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Patients operated for infective endocarditis (IE) are at high risk of developing an excessive systemic hyperinflammatory state, resulting in systemic inflammatory response syndrome and septic shock. Hemoadsorption (HA) by cytokine adsorbers has been successfully applied to remove inflammatory mediators. This randomized controlled trial investigates the effect of perioperative HA therapy on inflammatory parameters and hemodynamic status in patients operated for IE. A total of 20 patients were randomly assigned to either HA therapy or the control group. HA therapy was initiated intraoperatively and continued for 24 hours postoperatively. Cytokine levels (IL‐6, IL‐1b, TNF‐α), leukocytes, C‐reactive protein (CRP), and Procalcitonin (PCT) as well as catecholamine support, and volume requirement were compared between both groups. Operative procedures included aortic (n = 7), mitral (n = 6), and multiple valve surgery (n = 7). All patients survived to discharge. No significant differences concerning median cytokine levels (IL‐6 and TNF‐α) were observed between both groups. CRP and PCT baseline levels were significantly higher in the HA group (59.5 vs. 26.3 mg/dL, P = .029 and 0.17 vs. 0.05 µg/L, P = .015) equalizing after surgery. Patients in the HA group required significantly higher doses of vasopressors (0.093 vs. 0.025 µg/kg/min norepinephrine, P = .029) at 12 hours postoperatively as well as significantly more overall volume replacement (7217 vs. 4185 mL at 12 hours, P = .015; 12 021 vs. 4850 mL at 48 hours, P = .015). HA therapy did neither result in a reduction of inflammatory parameters nor result in an improvement of hemodynamic parameters in patients operated for IE. For a more targeted use of HA therapy, appropriate selection criteria are required.
Understanding the mechanisms of early invasion and epithelial defense in opportunistic mold infections is crucial for the evaluation of diagnostic biomarkers and novel treatment strategies. Recent studies revealed unique characteristics of the immunopathology of mucormycoses. We therefore adapted an alveolar Transwell® A549/HPAEC bilayer model for the assessment of epithelial barrier integrity and cytokine response to Rhizopus arrhizus, Rhizomucor pusillus, and Cunninghamella bertholletiae. Hyphal penetration of the alveolar barrier was validated by 18S ribosomal DNA detection in the endothelial compartment. Addition of dendritic cells (moDCs) to the alveolar compartment led to reduced fungal invasion and strongly enhanced pro-inflammatory cytokine response, whereas epithelial CCL2 and CCL5 release was reduced. Despite their phenotypic heterogeneity, the studied Mucorales species elicited the release of similar cytokine patterns by epithelial and dendritic cells. There were significantly elevated lactate dehydrogenase concentrations in the alveolar compartment and epithelial barrier permeability for dextran blue of different molecular weights in Mucorales-infected samples compared to Aspergillus fumigatus infection. Addition of monocyte-derived dendritic cells further aggravated LDH release and epithelial barrier permeability, highlighting the influence of the inflammatory response in mucormycosis-associated tissue damage. An important focus of this study was the evaluation of the reproducibility of readout parameters in independent experimental runs. Our results revealed consistently low coefficients of variation for cytokine concentrations and transcriptional levels of cytokine genes and cell integrity markers. As additional means of model validation, we confirmed that our bilayer model captures key principles of Mucorales biology such as accelerated growth in a hyperglycemic or ketoacidotic environment or reduced epithelial barrier invasion upon epithelial growth factor receptor blockade by gefitinib. Our findings indicate that the Transwell® bilayer model provides a reliable and reproducible tool for assessing host response in mucormycosis.
In der vorliegenden Arbeit wurde der Einfluss von verschiedenen in der Intensivtherapie der Sepsis etablierten Medikamenten auf die peripheren mononukleären Zellen gesunder Probanden und septischer Patienten untersucht. Als ein weiteres Ziel untersuchten wir, ob Plasma, das von septischen Patienten gewonnen wurde, ebenfalls eine die Mediatorproduktion der Zellen modifizierende Wirkung besitzt. Als Parameter dieser Studie dienten auf der einen Seite die proinflammatorischen Zytokine TNF-alpha, Interleukin-6 und Interferon-gamma, auf der anderen Seite Interleukin-10, dem antiinflammatorische Eigenschaften zugesprochen werden, und Procalcitonin.
