The Extracellular Matrix of the Lateral Pharyngeal Wall in Obstructive Sleep Apnea

Study Objectives: To compare the components of the extracellular matrix in the lateral pharyngeal muscular wall in patients with and without obstructive sleep apnea (OSA). This may help to explain the origin of the increased collapsibility of the pharynx in patients with OSA. Design: Specimens from the superior pharyngeal constrictor muscle, obtained during pharyngeal surgeries, were evaluated using histochemical and immunohistochemical analyses to determine the fractional area of collagen types I and II, elastic fibers, versican, fibronectin, and matrix metalloproteinases 1 and 2 in the endomysium. Setting: Academic tertiary center. Patiens: A total of 51 nonobese adult patients, divided into 38 patients with OSA and 13 nonsnoring control subjects without OSA. Interventions: Postintervention study performed on tissues from patients after elective surgery. Measurements and Results: Pharyngeal muscles of patients with OSA had significantly more collagen type I than pharyngeal muscles in control subjects. Collagen type I was correlated positively and independently with age. The other tested components of the extracellular matrix did not differ significantly between groups. In a logistic regression, an additive effect of both the increase of collagen type I and the increase in age with the presence of OSA was observed (odds ratio (OR), 2.06; 95% confidence interval (CI), 1.17-3.63), when compared with the effect of increased age alone (OR, 1.11; 95% CI, 1.03-1.20). Conclusion: Collagen type I in the superior pharyngeal constrictor muscle was more prevalent in patients with OSA and also increased with age. It was hypothesized that this increase could delay contractile-relaxant responses in the superior pharyngeal constrictor muscle at the expiratory-inspiratory phase transition, thus increasing pharyngeal collapsibility.

Comparison of the larynx-associated lymphoid tissue in the false vocal cords and subglottis in pediatric autopsies

The aim this work was to compare the distribution of cellular phenotypes of the LF in the FVC to the ones in the subglottic region in pediatric autopsy, relating this distribution to age and different causes of death. We analyzed 60 larynges of newborns and children autopsied in the period from 1993 to 2003. The fragments were prepared in order to perform histochemical and immunohistochemical techniques. The morphological analysis showed cases that presented LF only in FVC (35%), LF only in the subglottic region (20%), lack of LF in FVC (30%) and lymphoid aggregates, which did not characterize an LF (15%). The cases of LF in the subglottic region were significantly younger compared to the ones that presented LF in the FVC (p = 0.017). The LF in the subglottic region was bigger than the LF in the FVC (p = 0.020). There was no significant difference between the cause of death and cellular phenotype for both FVC and the subglottic region. In conclusion, the cells that make up the LF in the FVC in newborns and children younger than one year have functional characteristics similar to LF cells in the subglottic region, suggesting that there are similarities with LALT. (c) 2012 Elsevier GmbH. All rights reserved.

Effects of myenteric denervation on extracellular matrix fibers and mast cell distribution in normal stomach and gastric lesions

Abstract Background In this study the effect of myenteric denervation induced by benzalconium chloride (BAC) on distribution of fibrillar components of extracellular matrix (ECM) and inflammatory cells was investigated in gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Rats were divided in four experimental groups: non-denervated (I) and denervated stomach (II) without MNNG treatment; non-denervated (III) and denervated stomachs (IV) treated with MNNG. For histopathological, histochemical and stereological analysis, sections of gastric fragments were stained with Hematoxylin-Eosin, Picrosirius-Hematoxylin, Gomori reticulin, Weigert's Resorcin-Fuchsin, Toluidine Blue and Alcian-Blue/Safranin (AB-SAF). Results BAC denervation causes an increase in the frequency of reticular and elastic fibers in the denervated (group II) compared to the non-denervated stomachs (group I). The treatment of the animals with MNNG induced the development of adenocarcinomas in non-denervated and denervated stomachs (groups III and IV, respectively) with a notable increase in the relative volume of the stroma, the frequency of reticular fibers and the inflammatory infiltrate that was more intense in group IV. An increase in the frequency of elastic fibers was observed in adenocarcinomas of denervated (group IV) compared to the non-denervated stomachs (group III) that showed degradation of these fibers. The development of lesions (groups III and IV) was also associated with an increase in the mast cell population, especially AB and AB-SAF positives, the latter mainly in the denervated group IV. Conclusions The results show a strong association in the morphological alteration of the ECM fibrillar components, the increased density of mast cells and the development of tumors induced by MNNG in the non-denervated rat stomach or denervated by BAC. This suggests that the study of extracellular and intracellular components of tumor microenvironment contributes to understanding of tumor biology by action of myenteric denervation.

