI-1 Response in the Subsequent Polymicrobial Sepsis

Introduction: Plasminogen activator inhibitor 1 (PAI-1) is a key factor in trauma- and sepsis-induced coagulopathy. We examined how trauma-hemorrhage (TH) modulates PAI-1 responses in subsequent cecal ligation and puncture (CLP)-induced sepsis, and the association of PAI-1 with septic outcomes. Methods: Mice underwent TH and CLP 48 h later in three separate experiments. In experiment 1, mice were sacrificed pre-and post-CLP to characterize the trajectory of PAI-1 in plasma (protein) and tissues (mRNA). Post-CLP dynamics in TH-CL

Why do they die?:Comparison of selected aspects of organ injury and dysfunction in mice surviving and dying in acute abdominal sepsis

BACKGROUND: The mechanisms of sepsis mortality remain undefined. While there is some evidence of organ damage, it is not clear whether this damage alone is sufficient to cause death. Therefore, we aimed to examine contribution of organ injury/dysfunction to early deaths in the mouse abdominal sepsis. METHODS: Female OF-1 mice underwent either medium-severity cecal ligation and puncture (CLP-Only) or non-lethal CLP-ODam (CLP with cisplatin/carbontetrachloride to induce survivable hepatotoxicity and nephrotoxicity). In the first experiment, blood was collected daily from survivors (SUR; CLP-Only and CLP-ODam groups) or until early death (DIED; CLP-Only). In the second experiment (CLP-Only), early outcome was prospectively predicted based on body temperature (BT) and pairs of mice predicted to survive (P-SUR) and die (P-DIE) were sacrificed post-CLP. The overall magnitude of organ injury/dysfunction was compared in retrospectively and prospectively stratified mice. RESULTS: At day 7 post-CLP, survival in CLP-Only was 48%, while CLP-ODam was non-lethal. In CLP-Only mice within 24 h of death, urea increased to 78 (versus 40 mg/dl in SUR), ALT to 166 (vs. 108 U/l), LDH to 739 (vs. 438 U/l) and glucose declined to 43 (vs. 62 mg/dl). In CLP-ODam, hypoglycemia was exacerbated (by 1.5-fold) and ALT and LDH were 20- and 8-fold higher versus DIED (CLP-Only) mice. In CLP-Only, predicted deaths (P-DIE) were preceded by a significant rise only in cystatin C (268 vs. 170 ng/ml in P-SUR) but not in creatinine and troponin I. Respiratory function of mitochondria in the liver and kidney of P-SUR and P-DIE CLP-Only mice was not impaired (vs. controls) and ATP level in organs remained similar among all groups. Histologic injury scores in the liver, kidney, heart and lung showed no major disparities among dying, surviving and control mice. CONCLUSIONS: In CLP-Only mice, although the deregulation of parameters indicative of organ injury/dysfunction was greater in dying versus surviving mice, it never exceeded the changes in surviving CLP-ODam animals, and it was not followed by histopathological damage and/or mitochondrial dysfunction. This shows that interpretation of the contribution of the organ injury/dysfunction to early deaths in the CLP model is not straightforward and depends on the pathophysiological origin of the profiled disturbances

Relationship between Age/Gender-Induced Survival Changes and the Magnitude of Inflammatory Activation and Organ Dysfunction in Post-Traumatic Sepsis

<div><p>Age/gender may likely influence the course of septic complications after trauma. We aimed to characterize the influence of age/gender on the response of circulating cytokines, cells and organ function in post-traumatic sepsis. We additionally tested whether post-traumatic responses alone can accurately predict outcomes in subsequent post-traumatic sepsis. A mouse 2-hit model of trauma/hemorrhage (TH, 1^st hit) and cecal ligation and puncture (CLP, 2^nd hit) was employed. 3, 15 and 20 month (m) old female (♀) and male (♂) CD-1 mice underwent sublethal TH followed by CLP 2 days later. Blood was sampled daily until day 6 post-TH and survival was followed for 16 days. To compare general response patterns among groups, we calculated two scores: the inflammatory response (including KC, MIP-1α, TNFα, MCP-1, IFNγ, IL-1β,-5,-6,-10) and the organ dysfunction score (Urea, ALT, AST and LDH). Moreover, mice were retrospectively divided into survivors (SUR) and dying (DIE) based on post-CLP outcome. In general, females survived better than males and their survival did not correspond to any specific estrus cycle phase. <em>Pre-CLP phase</em>: the post-TH inflammatory score was weakest in 3 m♂ but there were no changes among remaining groups (similar lack of differences in the organ dysfunction score). TH induced a 40% increase of IFNγ, MIP-1α and IL-5 in 15 m♂ SUR (vs. DIE) but predictive accuracy for post-CLP outcomes was moderate. <em>Post-CLP phase</em>: while stable in males, inflammatory response score in 15 m and 20 m females decreased with age at day 1 and 2 post-CLP. SUR vs. DIE differences in inflammatory and organ dysfunction score were evident but their magnitude was comparable across age/gender. Nearly identical activation of the humoral inflammatory and organ function compartments, both across groups and according to sepsis severity, suggests that they are not directly responsible for the age/gender-dependent disparity in TH-CLP survival in the studied young-to-mature population.</p> </div

Rechnet sich die Harnsteinmetaphylaxe auch im Zeitalter des Diagnosis related groups (DRG)-Abrechnungssystem?