Multiple activities are ascribed to the cytokine tumor necrosis factor (TNF) in health and disease. In particular, TNF was shown to affect carcinogenesis in multiple ways. This cytokine acts via the activation of two cell surface receptors, TNFR1, which is associated with inflammation, and TNFR2, which was shown to cause anti-inflammatory signaling. We assessed the effects of TNF and its two receptors on the progression of pancreatic cancer by in vivo bioluminescence imaging in a syngeneic orthotopic tumor mouse model with Panc02 cells. Mice deficient for TNFR1 were unable to spontaneously reject Panc02 tumors and furthermore displayed enhanced tumor progression. In contrast, a fraction of wild type (37.5%), TNF deficient (12.5%), and TNFR2 deficient mice (22.2%) were able to fully reject the tumor within two weeks. Pancreatic tumors in TNFR1 deficient mice displayed increased vascular density, enhanced infiltration of CD4+ T cells and CD4+ forkhead box P3 (FoxP3)+ regulatory T cells (Treg) but reduced numbers of CD8+ T cells. These alterations were further accompanied by transcriptional upregulation of IL4. Thus, TNF and TNFR1 are required in pancreatic ductal carcinoma to ensure optimal CD8+ T cell-mediated immunosurveillance and tumor rejection. Exogenous systemic administration of human TNF, however, which only interacts with murine TNFR1, accelerated tumor progression. This suggests that TNFR1 has basically the capability in the Panc02 model to trigger pro-and anti-tumoral effects but the spatiotemporal availability of TNF seems to determine finally the overall outcome.
Receptor binding properties of four-helix-bundle growth factors deduced from electrostatic analysis
(1994)
Hormones of the hematopoietin class mediate signal transduction by binding to specific transmembrane receptors. Structural data show that the human growth hormone (hGH) forms a complex with a homodimeric receptor and that hGH is a member of a class of hematopoietins possessing an antiparallel 4-a-helix bundle fold. Mutagenesis experiments suggest that electrostatic interactions may have an important influence on hormonereceptor recognition. In order to examine the specificity of hormone-receptor complexation, an analysis was made of the electrostatic potentials of hGH, interleukin-2 (IL-2), interleukin-4 (IL-4), granulocyte colony-stimulating factor (G-CSF), granulocyte-macrophage colony-stimulating factor (GM-CSF), and the hGH and IL-4 receptors. The binding surfaces of hGH and its receptor, and of IL-4 and its receptor, show complementary electrostatic potentials. The potentials of the hGH and its receptor display approximately 2-fold rotational symmetry because the receptor subunits are identical. In contrast, the potentials of GM-CSF and IL-2 Iack such symmetry, consistent with their known high affinity for hetero-oligomeric receptors. Analysis of the electrostatic potentials supports a recently proposed hetero-oligomeric model for a high-affinity IL-4 receptor and suggests a possible new receptor binding mode for G-CSF; it also provides valuable information for guiding structural and mutagenesis studies of signal-transducing proteins and their receptors.
Background: The sympathetic nervous system is considered to modulate the endotoxin-induced activation of immune cells. Here we investigate whether thoracic epidural anesthesia with its regional symapathetic blocking effect alters endotoxin-induced leukocyte-endothelium activation and interaction with subsequent endothelial injury. Methods: Sprague Dawley rats were anesthetized, cannulated and hemodynamically monitored. E. coli lipopolysaccharide (Serotype 0127: B8, 1.5 mg x kg(-1) x h(-1)) or isotonic saline (controls) was infused for 300 minutes. An epidural catheter was inserted for continuous application of lidocaine or normal saline in endotoxemic animals and saline in controls. After 300 minutes we measured catecholamine and cytokine plasma concentrations, adhesion molecule expression, leukocyte adhesion, and intestinal tissue edema. Results: In endotoxemic animals with epidural saline, LPS significantly increased the interleukin-1 beta plasma concentration (48%), the expression of endothelial adhesion molecules E-selectin (34%) and ICAM-1 (42%), and the number of adherent leukocytes (40%) with an increase in intestinal myeloperoxidase activity (26%) and tissue edema (75%) when compared to healthy controls. In endotoxemic animals with epidural infusion of lidocaine the values were similar to those in control animals, while epinephrine plasma concentration was 32% lower compared to endotoxemic animals with epidural saline. Conclusions: Thoracic epidural anesthesia attenuated the endotoxin-induced increase of IL-1 beta concentration, adhesion molecule expression and leukocyte-adhesion with subsequent endothelial injury. A potential mechanism is the reduction in the plasma concentration of epinephrine.