Efeitos da oxigenoterapia hiperbárica em lesões actícas da veia cava e aorta infra-renal. Estudo experimental em ratos

Objetivos: Estudar o efeito da Oxigenoterapia Hiperbárica sobre as lesões causadas pela radiação ionizante na veia cava e aorta. Método: Foram utilizados 32 ratos adultos distribuídos aleatoriamente em 4 grupos: 3 no grupo controle; 10 no grupo oxigenoterapia hiperbárica (5 sessões e 3 ATA e após 3 dias, operados), 9 no grupo radioterapia (8 sessões de 5Gy, atingindo 54Gy, sendo operados após 7 dias), 10 no grupo radioterapia e oxigenoterapia hiperbárica (procedimento radioterapia, observação por 7 dias, submetidos a 5 sessões de oxigenoterapia hiperbárica e após 3 dias, são operados. Ressecamos a veia cava e aorta infrarenal para estudo morfológico (integridade endotelial, infiltrado inflamatório e integridade da vasa-vasorum), avaliação do ciclo celular pelo método do AgNOR e índice de apoptose nas distintas camadas dos vasos, através da Caspase-3 (imuno-histoquímica). Resultados: O grupo radioterapia – oxigenoterapia hiperbárica apresentou diminuição estatisticamente significativa das alterações morfológicas em vasa-vasorum, aumento da atividade celular na camada média e também diminuição do índice de apoptose nas camadas íntima e média da aorta infra-renal quando comparados ao grupo apenas submetidos à radiação ionizante. A lesões rádio induzidas na veia cava não apresentaram melhora significativa com a oxigenoterapia hiperbárica Conclusão: A oxigenoterapia hiperbárica é capaz de minimizar as lesões radio-induzidas em fase aguda em aorta.

Autophagic programmed cell death in the suspensor and endosperm of Vicia faba : An ultrastructural study

Programmed cell death (PCD) is a widely spread phenomenon among multi-cellular organisms. Without the deletion of cells no longer needed, the organism will not be able to develop in a predicted way. It is now belived that all cells have the capacity to self-destruct and that the survival of the cells is depending on the repression of this suicidal programme. PCD has turned out to show similarities in many different species and there are strong indications that the mechanisms running the programme might, at least in some parts, be evolutionarily conserced. PCD is a generic term for different programmes of cell destruction, such as apoptosis and autophagic PCD. An important tool to determine if a cell is undergoing PCD is the transmitting electron microscope. The aims of my study were to find out if, and in what way, the suspensor and endosperm in Vicia faba (Broad bean), which are short-lived structures, undergoes PCD. The endosperm degradation preceed the suspensor cell death and they differ to some extent ultrastructurally. The cell death occurs in both tissues about 13-14 days after pollination when the embryo proper is mature enough to support itself. It was found that both tissues are committed to autophagic PCD, a cell death characteristic of conspicuous formations of autophagic vacuoles. It was shown by histochemical staining that acid phosphatases are accumulated in these vacuoles but are also present in the cytoplasm. These vacuoles are similar to autophagic vacuoles formed in rat liver cells, indicating that autophagy is a widely spread phenomenon. DNA fragmentation is the first visible sign of PCD in both tissues and it is demonstrated by a labelling technique (TUNEL). In the endosperm nuclei the heterochromatin subsequently appears in the form of a network, while in the suspensor it is more conspicuous, with heterochromatin that forms large electron dense aggregates located close to the nuclear envelope. In the suspensor, the plastids develop into chromoplasts with lycopene crystals at the same time or shortly after DNA fragmentation. This is probably due to the fact that the suspensor plastids function as hormone producing organelles and support the embryo proper with indispensable growth factors. Later the embryo will be able to produce its own growth factors and the synthesis of these, in particular gibberelines, might be suppressed in the suspensor. The precursors can then be used for synthesis of lycopene instead. Both the suspensor and endosperm are going through autophagic PCD, but the process differs in some respects. This is probably due the the different function of the two tissues, and that the signals that trigger the process presumably are different. The embryo proper is probably the source of the death signal affecting the suspensor. The endosperm, which has a different origin and function, might be controlling the death signal within its own cell. The death might in this case be related to the age of the cell.

