Hinweise für die funktionelle und dynamische Mikrokompartimentierung von Caveolin-1, TRPV4 und Calcium-aktivierten Kaliumkanälen in den Caveolae von Endothelzellen

Noch immer zählen kardiovaskuläre Erkrankungen zu den häufigsten Todesursachen weltweit. Besonders die arterielle Hypertonie stellt nach wie vor einen Risikofaktor für Herz-Kreislauf-Erkrankungen dar. So geht Bluthochdruck stets mit einer endothelialen Dysfunktion, Athero- und Arteriosklerose, und pathologischen Gefäßveränderungen einher. Zur Regulation des Blutdruckes setzt das Endothel vasodilatatorische und vasokonstriktorische Faktoren frei und beeinflusst so den Kontraktionszustand der glatten Muskulatur. Zu den am längsten bekannten endothelabhängigen Vasodilatatoren gehören Prostazyklin (PGI2) und Stickstoffmonoxid (NO). Neben ihnen besitzt das Endothel ein drittes gefäßerweiterndes System, den endothelium-derived hyperpolarizing factor (EDHF). Während die Funktionen des endothelialen NO und des PGI2 für die lokale und systemische Blutdruckregulation heut zu Tage weitestgehend bekannt sind, sind die biologischen Mechanismen und Signaltransduktionskaskaden des EDHF noch nicht vollständig geklärt. Insbesondere Ca2+-aktivierte K+-Kanäle, zu denen KCa2.3 und KCa3.1 zählen, spielen eine entscheidende Rolle in der endothelabhängigen Hyperpolarisation und in der Regulation des Gefäßtonus und des Blutdruckes. Die Aktivierung dieser beiden KCa-Kanäle beruht auf einem Anstieg der intrazellulären Calciumkonzentration, die weitestgehend durch Ca2+- durchlässige Kationenkanäle, einschließlich dem transient receptor potential V4 (TRPV4), vermittelt werden. Es wir vermutet, dass KCa-Kanäle und Ca2+-durchlässige Kationenkanäle räumlich assoziiert sind, eine funktionelle Einheit bilden und die Caveolae der Zellmembran hier als Mikrokompartimente dienen. Caveolae können als mikroskopisch kleine, sackförmige Einstülpungen an der Oberfläche vieler Säugerzelltypen beobachtet werden. Sie haben eine einzigartige Lipidzusammensetzung und werden wegen ihres hohen Gehalts an Cholesterin und Sphingolipiden zu den „Lipid Rafts“ gezählt. Die Caveolae sind an zahlreichen zellulären und physiologischen Prozessen beteiligt, z.B. Endozytose, Transzytose, Fettstoffwechsel, Virus-Wirt-Interaktion, Krebs und Signaltransduktion. Das integrale Membranprotein Caveolin-1 stellt das Hauptstrukturprotein der Caveolae dar. Die vorliegende Arbeit hat sich mit der Frage beschäftigt, ob die endothelialen KCa-Kanäle KCa2.3 und KCa3.1 und der Ca2+-durchlässige Kationenkanäle TRPV4 in den Caveolae angereichert sind und welchen Effekt ein Fehlen von Caveolin-1, dem Strukturprotein der Caveolae, in vivo auf die genannten KCa-Ströme hat. Es konnte gezeigt werden, dass TRPV4 und KCa2.3 in den Caveolae humaner mikrovaskulärer Endothelzellen (HMEC-1) angereichert sind. Mit Hilfe von Immunpräzipitations-Studien und immunzytologischen Untersuchungen sowie hochauflösender Mikroskopie konnte gezeigt werden, dass in humanen mikrovaskulären Endothelzellen (HMEC-1) unter statischen Bedingungen eine physikalisch und Caveolae-abhängige Assoziation zwischen Caveolin-1, TRPV4 und dem KCa2.3 aber nicht mit KCa3.1 besteht. Zudem konnten wir eine Interaktion und Kolokalisation zwischen TRPV4 und KCa2.3 beobachten. Schließlich konnten wir nach Applikation laminarer Schubspannung eine de novo Kolokalisation von KCa3.1/Caveolin-1 und KCa3.1/TRPV4 nachweisen. Anhand von Patch-Clamp Versuchen an frisch isolierten Endothelzellen der Arteria carotis communis von Caveolin-1-defizienten Mäusen konnte eine 45 %-ige Reduktion der KCa-vermittelten Kaliumströme im Vergleich zu den Wildtyp-Kontrollen gezeigt werden (-54 % für KCa2.3 und -40 % für KCa3.1). Zusammenfassend liefert die vorliegende Arbeit Hinweise für eine dynamische Mikrokompartimentierung von TRPV4/KCa in Caveolae von Endothelzellen und zeigt die Bedeutung des Caveolin-1 für die endothelialen KCa -Funktionen.