Background
Natural surfactant preparations, commonly isolated from porcine or bovine lungs, are used to treat respiratory distress syndrome in preterm infants. Besides biophysical effectiveness, several studies have documented additional immunomodulatory properties. Within the near future, synthetic surfactant preparations may be a promising alternative. CHF5633 is a new generation reconstituted synthetic surfactant preparation with defined composition, containing dipalmitoyl-phosphatidylcholine, palmitoyl-oleoyl-phosphatidylglycerol and synthetic analogs of surfactant protein (SP-) B and SP-C. While its biophysical effectiveness has been demonstrated in vitro and in vivo, possible immunomodulatory abilities are currently unknown.
Aim
The aim of the current study was to define a potential impact of CHF5633 and its single components on pro- and anti-inflammatory cytokine responses in human CD4\(^+\) lymphocytes.
Methods
Purified human CD4\(^+\) T cells were activated using anti CD3/CD28 antibodies and exposed to CHF5633, its components, or to the well-known animal-derived surfactant Poractant alfa (Curosurf®). Proliferative response and cell viability were assessed using flow cytometry and a methylthiazolyldiphenyltetrazolium bromide colorimetric assay. The mRNA expression of IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 was measured by quantitative PCR, while intracellular protein expression was assessed by means of flow cytometry.
Results
Neither CHF5633 nor any of its phospholipid components with or without SP-B or SP-C analogs had any influence on proliferative ability and viability of CD4\(^+\) lymphocytes under the given conditions. IFNγ, IL-2, IL-17A, IL-22, IL-4, and IL-10 mRNA as well as IFNγ, IL-2, IL-4 and IL-10 protein levels were unaffected in both non-activated and activated CD4+ lymphocytes after exposure to CHF5633 or its constituents compared to non-exposed controls. However, in comparison to Curosurf®, expression levels of anti-inflammatory IL-4 and IL-10 mRNA were significantly increased in CHF5633 exposed CD4\(^+\) lymphocytes.
Conclusion
For the first time, the immunomodulatory capacity of CHF5633 on CD4\(^+\) lymphocytes was evaluated. CHF5633 did not show any cytotoxicity on CD4\(^+\) cells. Moreover, our in vitro data indicate that CHF5633 does not exert unintended pro-inflammatory effects on non-activated and activated CD4+ T cells. As far as anti-inflammatory cytokines are concerned, it might lack an overall reductive ability in comparison to animal-derived surfactants, potentially leaving pro- and anti-inflammatory cytokine response in balance.
Background: Extracorporeal hemadsorption eliminates proinflammatory mediators in critically ill patients with hyperinflammation. The use of a pumpless extracorporeal hemadsorption technique allows its early usage prior to organ failure and the need for an additional medical device. In our animal model, we investigated the feasibility of pumpless extracorporeal hemadsorption over a wide range of mean arterial pressures (MAP). Methods: An arteriovenous shunt between the femoral artery and femoral vein was established in eight pigs. The hemadsorption devices were inserted into the shunt circulation; four pigs received CytoSorb\(^®\) and four Oxiris\(^®\) hemadsorbers. Extracorporeal blood flow was measured in a range between mean arterial pressures of 45–85 mmHg. Mean arterial pressures were preset using intravenous infusions of noradrenaline, urapidil, or increased sedatives. Results: Extracorporeal blood flows remained well above the minimum flows recommended by the manufacturers throughout all MAP steps for both devices. Linear regression resulted in CytoSorb\(^®\) blood flow [mL/min] = 4.226 × MAP [mmHg] − 3.496 (R-square 0.8133) and Oxiris\(^®\) blood flow [mL/min] = 3.267 × MAP [mmHg] + 57.63 (R-square 0.8708), respectively. Conclusion: Arteriovenous pumpless extracorporeal hemadsorption resulted in sufficient blood flows through both the CytoSorb\(^®\) and Oxiris\(^®\) devices over a wide range of mean arterial blood pressures and is likely an intriguing therapeutic option in the early phase of septic shock or hyperinflammatory syndromes.
Systemic sclerosis (SSc) is a severe chronic disease with a broad spectrum of clinical manifestations. SSc displays disturbed lymphocyte homeostasis. Immunosuppressive medications targeting T or B cells can improve disease manifestations. SSc clinical manifestations and immunosuppressive medication in itself can cause changes in lymphocyte subsets. The aim of this study was to investigate peripheral lymphocyte homeostasis in SSc with regards to the immunosuppression and to major organ involvement. 44 SSc patients and 19 healthy donors (HD) were included. Immunophenotyping of peripheral whole blood by fluorescence-activated cell sorting was performed. Cytokine secretions of stimulated B cell cultures were measured. SSc patients without immunosuppression compared to HD displayed lower γδ T cells, lower T helper cells (CD3+/CD4+), lower transitional B cells (CD19+/CD38++/CD10+/IgD+), lower pre-switched memory B cells (CD19+/CD27+/IgD+), and lower post-switched memory B cells (CD19+/CD27+/IgD-). There was no difference in the cytokine production of whole B cell cultures between SSc and HD. Within the SSc cohort, mycophenolate intake was associated with lower T helper cells and lower NK cells (CD56+/CD3-). The described differences in peripheral lymphocyte subsets between SSc and HD generate further insight in SSc pathogenesis. Lymphocyte changes under effective immunosuppression indicate how lymphocyte homeostasis in SSc might be restored.
