Enlarged infarct volume and loss of BDNF mRNA induction following brain ischemia in mice lacking FGF-2

FGF-2, a potent multifunctional and neurotrophic growth factor, is widely expressed in the brain and upregulated in cerebral ischemia. Previous studies have shown that intraventricularly or systemically administered FGF-2 reduces the size of cerebral infarcts. Whether endogenous FGF-2 is beneficial for the outcome of cerebral ischemia has not been investigated. We have used mice with a null mutation of the fgf2 gene to explore the relevance of endogenous FGF-2 in brain ischemia. Focal cerebral ischemia was produced by occlusion of the middle cerebral artery (MCAO). We found a 75% increase in infarct volume in fgf2 knock-out mice versus wild type littermates (P < 0.05). This difference in the extent of ischemic damage was observed after 24 h, and correlated with decreased viability in fgf2 mutant mice following MCA occlusion. Increased infarct volume in fgf2 null mice was associated with a loss of induction in hippocampal BDNF and trkB mRNA expression. These findings indicate that signaling through trkB may contribute to ameliorating brain damage following ischemia and that bdnf and trkB may be target genes of FGF-2. Together, our data provide the first evidence that endogenous FGF-2 is important in coping with ischemic brain damage suggesting fgf2 as one crucial target gene for new therapeutic strategies in brain ischemia

Species-specific responses of herbivores to within-plant and environmentally mediated between-plant variability in plant chemistry

Allocation of resources to growth and defense against herbivores crucially affects plant competitiveness and survival, resulting in a specific distribution of assimilates and defense compounds within plant individuals. Additionally, plants rarely experience stable environmental conditions, and adaptations to abiotic and biotic stresses may involve shifts in resistance to herbivores. We studied the allocation of phytochemicals in Brassica oleracea (Brussels sprouts) due to leaf age, drought stress and herbivore damage and assessed effects on two lepidopteran herbivores differing in diet breadth: the generalist Spodoptera littoralis and the specialist Pieris brassicae. Glucosinolates as secondary defense compounds and total nitrogen and carbon were quantified and linked to plant palatability, i.e., herbivore feeding preference. Herbivore responses were highly species-specific and partially related to changes in phytochemicals. Spodoptera littoralis preferred middle-aged leaves with intermediate levels of glucosinolates and nitrogen over young, glucosinolate and nitrogen rich leaves, as well as over old leaves, poor in glucosinolates and nitrogen. In contrast, P. brassicae preferred young leaves. Both species preferred severely drought-stressed plants to the well-watered control, although analyzed glucosinolate concentrations did not differ. Both S. littoralis and P. brassicae feeding induced an increase of indole glucosinolate levels, which may explain a reduced consumption of damaged plants detected for S. littoralis but not for P. brassicae. By revealing distinct, sometimes contrasting responses of two insect herbivores to within-plant and stress-mediated intraspecific variation in phytochemistry of B. oleracea, this study emphasizes the need to consider specific herbivore responses to understand and predict the interactions between herbivores and variable plant

MiR-124 is differentially expressed in derivatives of the sympathoadrenal cell lineage and promotes neurite elongation in chromaffin cells

The neural-crest-derived sympathoadrenal cell lineage gives rise to sympathetic neurons and to endocrine chromaffin cells of the adrenal medulla. Both cell types express a largely overlapping set of genes, including those coding for the molecular machinery related to the synthesis and exocytotic release of catecholamines. During their early development, sympathetic neurons and chromaffin cells rely on a shared transcription factor network that controls the establishment of these common features. Despite many similarities, mature sympathetic neurons and chromaffin cells significantly differ regarding their morphology and function. Most prominently, sympathetic neurons possess axons that are absent in mammalian adrenal chromaffin cells. The molecular mechanism underlying the divergent development of sympathoadrenal cells into neuronal and endocrine cells remains elusive. Mutational inactivation of the ribonuclease dicer hints at the importance of microRNAs in this diversification. We show here that miR-124 is detectable in developing sympathetic neurons but absent in chromaffin cell precursors. We further demonstrate that miR- 124 promotes neurite elongation when transfected into cultured chromaffin cells indicating its capability to support the establishment of a neuronal morphology in non- neuronal sympathoadrenal cells. Our results also show that treatment of PC12 cells with the neurotrophin nerve growth factor leads to an upregulation of miR-124 expression and that inhibition of miR-124 reduces nerve-growth-factor-induced neurite outgrowth in PC12 cells. Thus, our data indicate that miR-124 contributes to the establishment of specific neuronal features in developing sympathoadrenal cells