Serum cytokines MCP-1 and GCS-F as potential biomarkers in pediatric inflammatory bowel disease

Background Inflammatory bowel diseases (IBDs) with the subtypes ulcerative colitis (UC) and Crohn disease (CD), are chronic autoimmune inflammatory disorders of the gastrointestinal tract. Cytokines are associated with the development and progression in pediatric IBD. We measured cytokine levels in pediatric IBD patients to assess their potential function as biomarkers in disease assessment. Method In this prospective cohort study, we enrolled 33 children with IBD. All patients were in stable remission for 3 months on enrollment. Patients who developed a relapse within six months after enrollment were classified as relapsers. Blood sampling was performed at enrolment and for relapsers in relapse and post-relapse. Serum concentrations of 14 cytokines, chemokines and growth factors (IL-1α, IL-1β, IL-6, IL-12p40, IP-10, TNF-α, IFN-γ, IL-10, IL-8, MIP-1α, MCP-1, MCP-3, G-CSF, GM-CSF) were measured simultaneously using multiplex bead-based sandwich immunoassay on Luminex 100 system. Results MCP-1 was significantly higher in CD patients compared to UC patients at each disease stage: stable remission (P<0.048), unstable remission (P<0.013), relapse (P<0.026) and post-relapse (P<0.024). G-CSF was significantly increased in UC patients developing a relapse and in post-relapse stage compared to UC patients in remission (P<0.02 and p<0.03, respectively). Conclusion MCP-1 showed potential as a diagnostic biomarker in CD patients independent of disease activity as it was able to discriminate between subtypes of pediatric IBD. In UC patients, G-CSF was significantly elevated in relapsers indicating its use and role as a potential prognostic biomarker

Immunization of preterm infants: current evidence and future strategies to individualized approaches

Preterm infants are at particularly high risk for infectious diseases. As this vulnerability extends beyond the neonatal period into childhood and adolescence, preterm infants beneft greatly from infection-preventive measures such as immunizations. However, there is an ongoing discussion about vaccine safety and efcacy due to preterm infants’ distinct immunological features. A signifcant proportion of infants remains un- or under-immunized when discharged from primary hospital stay. Educating health care professionals and parents, promoting maternal immunization and evaluating the potential of new vaccination tools are important means to reduce the overall burden from infectious diseases in preterm infants. In this narrative review, we summarize the current knowledge about vaccinations in premature infants. We discuss the specifcities of early life immunity and memory function, including the role of polyreactive B cells, restricted B cell receptor diversity and heterologous immunity mediated by a cross-reactive T cell repertoire. Recently, mechanistic studies indicated that tissue-resident memory (Trm) cell populations including T cells, B cells and macrophages are already established in the fetus. Their role in human early life immunity, however, is not yet understood. Tissue-resident memory T cells, for example, are diminished in airway tissues in neonates as compared to older children or adults. Hence, the ability to make specifc recall responses after secondary infectious stimulus is hampered, a phenomenon that is transcriptionally regulated by enhanced expression of T-bet. Furthermore, the microbiome establishment is a dominant factor to shape resident immunity at mucosal surfaces, but it is often disturbed in the context of preterm birth. The proposed function of Trm T cells to remember benign interactions with the microbiome might therefore be reduced which would contribute to an increased risk for sustained infammation. An improved understanding of Trm interactions may determine novel targets of vaccination, e.g., modulation of T-bet responses and facilitate more individualized approaches to protect preterm babies in the future

Bedside Measurement of Volatile Organic Compounds in the Atmosphere of Neonatal Incubators Using Ion Mobility Spectrometry

Background: Early and non-invasive diagnosis of common diseases is of great importance in the care of preterm infants. We hypothesized that volatile organic compounds (VOC) can be successfully measured in the neonatal incubator atmosphere. Methods: This is a feasibility study to investigate whether the discrimination of occupied and unoccupied neonatal incubators is possible by bedside measurement of volatile organic compounds (VOCs) on the neonatal intensive care unit. VOC profiles were measured in the incubator air using ion mobility spectrometry coupled to multi-capillary columns (BreathDiscovery B&S Analytik GmbH, Dortmund, Germany). Results: Seventeen incubators occupied by preterm infants (50 measurements) and nine unoccupied neonatal incubators were sampled, using 37 room air measurements as controls. Three VOC signals that allow the discrimination between occupied and unoccupied incubators were identified. The best discrimination was reached by peak P20 exhibiting a sensitivity, specificity, positive predictive value and negative predictive value of 94.0, 88.9, 97.3, and 72.3%, respectively. Use of a decision tree improved these values to 100.0, 88.9, 98.0, and 100.0%, respectively. Discussion: A bedside method that allows the characterization of VOC profiles in the neonatal incubator atmosphere using ion mobility spectrometry was established. Occupied and unoccupied incubators could be discriminated by characterizing VOC profiles. This technique has the potential to yield results within minutes. Thus, future studies are recommended to test the hypothesis that VOCs within neonatal incubators are useful biomarkers for non-invasive diagnostics in preterm neonates