Background
Surfactant replacement therapy is the standard of care for the prevention and treatment of neonatal respiratory distress syndrome. New generation synthetic surfactants represent a promising alternative to animal-derived surfactants. CHF5633, a new generation reconstituted synthetic surfactant containing SP-B and SP-C analogs and two synthetic phospholipids has demonstrated biophysical effectiveness in vitro and in vivo. While several surfactant preparations have previously been ascribed immunomodulatory capacities, in vitro data on immunomodulation by CHF5633 are limited, so far. Our study aimed to investigate pro- and anti-inflammatory effects of CHF5633 on native and LPS-stimulated human adult monocytes.
Methods
Highly purified adult CD14\(^{+}\) cells, either native or simultaneously stimulated with LPS, were exposed to CHF5633, its components, or poractant alfa (Curosurf\(^{®}\)). Subsequent expression of TNF-α, IL-1β, IL-8 and IL-10 mRNA was quantified by real-time quantitative PCR, corresponding intracellular cytokine synthesis was analyzed by flow cytometry. Potential effects on TLR2 and TLR4 mRNA and protein expression were monitored by qPCR and flow cytometry.
Results
Neither CHF5633 nor any of its components induced inflammation or apoptosis in native adult CD14\(^{+}\) monocytes. Moreover, LPS-induced pro-inflammatory responses were not aggravated by simultaneous exposure of monocytes to CHF5633 or its components. In LPS-stimulated monocytes, exposure to CHF5633 led to a significant decrease in TNF-α mRNA (0.57 ± 0.23-fold, p = 0.043 at 4h; 0.56 ± 0.27-fold, p = 0.042 at 14h). Reduction of LPS-induced IL-1β mRNA expression was not significant (0.73 ± 0.16, p = 0.17 at 4h). LPS-induced IL-8 and IL-10 mRNA and protein expression were unaffected by CHF5633. For all cytokines, the observed CHF5633 effects paralleled a Curosurf®-induced modulation of cytokine response. TLR2 and TLR4 mRNA and protein expression were not affected by CHF5633 and Curosurf®, neither in native nor in LPS-stimulated adult monocytes.
Conclusion
The new generation reconstituted synthetic surfactant CHF5633 was tested for potential immunomodulation on native and LPS-activated adult human monocytes. Our data confirm that CHF5633 does not exert unintended pro-inflammatory effects in both settings. On the contrary, CHF5633 significantly suppressed TNF-α mRNA expression in LPS-stimulated adult monocytes, indicating potential anti-inflammatory effects.
Developmentally regulated features of innate immunity are thought to place preterm and term infants at risk of infection and inflammation-related morbidity. Underlying mechanisms are incompletely understood. Differences in monocyte function including toll-like receptor (TLR) expression and signaling have been discussed. Some studies point to generally impaired TLR signaling, others to differences in individual pathways. In the present study, we assessed mRNA and protein expression of pro- and anti-inflammatory cytokines in preterm and term cord blood (CB) monocytes compared with adult controls stimulated ex vivo with Pam3CSK4, zymosan, polyinosinic:polycytidylic acid, lipopolysaccharide, flagellin, and CpG oligonucleotide, which activate the TLR1/2, TLR2/6, TLR3, TLR4, TLR5, and TLR9 pathways, respectively. In parallel, frequencies of monocyte subsets, stimulus-driven TLR expression, and phosphorylation of TLR-associated signaling molecules were analyzed. Independent of stimulus, pro-inflammatory responses of term CB monocytes equaled adult controls. The same held true for preterm CB monocytes—except for lower IL-1β levels. In contrast, CB monocytes released lower amounts of anti-inflammatory IL-10 and IL-1ra, resulting in higher ratios of pro-inflammatory to anti-inflammatory cytokines. Phosphorylation of p65, p38, and ERK1/2 correlated with adult controls. However, stimulated CB samples stood out with higher frequencies of intermediate monocytes (CD14\(^+\)CD16\(^+\)). Both pro-inflammatory net effect and expansion of the intermediate subset were most pronounced upon stimulation with Pam3CSK4 (TLR1/2), zymosan (TR2/6), and lipopolysaccharide (TLR4). Our data demonstrate robust pro-inflammatory and yet attenuated anti-inflammatory responses in preterm and term CB monocytes, along with imbalanced cytokine ratios. Intermediate monocytes, a subset ascribed pro-inflammatory features, might participate in this inflammatory state.
