Neurotrophic dependency of sensory neurons in a co-culture model of porcine DRGs and human skin cells

Im Rahmen dieser Arbeit wurde der Einfluss von dermalen und epidermalen Hautzellen auf das Wachstum von peripheren sensorischen Nerven in einem neuen in-vitro Kokultur-Modell untersucht. In diesem Modell werden neuronale Zellkörper und ihre peripheren Fortsätze örtlich getrennt in separaten, flüssigkeitsdichten Kammern kultiviert. Entsprechend den Gegebenheiten in-vivo, wurden dermale und epidermale Hautzellen ausschließlich mit den neuronalen Fortsätzen in der „peripheren“ Seitenkammer kultiviert. Dieses spezielle Setup ermöglicht die Untersuchung des peripheren neuronalen Netzwerks in der Haut in-vitro. Im ersten Schritt der Arbeit wurde das Kokultur-Modell etabliert und charakterisiert. Die Sensitivität des Systems auf neurotrophe Faktoren wurde mittels humanem NGFβ analysiert. Rekombinantes huNGFβ induzierte dosis-abhängiges peripheres Neuritenwachstum in einem Bereich von wenigen pg/mL bis hin zu 10 ng/mL. Die größte Zunahme an der gesamten peripheren Neuriten Länge (cumulative peripheral neurite length, CPNL) wurde zwischen dem sechsten und zehnten Tag der Kultivierung beobachtet. Die von huNGFβ induzierten peripheren Fortsätze wurden im Anschluss mittels Immunfluoreszenzfärbung charakterisiert und quantifiziert. Detektiert wurden die Marker für sensorische, nozizeptive, peptiderge Neurone Calcitonin-gene-related-peptide (CGRP) und Receptor tyrosine kinase TrkA, der sensorische, nozizeptive, nicht-peptiderge Marker Isolectin B4 (IB4) und der Vanilloid Rezeptor TRPV1. Die Espression von IB4 und CGRP war mit 63,8 % und 54,7 % am stärksten detektiertbar, gefolgt von TrkA mit 17,8 % und TRPV1 mit 4,6 %. Der positive Nachweis dieser Marker bestätigt die Präsenz von sensorischen, nozizeptiven Fasern im vorliegenden Modell. Dabei kann zum ersten Mal eine quantitative Aussage über das Expressionsmuster dieser Marker in den peripheren Fasern, abhängig von den Wachstumsbedingungen gemacht werden. Die Funktionalität der kultivierten Neuronen wurde mittels unspezifischer Stimulation mit 50 mM Kalium und spezifischer Stimulation mit dem TRPV1 Agonisten Capsaicin (CPS), sowie Inhibition mit dem Antagonisten Capsazepin (CPZ) bestätigt. Sowohl unspezifische als auch spezifische Stimulation der neuronalen Somata in der Mittelkammer bewirkten die efferente Ausschüttung des Neuropeptids CGRP in der peripheren Seitenkammer. Die Stimulation von TRPV1 mittels CPS wurde erfolgreich durch den Rezeptor-spezifischen Antagonisten CPZ inhibiert und bestätigt erneut die Funktionalität der kultivierten Neurone. Schließlich wurde die neurotrophe Wirkung von Fibroblasten und Keratinozyten auf das Wachstum der peripheren neuronalen Fortsätze untersucht. Beide Zelltypen induzierten erhebliches Neuritenwachstum, wobei Keratinozyten sogar höhere CPNL Werte erzielte als recombinantes huNGFβ. Bei gleichzeitiger Anwendung eines anti-NGFβ Antikörpers wurde das von Fibroblasten induzierte Wachstum auf Kontrollniveau reduziert, wohingegen Keratinozyten vermitteltes Wachstum nur auf 42,9 % verringert wurde. Diese Ergebnisse deuten darauf hin, dass Fibroblasten induziertes Neuritenwachstum größtenteils durch NGF vermittelt wird. Keratinozyten scheinen hingegen zumindest einen zustätzlichen, sekretierten Faktor zu produzieren, der für das verbleibende, NGF unabhängige Wachstum verantwortlich ist. Im Vergleich zwischen Keratinozyten vermitteltem Neuritenwachstum mit und ohne anti-NGFβ Antikörper, zeigte sich keinerlei Unterschied in den CPNL Werten am Tag 4 und 6. Da Neurite üblicherweise die Keratinozytenschicht am Tag 6 erreichen, scheint NGF nicht notwendig für das Auswachsen und Erreichen der Keratinozyten zu sein. NGF spielt möglicherweise eine größere Rolle im Längenwachstum zwischen Tag 6 und 10, wo sich ein Unterschied von minus 57,1 % ohne NGF manifestiert. Mit dieser Annahme gehen auch Beobachtungen der Morphology der Neurite konform. Keratinozyten vermittelten Fortsätze mit einem hohen Grad an Verzweigungen, die häufig einzelne Zellen umwickelten. Hingegen wiesen Fibroblasten induzierte Fasern einen geradlinigen, gering verzweigten Phänotyp auf.In this work, the local influence of dermal and epidermal skin cells on sensory afferent nerve growth was investigated using a novel in-vitro co-culture system of porcine dorsal root ganglion cells (DRGs) and human primary skin cells. The co-culture system allows for the cultivation of cell somata and peripheral neuronal processes in spatially separated compartments that are liquid-impermeable. In accordance with the in-vivo situation, only the axonal processes of sensory neurons were co-cultivated with dermal and epidermal skin cells in the „peripheral“ compartment. This approach allows to study the neuronal cutaneous network in-vitro. In a first attempt, the co-culture system was established and characterized. The sensitivity of the system in response to the neurotrophic factor human NGFβ was examined. Recombinant huNGFβ induced a dose-dependent outgrowth of peripheral sensory fibers in a range between several pg/mL to 10 ng/mL. The major increase in cumulative peripheral neurite length (CPNL) was detected between day 6 and 10 of cells in culture. Subsequently, subtypes of peripheral sensory fibers grown in response to huNGFβ were characterized and quantified via immunofluorescent staining for the nociceptive, peptidergic markers calcitonin-gene-related-peptide (CGRP) and receptor tyrosine kinase TrkA, the non-peptidergic marker Isolectin B4 (IB4) and the vanilloid receptor TRPV1. IB4 and CGRP expressions were prevailing with 63,8 % and 54,7 %, followed by TrkA with 17,8 % and TRPV1 with 4,6 % of CPNL, thus confirming the nociceptive sensory phenotype of the grown fibers. The functionality of the cultured sensory neurons was verified via unspecific stimulation with high potassium (50mM) and specific stimulation with the TRPV1 agonist capsaicin (CPS) and inhibition with the antagonist capsazepine (CPZ), respectively. Unspecific and specific stimulation of the somata in the middle compartment resulted in an efferent release of the neuropeptide CGRP in the peripheral side compartment. The CPS mediated stimulation of TRPV1 was successfully inhibited by the receptor specific antagonist CPZ, further emphasizing the functionality of the cultured neurons. Finally, the neurotrophic effects of keratinocytes and fibroblasts on peripheral sensory fiber outgrowth was investigated. Both cell types were sufficient to induce substantial sensory fiber outgrowth. Surprisingly, keratinocyte mediated CPNL exceeded the outgrowth mediated by recombinant huNGFβ. Additionally, co-application of an anti-NGFβ antibody decreased fibroblast mediated growth to negative control level, whereas keratinocytes mediated growth was only reduced to 42,9 %. These results indicate that fibroblasts induce fiber growth mainly via NGF, whereas keratinocytes produce at least one additional soluble factor that accounts for the NGF independent fiber growth. Comparison of keratinocyte mediated outgrowth with and without anti-NGFβ antibody revealed no difference in CPNL values at day 4 and 6. As neuronal fibers usually reached the keratinocyte layer at day 6, NGF seems not necessary for neurites to enter the side compartment, extend and reach the keratinocytes. NGF may rather play a role for the increase in length between day 6 and 10, consistent with the reduction of 57,1 % of CPNL without NGF. Further consistent with this hypothesis was the analysis of fiber morphology. Neurites stimulated by keratinocytes exhibited extensive branching and frequently wrapped around individual cells. On the contrary, fibroblasts mediated fibers of more unbranched and straight phenotype