Detection of volatile organic compounds in headspace of Klebsiella pneumoniae and Klebsiella oxytoca colonies

Introduction: Early diagnosis of infections and sepsis is essential as adequate therapy improves the outcome. Unfortunately, current diagnostics are invasive and time-consuming, making diagnosis difficult, especially in neonatology. Novel non-invasive analytical methods might be suitable to detect an infection at an early stage and might even allow identification of the pathogen. Our aim is to identify specific profiles of volatile organic compounds (VOCs) of bacterial species. Methods: Using multicapillary column-coupled ion mobility spectrometry (MCC/ IMS), we performed headspace measurements of bacterial cultures from skin and anal swabs of premature infants obtained during weekly screening for bacterial colonization according to KRINKO. We analyzed 25 Klebsiella pneumoniae (KP) cultures on MacConkey (MC) agar plates, 25 Klebsiella oxytoca (KO) cultures on MC agar and 25 bare MC agar plates as a control group. Results: Using MCC/IMS, we identified a total of 159 VOC peaks. 85 peaks allowed discriminating KP and bare MC agar plates, and 51 peaks comparing KO and bare MC agar plates and 6 peaks between KP and KO (significance level of p < 0.05 after Bonferroni post hoc analysis), respectively. Peaks P51 (n-Decane) and P158 (Phenylethyl Alcohol), showed the best sensitivity/specificity/ positive predictive value/negative predictive value of 99.9% each (p < 0.001) for KP. P158 showed the best sensitivity/specificity/positive predictive value/negative predictive value of 99.9% each (p < 0.001) for KO. Comparing KP and KO, best differentiation was enabled using peaks P72, P97 and P16 with sensitivity/specificity/positive predictive value/negative predictive value of 76.0%, 84.0%, 82.6%, 77.8%, respectively (p < 0.05)

Attenuated asthma phenotype in mice with a fetal-like antigen receptor repertoire

We hypothesized that the scarcity of N-nucleotides might contribute to the inability of the neonate to mount a robust allergic immune response. To test this, we used terminal deoxyribunucleotidyl Transferase defcient (TdT−/−) mice, which express “fetal-like” T cell receptor and immunoglobulin repertoires with largely germline-encoded CDR3 regions. Intraperitoneal sensitization was followed by aerosol provocation with either PBS or the allergen OVA in both TdT−/− mice and wild-type mice to develop allergic respiratory infammation. The efects of this procedure were investigated by lung function test, immunological analysis of serum and brochoalveolar lavage. The local TH2 cytokine milieu was signifcantly attenuated in TdT−/− mice. Within this group, the induction of total IgE levels was also signifcantly reduced after sensitization. TdT−/− mice showed a tendency toward reduced eosinophilic infow into the bronchial tubes, which was associated with the elimination of respiratory hyperreactivity. In conclusion, in a murine model of allergic airway infammation, the expression of fetal-like antigen receptors was associated with potent indications of a reduced ability to mount an asthma phenotype. This underlines the importance of somatically-generated antigen-receptor repertoire diversity in type one allergic immune responses and suggests that the fetus may be protected from allergic responses, at least in part, by controlling N addition

Patterns of volatile organic compounds in excrements of preterm neonates

Background: As neonates are susceptible for many diseases, establishing noninvasive diagnostic methods is desirable. We hypothesized that volatile organic compounds (VOCs) could be successfully measured in diaper samples. Methods: We performed a feasibility study to investigate whether ambient airindependent headspace measurements of the VOC profiles of diapers from premature infants can be conducted using ion mobility spectrometer coupled with multi-capillary columns (B & S Analytik GmbH). Results: We analysed 39 diapers filled with stool (n = 10) or urine (n = 20) respectively, using empty diapers as a control (n = 9). A total of 158 different VOCs were identified, and we classified the content of the diapers (urine or stool) according to their VOC profiles with a significance level of p<0.05. Conclusions: We have developed a novel method to study headspace VOC profiles of biosamples using ion mobility spectrometry coupled with multi-capillary columns. Using this method, we have characterized the VOC profiles of stool and urine of preterm neonates. Future studies are warranted to characterize specific VOC profiles in infections and other diseases of the preterm neonate, thus establishing quick and noninvasive diagnostics in the routine care of the highly vulnerable preterm and term neonates

Detection of volatile organic compounds in headspace of Klebsiella pneumoniae and Klebsiella oxytoca colonies