Der menschliche Körper besitzt Anpassungsmechanismen, die es ihm ermöglichen, sich an verschiedene Belastungssituationen anzupassen. Es gab in letzter Zeit mehrere Hinweise darauf, dass diese Mechanismen durch die Ausschüttung von Zytokinen, bzw. Myokinen durch die betroffenen Zellen selbst ausgelöst werden. In dieser Arbeit wurden die Serumkonzentration von Myostatin, Follistatin, Follistatin-like-3, Interleukin 6, Interleukin 8 und Klotho vor und nach einer kurzen körperlichen Belastung bestimmt. Dabei konnte allerdings keine signifikante Änderung der Konzentrationen nachgewiesen werden, was die Frage aufwirft, ob mesenchymales Gewebe, insbesondere Muskelgewebe, überhaupt über einen klassischen endokrinen Sekretionsmechanismus verfügt.
Beeinflussung der Aktivität von NF-kappaB durch gram-negative Bakterien - ein Beitrag zur Pathogenese der reaktiven Arthritis Als Beitrag zur Pathogenese der reaktiven Arthritis wurde die Aktivierung von NF-kB durch gram-negative Bakterien untersucht. Mittelpunkt dieser Arbeit war die Beobachtung, dass die Aktivität des Transkriptionsfaktors NF-kB in Synovialzellen nach Infektion stimuliert wird. Die Erkrankung steht im klinischen Zusammenhang mit einer Infektion durch gram-negative Darmbakterien und weiteren Erregern. TNF-a spielt eine wichtige Rolle bei der Erregerantwort der infizierten Zellen, in welchen erhöhte TNF-a-Titer gemessen wurden. Das bekannte Mitwirken von NF-kB in immunologischen Prozessen ließ vermuten, dass dieser Transkriptionsfaktor an der Pathogenese der reaktiven Arthritis beteiligt ist. Dies steht in engem Zusammenhang mit der Induktion von TNF-a, ein Zytokin, das gleichzeitig ein wichtiger Induktor von NF-kB darstellt. In unseren Experimenten wurde ein Unterschied zwischen apathogenen und pathogenen Keimen in der zeitlichen Aktivierung von NF-kB beobachtet. Die Vertreter pathogener Erreger waren Yersinia enterocolitica O.3 und Salmonella enteritidis. Diese induzierten NF-kB zwischen 4 und 6 Stunden post infectionem, im Unterschied zu dem apathogenen Bakterium Escherichia coli, das den Transkriptionsfaktor schon nach 1 - 2 Stunden induzierte. Als Folge dieser Differenz könnte die Immunantwort der Zelle zu unterschiedlichen Reaktionen in der Lage sein und die Erreger abtöten oder eine Persistenz zulassen. Zusätzlich wurde das Augenmerk auf die einzelnen Zellbestandteile oder –produkte gelenkt. Im Vergleich zu intakten Bakterien wurde die Wirkung des Überstandes und die von UV-inaktivierten Keimen untersucht. Die Induktionsstärke war bei unbehandelten Erregern am größten und fiel dann bei UV-inaktivierten Bakterien deutlich ab. Ein weiteres Abfallen der Aktivierung war bei der Infektion mit dem bloßen Überstand zu verzeichnen. Mit diesen Ergebnissen wurde deutlich, dass NF-kB bei der Etablierung der reaktiven Arthritis eine Rolle spielen könnte. Noch bleibt offen, in welcher Art der Transkriptionsfaktor in die intrazellulären Prozesse eingreift und welche medikamentösen Behandlungsmöglichkeiten sich daraus ergeben könnten.
Es konnte erstmals gezeigt werden, dass Tumor Nekrose Faktor-α (TNF- α) (TNF-α) in pathophysiologisch relevanten Konzentrationen neben seiner bekannten negativ inotropen Wirkung einen deutlichen Effekt auf die myokardiale Energetik im Myokard der Ratte besitzt. Dieser wurde anhand des Sauerstoffverbrauchs an rechtsventrikulären Muskelstreifenpräparaten quantifiziert. Der erhöhte Energieumsatz bei gleichzeitig reduzierter myokardialer Arbeit, d.h. der gesteigerte spezifische Sauerstoffverbrauch, basiert auf einer verschlechterten Ökonomie des Kontraktionsprozesses. Diese schnelle Wirkung auf die myokardiale Energetik ist durch einen Sphingolipid Signaltransduktionsweg vermittelt. Dagegen spielt wohl für den mechanischen Effekt von TNF-α sowohl NO, als auch Sphingosin eine Rolle.