Induction of Matrixmetalloproteinases and Interleukin-18 by Borrelia burgdorferi in vivo and

Titelblatt, Widmung, Inhaltsverzeichnis, Abkürzungsverzeichnis, Lebenslauf, Danksagung, Selbständigkeitserklärung 1 Einleitung 2 Literaturübersicht 3 Material und Methoden 4 Ergebnisse 5 Diskussion 6 Zusammenfassung und 7 Summary 8 Anhang 9 LiteraturverzeichnisDer Erreger der Lyme-Borreliose Borrelia burgdorferi ist in der Lage, über viele, großteils noch ungeklärte Pathogenitätsmechanismen ein multisystemisches Krankheitsbild mit Beteiligung von Haut, Bewegungsapparat, Herz und Nervensystem zu verursachen. Die spezifische Immunantwort gegen B. burgdorferi ist durch eine präferentielle Th1-Antwort mit Produktion von IFN- gamma gekennzeichnet. IL-18 ist bekannt als IFN-gamma induzierender Faktor. In der Arbeit wurde deshalb die Rolle von IL-18 bei der Lyme-Borreliose untersucht. Darüber hinaus ist es bis heute nicht klar, wie sich die Borrelien innerhalb des Wirtsorganismus ausbreiten. Die mögliche Induktion von MMPs durch B. burgdorferi wurde deshalb näher untersucht. Anhand der Untersuchung von 100 Seren von Lyme-Borreliose-Patienten (Lyme-Arthritis n = 65; andere Manifestationsformen n = 35) mittels ?Enzyme-linked immunosorbent assay? (ELISA) konnte gezeigt werden, dass bei Borreliosepatienten im Vergleich zu Normalspenderseren (n = 37) signifikant erhöhte IL-18-Konzentrationen im Serum vorliegen. MMP-1 und MMP-3 sowie deren physiologischer Antagonist TIMP-1 zeigten keine Unterschiede in Seren von Lyme-Borreliose-Patienten verglichen mit Seren von gesunden Spendern. Der signifikante Anstieg von IL-18 in Seren von Patienten mit Lyme-Borreliose weist auf eine pathophysiologische Rolle dieses Zytokins hin. Zusätzliche Untersuchungen müssen zeigen, ob IL-18 als Marker in der Diagnose und Therapie der Lyme-Borreliose dienen kann. Die potentielle Sekretion und Induktion von IL-18, verschiedener MMPs und TIMP-1 bei der Lyme-Arthritis wurde anhand eines neuen dreidimensionalen Zellkulturmodells untersucht. 19 humane Synovialgewebsproben wurden mit B. burgdorferi infiziert und bis zu 72 Stunden inkubiert. Immunhistochemisch konnte nachgewiesen werden, dass die Borrelien in das Synovialgewebe einwandern. Im Überstand wurden Proteinkonzentrationen von IL-18, MMP-1, MMP-3 und TIMP-1 mittels ELISA gemessen. MMP-2 und MMP-9 wurde zymographisch nachgewiesen. Aus den Geweben wurde darüber hinaus mittels semiquantitativer Realtime-PCR die Induktion von TNF-alpha, IL-1beta, IL-18, MMP-1, MMP-2, MMP-3 und MMP-9 bestimmt. In infizierten Kulturen war nach 72 Stunden im Verhältnis zur Konzentration nach 24 Stunden die 1,3fache Menge an IL-18 vorhanden. MMP-1 stieg im Vergleich zu den Kontrollen an, jedoch konnte auch in uninfizierten Kontrollen ein geringgradiger Anstieg gemessen werden, bei MMP-3 war der Anstieg weniger ausgeprägt. TIMP-1 zeigte in infizierten Gewebekulturüberständen einen Abfall der Konzentrationen nach 72 Stunden. Die Ergebnisse der RT-PCR zeigten eine Induktion durch B. burgdorferi von MMP-1, MMP-3, TNF-alpha und IL-1beta. Zymographisch konnte ein geringgradiger Anstieg von MMP-9 in infizierten Kulturüberständen nachgewiesen werden, die Banden von MMP-2 waren in allen Kulturüberständen etwa gleich stark ausgeprägt. Spirochäten alleine produzierten weder MMP-2 noch MMP-9. Die mRNA-Analyse von IL-18 ergab kein eindeutiges Ergebnis der 4 Gewebepools, das gleiche gilt für MMP-2 und MMP-9. B. burgdorferi ist in der Lage, wirtseigene proteolytische Enzyme zu induzieren. Darüber hinaus wird die TIMP-1-Produktion reduziert, wodurch das normalerweise herrschende Gleichgewicht zwischen MMP-1 und TIMP-1 zu Gunsten von MMP-1 verschoben ist. Dieser Pathogenitätsmechanismus kann den Borrelien in vivo dazu