IntroductionEarly diagnosis of infections and sepsis is essential as adequate therapy improves the outcome. Unfortunately, current diagnostics are invasive and time-consuming, making diagnosis difficult, especially in neonatology. Novel non-invasive analytical methods might be suitable to detect an infection at an early stage and might even allow identification of the pathogen. Our aim is to identify specific profiles of volatile organic compounds (VOCs) of bacterial species.MethodsUsing multicapillary column-coupled ion mobility spectrometry (MCC/IMS), we performed headspace measurements of bacterial cultures from skin and anal swabs of premature infants obtained during weekly screening for bacterial colonization according to KRINKO. We analyzed 25 Klebsiella pneumoniae (KP) cultures on MacConkey (MC) agar plates, 25 Klebsiella oxytoca (KO) cultures on MC agar and 25 bare MC agar plates as a control group.ResultsUsing MCC/IMS, we identified a total of 159 VOC peaks. 85 peaks allowed discriminating KP and bare MC agar plates, and 51 peaks comparing KO and bare MC agar plates and 6 peaks between KP and KO (significance level of p &lt; 0.05 after Bonferroni post hoc analysis), respectively. Peaks P51 (n-Decane) and P158 (Phenylethyl Alcohol), showed the best sensitivity/specificity/ positive predictive value/negative predictive value of 99.9% each (p &lt; 0.001) for KP. P158 showed the best sensitivity/specificity/positive predictive value/negative predictive value of 99.9% each (p &lt; 0.001) for KO. Comparing KP and KO, best differentiation was enabled using peaks P72, P97 and P16 with sensitivity/specificity/positive predictive value/negative predictive value of 76.0%, 84.0%, 82.6%, 77.8%, respectively (p &lt; 0.05).DiscussionWe developed a method for the analysis of VOC profiles of bacteria. Using MCC/IMS, we demonstrated that VOCs derived from bacteria are clearly distinguishable from a bare agar plate. Characteristic peaks obtained by MCC/IMS are particularly suitable for the species-specific identification and differentiation of KP and KO. Thus, MCC/IMS might be a useful tool for in vitro diagnostics. Future studies must clarify whether similar patterns of VOCs can be detected in vivo in patients that are colonized or infected with KP or KO to enable rapid and accurate diagnosis of bacterial colonization

Sex Differences in the Frequencies of B and T Cell Subpopulations of Human Cord Blood

Cord blood represents a link between intrauterine and early extrauterine development. Cord blood cells map an important time frame in human immune imprinting processes. It is unknown whether the sex of the newborn affects the lymphocyte subpopulations in the cord blood. Nine B and twenty-one T cell subpopulations were characterized using flow cytometry in human cord blood from sixteen male and twenty-one female newborns, respectively. Except for transitional B cells and naïve B cells, frequencies of B cell counts across all subsets was higher in the cord blood of male newborns than in female newborns. The frequency of naïve thymus-negative Th cells was significantly higher in male cord blood, whereas the remaining T cell subpopulations showed a higher count in the cord blood of female newborns. Our study is the first revealing sex differences in the B and T cell subpopulations of human cord blood. These results indicate that sex might have a higher impact for the developing immune system, urging the need to expand research in this area

Detection of volatile organic compounds as potential novel biomarkers for chorioamnionitis - proof of experimental models

Background: Histologic chorioamnionitis is only diagnosed postnatally which prevents interventions. We hypothesized that volatile organic compounds (VOCs) in the amniotic fluid might be useful biomarkers for chorioamnionitis and that VOC profiles differ between amnionitis of different origins. Methods: Time-mated ewes received intra-amniotic injections of media or saline (controls), or live Ureaplasma parvum serovar 3 (Up) 14, 7 or 3d prior to c-section at day 124 gestational age (GA). 100 μg recombinant ovine IL-1α was instilled at 7, 3 or 1d prior to delivery. Headspace VOC profiles were measured from amniotic fluids at birth using ion mobility spectrometer coupled with multi-capillary columns. Results: 127 VOC peaks were identified. 27 VOCs differed between samples from controls and Up- or IL-1α induced amnionitis. The best discrimination between amnionitis by Up vs. IL-1α was reached by 2-methylpentane, with a sensitivity/specificity of 96/95% and a positive predictive value/negative predictive values of 96 and 95%. The concentration of 2-methylpentane in VOCs peaked 7d after intra-amniotic instillation of Up. Discussion: We established a novel method to study headspace VOC profiles of amniotic fluids. VOC profiles may be a useful tool to detect and to assess the duration of amnionitis induced by Up. 2-methylpentane was previously described in the exhalate of women with pre-eclampsia and might be a volatile biomarker for amnionitis. Amniotic fluids analyzed by ion mobility spectrometry coupled with multi-capillary columns may provide bedside diagnosis of amnionitis and understanding inflammatory mechanisms during pregnancy