The prevalence of chronic heart failure is still increasing making it a major health issue in the 21st century. Tremendous evidence has emerged over the past decades that heart failure is associated with a wide array of mechanisms subsumed under the term "inflammation". Based on the great success of immuno-suppressive treatments in auto-immunity and transplantation, clinical trials were launched targeting inflammatory mediators in patients with chronic heart failure. However, they widely lacked positive outcomes. The failure of the initial study program directed against tumor necrosis factor-a led to the search for alternative therapeutic targets involving a broader spectrum of mechanisms besides cytokines. We here provide an overview of the current knowledge on immune activation in chronic heart failure of different etiologies, summarize clinical studies in the field, address unresolved key questions, and highlight some promising novel therapeutic targets for clinical trials from a translational basic science and clinical perspective.
Each positive well in ELISPOT assays contains spots of variable sizes that can range from tens of micrometers up to a millimeter in diameter. Therefore, when it comes to counting these spots the decision on setting the lower and the upper spot size thresholds to discriminate between non-specific background noise, spots produced by individual T cells, and spots formed by T cell clusters is critical. If the spot sizes follow a known statistical distribution, precise predictions on minimal and maximal spot sizes, belonging to a given T cell population, can be made. We studied the size distributional properties of IFN-γ, IL-2, IL-4, IL-5 and IL-17 spots elicited in ELISPOT assays with PBMC from 172 healthy donors, upon stimulation with 32 individual viral peptides representing defined HLA Class I-restricted epitopes for CD8 cells, and with protein antigens of CMV and EBV activating CD4 cells. A total of 334 CD8 and 80 CD4 positive T cell responses were analyzed. In 99.7% of the test cases, spot size distributions followed Log Normal function. These data formally demonstrate that it is possible to establish objective, statistically validated parameters for counting T cell ELISPOTs.
Die Leber verfügt über einzigartige immunologische Eigenschaften, wobei die Lebertransplantat-Spontantoleranz eine der beeindruckensten ist. Dies steht im Gegensatz zum zentralen Dogma der Transplantationsimmunologie, wonach MHC-differente Transplantate ohne Immunsuppression abgestoßen werden. Die Spontantoleranz entwickelt sich aus der vom Lebertransplantat ausgelösten Immunantwort, während andere MHC-differente Organ-transplantate im Rahmen einer solchen Immunantwort irreversibel zerstört werden. Zielsetzung dieser Arbeit war, die immunologischen Vorgänge im Lebertransplantat bei Abstoßung und Spontantoleranz näher zu untersuchen. Deshalb wurden die intrahepatischen T-Lymphozyten auf ihr Zytokinprofil (Th1/Th2 Zytokine) und ihre CD45RC Expression hin untersucht. Dies geschah in der Frühphase bis Tag 7 und in der Spätphase bis Tag 100 nach Lebertransplantation. Mit diesem experimentellen Design wurde überprüft, ob aus der frühen Aktivierung nach Lebertransplantation je nach Präsenz von Th1- oder Th2-Zytokinen entweder Abstoßung oder Spontantoleranz entsteht. Das Phänomen der Lebertransplantat-Spontantoleranz wurde in der Ratte mit der Zuchtlinie Dark Agouti (DA) nach Übertragung von Lebertransplantaten aus Lewis-Spendertieren (LEW) untersucht. Andere vaskularisierte Organe aus LEW Spendertieren wurden dagegen von ihren DA-Empfängern abgestoßen. Dienten DA-Tiere als Leberspender und LEW-Tiere als Empfänger, so war ebenso eine akute Transplantatabstoßung zu beobachten. Die Quantifizierung und Phänotypisierung der Leukozyten aus den Lebertransplantaten der Spontantoleranzgruppe und der Abstoßungsgruppe zeigten, dass es bereits in der Frühphase nach Transplantation zu einer starken Infiltration mit Leukozyten des Empfängers kam. Bereits am Tag 3 p.op. waren in diesen Lebertransplantaten nahezu nur noch Leukozyten der Empfänger nachzuweisen. Auch in spontantolerierten LEW Lebertransplantaten