Optimizing Nervous System-Specific Gene Targeting with Cre Driver Lines: Prevalence of Germline Recombination and Influencing Factors.

The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities

Secular changes in body height and weight of Portuguese boys over one century

This study examines secular changes in attained height and weight in student boys from a military boarding school (Colégio Militar) in Lisbon, Portugal. Data for 1899-1906, 1929-1936, 1961-1966, and 1999-2006 obtained from the archives and medical files of the Colégio Militar were used in this study. In a century Portuguese boys increased in height by 10.5 cm at age 10 to 19.1 cm at age 14, at a mean increase of 1.54 cm per decade. The gain in weight was between 8.7 and 18.9 kg for 10- and 14-year-old boys, respectively, at a mean increase of 1.54 kg per decade. In the same period, age at peak height and weight velocity advanced sim2 years, showing an acceleration of developmental tempo. However, most of the real gain in height and weight and the decrease in pubertal age occurred after 1961-1966. This mirrors major improvements in social and economic conditions that initiated in Portugal in the 1960s, and then by political events in the 1970s that promoted further progress. Am. J. Hum. Biol., 2008. © 2008 Wiley-Liss, Inc

Evaluation of an inactivated Ross River virus vaccine in active and passive mouse immunization models and establishment of a correlate of protection

Ross River Virus has caused reported outbreaks of epidemic polyarthritis, a chronic debilitating disease associated with significant long-term morbidity in Australia and the Pacific region since the 1920s. To address this public health concern, a formalin- and UV-inactivated whole virus vaccine grown in animal protein-free cell culture was developed and tested in preclinical studies to evaluate immunogenicity and efficacy in animal models. After active immunizations, the vaccine dose-dependently induced antibodies and protected adult mice from viremia and interferon α/β receptor knock-out (IFN-α/βR(-/-)) mice from death and disease. In passive transfer studies, administration of human vaccinee sera followed by RRV challenge protected adult mice from viremia and young mice from development of arthritic signs similar to human RRV-induced disease. Based on the good correlation between antibody titers in human sera and protection of animals, a correlate of protection was defined. This is of particular importance for the evaluation of the vaccine because of the comparatively low annual incidence of RRV disease, which renders a classical efficacy trial impractical. Antibody-dependent enhancement of infection, did not occur in mice even at low to undetectable concentrations of vaccine-induced antibodies. Also, RRV vaccine-induced antibodies were partially cross-protective against infection with a related alphavirus, Chikungunya virus, and did not enhance infection. Based on these findings, the inactivated RRV vaccine is expected to be efficacious and protect humans from RRV diseas