dienen, Gewebebarrieren zu überwinden. Außerdem kann die lokale Freisetzung von MMPs zur Pathophysiologie der Lyme-Arthritis beitragen, wie es auch bei anderen entzündlichen Gelenkerkrankungen, beispielsweise der Rheumatoiden Arthritis, der Fall ist.Lyme borreliosis is a systemic disease involving the skin, the musculoskeletal system, the heart and the central nervous system. The shift of the cytokine pattern towards the Th1 response is one characteristic feature of the disease. Recently, interleukin-18 has been described to induce interferon-gamma. Therefore the potential role of IL-18 in Lyme disease has been studied. In addition, it is not clear how the spirochetes can disseminate within the host organism. Therefore, the potential induction of metalloproteinases (MMPs) by B. burgdorferi was observed. Serum samples from patients with Lyme disease (n = 100) were collected (Lyme arthritis; n = 65, other manifestations of Lyme disease n = 35). Normal blood donors (n = 37) and patients with rheumatoid arthritis (RA; n = 12) served as controls. IL-18 levels were determined by ELISA. In patients with Lyme arthritis IL-18 was significantly elevated (median value 179 pg/ml; p < 0.01) as compared to normal controls (0,05 pg/ml). Patients with RA had significantly higher levels compared to normal controls, too (162 pg/ml). Collagenase-1 (MMP-1) and stromelysin (MMP-3) as well as tissue inhibitor of metalloproteinases-1 (TIMP-1) had no significant different levels in patients with Lyme Borreliosis compared to healthy donors. The significant elevation of IL-18 in sera of patients with Lyme disease implies a pathophysiological role of this cytokine. Additional studies will have to show if IL-18 reflects the activity of the disease and may be useful as a novel marker in diagnosis and therapy of Lyme borreliosis. The potential secretion and induction of IL-18, MMPs and TIMP-1 by Borrelia burgdorferi was tested using a novel threedimensional in-vitro model of Lyme arthritis. Explant cultures of human synovial tissue were infected with B. burgdorferi. and co-cultered for up to 72 hours. The concentrations of IL-18, MMP-1, MMP-3 and of TIMP-1 were measured by ELISA. The activities of gelatinase A (MMP-2) and B (MMP-9) were assessed by zymography. Moreover, a semiquantitative RT-PCR was used to determine the induction of TNF-alpha, IL-1beta, IL-18, MMP-1, MMP-2, MMP-3 and MMP-9 mRNA. After 72 hours, the infected cultures showed an increase of IL-18 concentrations up to the 1.3 fold of the levels at 24 hours after infection. In uninfected controls the IL-18 level after 72 hours was reduced to 80 % of the level at 24 hours. Furthermore, in the infected cultures there was an increase of MMP-1 levels compared to the uninfected cultures, whereas no increase of MMP-3 concentrations was observed. Of note, there was a reduction of TIMP-1 levels in the infected cultures. The RT-PCR results showed MMP-1 and MMP-3 to be induced by B. burgdorferi. By zymography, a slightly increased activity of MMP-9 in the infected cultures could be demonstrated, MMP-2 had activity in all supernatants. Spirochetes themselves did not produce MMP-2 or MMP-9. An increase of IL-18 could be demonstrated in 2/4 cultures infected with B. burgdorferi. B. burgdorferi is able to induce proteolytic enzymes of the host. Moreover, the spirochetes reduce the TIMP-1 production, pointing towards an imbalance between the proteases and their inhibitor. This may reflect a mechanism potentially operative in vivo that enables the microorganism to penetrate host barriers. In addition, the local release of MMPs within the joint may contribute to the pathophysiology of Lyme arthritis like in other inflammatory arthritides such as rheumatoid arthritis