wurde dies beobachtet. Weiter wurde die Expression des Oberflächenmoleküls CD45RC auf den CD4+ T-Lymphozyten untersucht, da sich hierdurch naive von aktivierten Zellen unterscheiden lassen. Während der Frühphase zeigte sich ein dynamisches Verhältnis von CD45RCpos (naive T-Lymphozyten) und CD45RCneg (aktivierte T-Lymphozyten). In beiden allogenen Gruppen stieg dieses Verhältnis innerhalb von 3 Tagen nach Transplantation von 0,5 auf über 3 an. Bereits zwischen Tag 5 und 7 p.op. verringert sich dieses Verhältnis wieder in beiden Gruppen. In der Spätphase um den Tag 30 nach Lebertransplantation erhöhte sich in der Spontantoleranzgruppe das Verhältnis von aktivierten zu naiven T-Lymphozyten noch einmal, bevor es sich schließlich dem Niveau der Syngenen Kontrollgruppe annäherte. Für die intrahepatischen CD4+ T-Lymphozyten beider allogener Gruppen wurde in der Frühphase nach Transplantation eine deutliche Anwesenheit von IL-2 und IL-2 mRNA nachgewiesen. In dieser Phase sezernierten sie auch die Th2- Zytokine IL-4 und IL-10. Dies war unabhängig davon, ob die T-Lymphozyten aus Lebertransplantaten der Abstoßungs- oder Spontantoleranzgruppe stammten. Somit konnte nicht eindeutig gezeigt werden, dass die Anwesenheit von Th2-Zytokinen für die Induktion von Spontantoleranz notwendig ist bzw. ihre Abwesenheit zur Transplantatabstoßung führt. In der Spätphase nach Transplantation wurden aus den spontantoleranten Lebertransplantaten CD4+ T-Lymphozyten isoliert, für die nach einer in vitro- Stimulierung IL-13 mRNA nachzuweisen war. Diese Fähigkeit ließ sich bis zum Versuchsende am Tag 100 verfolgen. Hingegen brachten Analysen von CD4+ TLymphozyten, die zum gleichen Zeitpunkt aus den Milzen dieser Tiere isoliert wurden, für dieses Zytokin kein Ergebnis. Auch in der Syngenen Kontrollgruppe waren die CD4+ T-Lymphozyten, die aus den Lebertransplantaten und Milzen isoliert wurden, nicht in der Lage, dieses Zytokin zu produzieren. IL-13 zählt zu den Th2-Zytokinen, das regulierend auf immunkompetente Zellen wirkt und die Produktion verschiedener inflammatorischer Zytokine hemmt. Diese IL-13-positiven CD4+ T-Lymphozyten stellen somit attraktive Kandidaten für Zellen mit einem regulatorischen Potential dar, die zur Erhaltung der Langzeitfunktion von Lebertransplantaten entscheidend sein könnten. Die Leber mit ihren einzigartigen immunologischen Fähigkeiten verbirgt noch zahlreiche Geheimnisse. Unstrittig ist, dass weiterführende Untersuchungen zu neuen Erkenntnissen über die Immunbiologie IL-13-positiver T-Lymphozyten im Besonderen und zur Leberimmunologie im Allgemeinen führen werden.
Aging is an independent risk factor for cardiovascular diseases and therefore of particular interest for the prevention of cardiovascular events. However, the mechanisms underlying vascular aging are not well understood. Since carcinoembryonic antigen‐related cell adhesion molecule 1 (CEACAM1) is crucially involved in vascular homeostasis, we sought to identify the role of CEACAM1 in vascular aging. Using human internal thoracic artery and murine aorta, we show that CEACAM1 is upregulated in the course of vascular aging. Further analyses demonstrated that TNF‐α is CEACAM1‐dependently upregulated in the aging vasculature. Vice versa, TNF‐α induces CEACAM1 expression. This results in a feed‐forward loop in the aging vasculature that maintains a chronic pro‐inflammatory milieu. Furthermore, we demonstrate that age‐associated vascular alterations, that is, increased oxidative stress and vascular fibrosis, due to increased medial collagen deposition crucially depend on the presence of CEACAM1. Additionally, age‐dependent upregulation of vascular CEACAM1 expression contributes to endothelial barrier impairment, putatively via increased VEGF/VEGFR‐2 signaling. Consequently, aging‐related upregulation of vascular CEACAM1 expression results in endothelial dysfunction that may promote atherosclerotic plaque formation in the presence of additional risk factors. Our data suggest that CEACAM1 might represent an attractive target in order to delay physiological aging and therefore the transition to vascular disorders such as atherosclerosis.