BACKGROUND: Mild therapeutic hypothermia following trauma has been introduced in several studies to reduce the posttraumatic inflammation and organ injury. In this study, we analyzed the effects of induced mild hypothermia (34°C) on the inflammation of the shock organs liver and kidney. METHODS: In a porcine model of multiple trauma including blunt chest trauma, liver laceration, and hemorrhagic shock followed by fluid resuscitation, the influence of induced hypothermia on hepatic and renal damage and organ-specific inflammation were evaluated. A total of 40 pigs were randomly assigned to four groups, which were sham (anesthesia only) or trauma groups receiving either hypothermia or normothermia. The parameters analyzed were laboratory parameters (aspartate transaminase [AST], lactate dehydrogenase, urea, creatinine) as well as hepatic and renal cytokine expression determined by real-time polymerase chain reaction (interleukin 6 [IL-6], IL-8). Blinded analysis of histologic changes in the liver and kidney was performed. RESULTS: Fifteen and a half hours following combined trauma, hepatic cytokine expression and liver damage were significantly increased in animals with normothermia compared with the respective sham group. Hypothermia, however, resulted in a fivefold reduced hepatic expression of IL-8 (mean ± SE, 2.4 ± 1.3; p = 0.01) when compared with the normothermic trauma group (IL-8, 12.8 ± 4.7). Accordingly, granulocyte infiltration and a histologic, semiquantitative score for liver injury were significantly higher in the normothermic trauma group. Serum AST levels raised significantly after trauma and normothermia compared with the respective sham group, while AST levels showed no difference from the sham groups in the hypothermic trauma group. In contrast, neither trauma nor hypothermia influenced the expression of IL-6 and IL-8 and tissue injury in the kidney. CONCLUSION: Therapeutic hypothermia seems to attenuate the hepatic inflammatory response and the associated liver injury after severe trauma. Therefore, induced hypothermia might represent a potential therapeutic strategy to avoid posttraumatic organ dysfunction

A Non-Lethal Traumatic/Hemorrhagic Insult Strongly Modulates the Compartment-Specific PAI-1 Response in the Subsequent Polymicrobial Sepsis

<div><h3>Introduction</h3><p>Plasminogen activator inhibitor 1 (PAI-1) is a key factor in trauma- and sepsis-induced coagulopathy. We examined how trauma-hemorrhage (TH) modulates PAI-1 responses in subsequent cecal ligation and puncture (CLP)-induced sepsis, and the association of PAI-1 with septic outcomes.</p> <h3>Methods</h3><p>Mice underwent TH and CLP 48 h later in three separate experiments. In experiment 1, mice were sacrificed pre- and post-CLP to characterize the trajectory of PAI-1 in plasma (protein) and tissues (mRNA). Post-CLP dynamics in TH-CLP (this study) and CLP-Only mice (prior study) were then compared for modulatory effects of TH. In experiment 2, to relate PAI-1 changes to outcome, mice underwent TH-CLP and were sampled daily and followed for 14 days to compare non-survivors (DEAD) and survivors (SUR). In experiment 3, plasma and tissue PAI-1 expression were compared between mice predicted to die (P-DIE) and to live (P-LIVE).</p> <h3>Results</h3><p>In experiment 1, an early post-TH rise of circulating PAI-1 was contrasted by a delayed (post-TH) decrease of PAI-1 mRNA in organs. In the post-CLP phase, profiles of circulating PAI-1 were similar between TH-CLP and CLP-Only mice. Conversely, PAI-1 mRNA declined in the liver and heart of TH-CLP mice versus CLP-Only. In experiment 2, there were no DEAD/SUR differences in circulating PAI-1 prior to CLP. Post-CLP, circulating PAI-1 in DEAD was 2–4-fold higher than in SUR. PAI-1 increase heralded septic deaths up to 48 h prior but DEAD/SUR thrombomodulin (endothelial injury marker) levels were identical. In experiment 3, levels of circulating PAI-1 and its hepatic gene expression were higher in P-DIE versus P-LIVE mice and those increases closely correlated with liver dysfunction.</p> <h3>Conclusions</h3><p>Trauma modulated septic PAI-1 responses in a compartment-specific fashion. Only post-CLP increases in circulating PAI-1 predicted septic outcomes. In posttraumatic sepsis, pre-lethal release of PAI-1 was mostly of hepatic origin and was independent of endothelial injury.</p> </div