Alien Registration- Unsicker, George (Fairfield, Somerset County)

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TGFβ induces GDNF responsiveness in neurons by recruitment of GFRα1 to the plasma membrane

We have previously shown that the neurotrophic effect of glial cell line–derived neurotrophic factor (GDNF) in vitro and in vivo requires the presence of transforming growth factor (TGF)β. Using primary neurons (chick E8 ciliary) we show that the combination of GDNF plus TGFβ promotes survival, whereas the single factors do not. This cooperative effect is inhibited by blocking the extracellular signal-regulated kinase (ERK)/MAPK pathway, but not by interfering with the PI3 kinase signaling cascade. Although there is no functional GDNF signaling in the absence of TGFβ, pretreatment with TGFβ confers GDNF responsiveness to the cells. This is not due to upregulation of GDNF receptors mRNA and protein, but to TGFβ-induced recruitment of the glycosyl-phosphatidylinositol-anchored GDNF receptor (GFR)α1 to the plasma membrane. This is supported by the fact that GDNF in the presence of a soluble GFRα1 can promote survival in the absence of TGFβ. Our data suggest that TGFβ is involved in GFRα1 membrane translocation, thereby permitting GDNF signaling and neurotrophic effects

Complementarity effects through dietary mixing enhance the performance of a generalist insect herbivore

The ontogenetic niche concept predicts that resource use depends on an organism’s developmental stage. This concept has been investigated primarily in animals that show differing resource use strategies as juveniles and as adults, such as amphibians. We studied resource use and performance in the grasshopper Chorthippus parallelus (Orthoptera, Acrididae) provided with food plant mixtures of either one, three or eight plant species throughout their development. C. parallelus survival and fecundity was highest in the food plant mixture with eight plant species and lowest in the treatments where only one single plant species was offered as food. C. parallelus’ consumption throughout its ontogeny depended on sex, and feeding on different plant species was dependent on a grasshopper’s developmental stage. To depict grasshopper foraging in food plant mixtures compared to foraging on single plant species, we introduce the term “relative forage total” (RFT) based on an approach used in biodiversity research by Loreau and Hector (Nature 413:548–274, 2001). RFT of grasshoppers in food plant mixtures was always higher than what would have been expected from foraging in monocultures. The increase in food consumption was due to an overall increase in feeding on plant species in mixtures compared to consumption of the same species offered as a single diet. Thus we argue that grasshopper foraging exhibits complementarity effects. Our results reinforce the necessity to consider development-related changes in insect herbivore feeding. Thorough information on the feeding ontogeny of insect herbivores could not only elucidate their nutritional ecology but also help to shed light on their functional role in plant communities