Background
The role of cytokines in the pathophysiology, diagnosis, and prognosis of small fiber neuropathy (SFN) is incompletely understood. We studied expression profiles of selected pro- and anti-inflammatory cytokines in RNA from white blood cells (WBC) of patients with a medical history and a clinical phenotype suggestive for SFN and compared data with healthy controls.
Methods
We prospectively recruited 52 patients and 21 age- and sex-matched healthy controls. Study participants were characterized in detail and underwent complete neurological examination. Venous blood was drawn for routine and extended laboratory tests, and for WBC isolation. Systemic RNA expression profiles of the pro-inflammatory cytokines interleukin (IL)-1ß, IL-2, IL-8, tumor necrosis factor-alpha (TNF) and the anti-inflammatory cytokines IL-4, IL-10, transforming growth factor beta-1 (TGF) were analyzed. Protein levels of IL-2, IL-8, and TNF were measured in serum of patients and controls. Receiver operating characteristic (ROC)-curve analysis was used to determine the accuracy of IL-2, IL-8, and TNF in differentiating patients and controls. To compare the potential discriminatory efficacy of single versus combined cytokines, equality of different AUCs was tested.
Results
WBC gene expression of IL-2, IL-8, and TNF was higher in patients compared to healthy controls (IL-2: p = 0.02; IL-8: p = 0.009; TNF: p = 0.03) and discriminated between the groups (area under the curve (AUC) ≥ 0.68 for each cytokine) with highest diagnostic accuracy reached by combining the three cytokines (AUC = 0.81, sensitivity = 70%, specificity = 86%). Subgroup analysis revealed the following differences: IL-8 and TNF gene expression levels were higher in female patients compared to female controls (IL-8: p = 0.01; TNF: p = 0.03). The combination of TNF with IL-2 and TNF with IL-2 and IL-8 discriminated best between the study groups. IL-2 was higher expressed in patients with moderate pain compared to those with severe pain (p = 0.02). Patients with acral pain showed higher IL-10 gene expression compared to patients with generalized pain (p = 0.004). We further found a negative correlation between the relative gene expression of IL-2 and current pain intensity (p = 0.02). Serum protein levels of IL-2, IL-8, and TNF did not differ between patients and controls.
Conclusions
We identified higher systemic gene expression of IL-2, IL-8, and TNF in SFN patients than in controls, which may be of potential relevance for diagnostics and patient stratification.
The pathophysiological mechanisms of pain in small fiber neuropathy (SFN) are unclear. Based on experimental and clinical studies, sensitized nociceptors in the skin are reported to be involved in pain development. These nociceptors may be sensitized by cutaneous and systemic pain mediators e.g. pro- and anti-inflammatory cytokines. The aim of our study was, to measure the systemic and local gene expression of pro- and anti-inflammatory cytokines in white blood cells (WBC) as well as in primary fibroblasts and keratinocytes obtained from human skin of patients with SFN. Furthermore, gene expression levels of axon guidance molecules and their receptors, as potential regulators of the intraepidermal nerve fiber density (IENFD), were investigated. 55 patients and 31 healthy controls were prospectively recruited. Participants underwent extensive clinical phenotyping and blood sampling, 6-mm skin punch biopsies were taken from the right lateral calf and the upper thigh. Systemic relative gene expression levels (ΔG) of the interleukin (IL)-1β, IL-2, IL-6, IL-8, and tumor necrosis factor (TNF) was measured in WBC. Skin punch biopsies were taken to determine the IENFD and to obtain primary fibroblast and keratinocyte cell cultures. Skin cells were then used for investigation of ΔG in axon guidance molecules netrin 1 (NTN1) and ephrin A4 (EPHA4) as well as their receptors Unc5b receptor, and ephrin A4 (EFNA4) as well as cytokines IL-1β, IL-4, IL-6, IL-8, IL-10, TNF, and transforming growth factor (TGF). Systemically, gene expression of IL-2, IL-8, and TNF was higher in SFN patients compared to healthy controls. In keratinocytes, higher expression levels of NTN1 and TGF were found when comparing the SFN patients to the controls. In fibroblasts higher gene expression was shown in NTN1, Unc5b, IL-6, and IL-8 when comparing patients to healthy controls. The systemically and local elevated levels of pro-inflammatory, algesic cytokines in SFN patients compared to healthy controls, confirms a potential pathophysiological role in the development of neuropathic pain. Data also indicate fibroblasts and keratinocytes to influence subepidermal and intraepidermal nerve fiber growth through the expression of NTN1 and Unc5b. Thus, skin cells may contribute to the development of neuropathic pain through local denervation.