Untersuchungen zum Mechanismus der Reblausresistenz der Unterlagsrebsorte `Börner´

Der Resistenzmechanismus der Unterlagsrebsorte `Börner´ Vitis riparia x V. cinerea gegenüber dem Reblausangriff Daktulosphaira vitifoliae (Fitch) wurde histologisch, cytologisch, histochemisch und biochemisch untersucht. Je nach dem Ausmaß der Differenzierung der betroffenen Wurzelgewebe gibt es zwei verschiedene Nekrosetypen. Die Blätter reagieren auf den Reblaus-Einstich, abhängig vom Blattalter bzw. vom Differenzierungsgrad der betroffenen Gewebe, nicht nur mit zwei verschiedenen Nekrosebildungen, sondern auch mit unvollständigen Gallbildungen. Die erhobenen cytologischen, ultrastrukturellen und histochemischen Daten zeigen, dass es sich hierbei um eine Hypersensitivitäts-Reaktion bzw. um Apoptose (PCD) handelt.Nekrosen und Gallen konnten experimentell nicht mechanisch sondern nur chemisch induziert werden. IES spielt eine wichtige Rolle bei den Abwehrreaktion der Unterlagsrebsorte `Börner´ gegen Reblausangriffe.Die vorliegenden Ergebnisse zeigen, daß die Unterlagsrebsorte `Börner` auf den Reblausangriff mit verschiedenen bzw. abgestuften Abwehrreaktionen sowohl der Wurzeln als auch der Blätter reagiert. Das deutet darauf hin, daß die Abwehrreaktionen der Unterlagsrebsorte `Börner´ von mehr als einem Gen abhängen bzw. reguliert werden. Es bleibt weiteren Forschungen vorbehalten, diese Gene und Signale zu identifizieren, um dieses Wissen gezielt in der Züchtung einsetzen zu können.

Neuroglobin, Cytoglobin und Myoglobin in der Blindmaus Spalax ehrenbergi: Adaptation an „unterirdische Verhältnisse“

Hypoxie ist ein Zustand des Sauerstoffmangels, hervorgerufen durch fehlende Verfügbarkeit von Sauerstoff in der Umgebung eines Organismus oder durch pathologisch bedingte unzureichende Nutzbarkeit des Sauerstoffs von Geweben. Die Sensitivität gegenüber Hypoxie variiert enorm im Tierreich zwischen verschiedenen Phyla und Spezies. Die meisten Säugetiere sind nur unzureichend an niedrige Sauerstoffkonzentrationen angepasst, wohingegen einige unterirdisch lebende Säuger sehr resistent gegen Hypoxiestress sind. Um die molekulare Basis der Hypoxietoleranz zu bestimmen, wurden in der vorliegenden Arbeit Globine untersucht, die potenziell in der Lage sind, als respiratorische Proteine zur Hypoxietoleranz von Tieren beizutragen. Dazu wurde die Expression der Globine in der hypoxieresistenten, in Israel lebenden Blindmaus Spalax ehrenbergi mit der Genexpression in der hypoxiesensitiven Ratte (Rattus norvegicus) verglichen. In der vorliegenden Arbeit wurden die erst vor wenigen Jahren entdeckten Globine Neuroglobin und Cytoglobin untersucht, deren exakte physiologische Rolle noch unklar ist, und mit Daten des viel detaillierter untersuchten Myoglobins verglichen. Beim Vergleich der Expression von Cytoglobin und Neuroglobin in Spalax versus Ratte fällt auf, dass Neuroglobin und Cytoglobin bereits unter normoxischen Bedingungen auf mRNA- und Proteinebene in der Blindmaus um einen Faktor von mindesten 2 bis 3 verstärkt exprimiert werden. Bei Myoglobin (als dem Kontrollgen mit bekannter Funktion) konnte auf mRNA-Ebene eine noch weitaus stärkere Expression in Spalax vs. Ratte gefunden werden. Das übergreifende Phänomen der verstärkten Genexpression von Globinen in Spalax kann im Sinne einer Präadaptation an das unterirdische, häufig hypoxische Leben der Blindmaus interpretiert werden. Einen weiteren Hinweis auf eine besondere, spezialisierte Funktion von Neuroglobin in Spalax geben immunhistochemische Daten, die zeigen, dass Neuroglobin im Gehirn von Spalax im Gegensatz zur Ratte nicht nur in Neuronen, sondern auch in Gliazellen exprimiert wird. Dies impliziert Änderungen des oxidativen Stoffwechsels im Nervensystem der hypoxietoleranten Spezies. Die zellulären Expressionsmuster von Cytoglobin erscheinen hingegen in beiden Säugerspezies weitgehend identisch. Es wurde der Frage nachgegangen, ob und wie experimentell induzierte Hypoxie die Genexpression der Globine verändert. Dabei zeigten sich für Neuroglobin und Cytoglobin unterschiedliche Expressionsmuster. Neuroglobin wird unter diversen Sauerstoffmangelbedingungen sowohl in der Ratte als auch in Spalax auf mRNA- und Proteinebene herunterreguliert. Ein ähnliches Regulationsverhalten wurde auch für Myoglobin beobachtet. Die verminderte Expression von Neuroglobin (und evtl. auch Myoglobin) unter Hypoxie ist mit einer gezielten Verringerung der Sauerstoff-Speicherkapazität in Abwesenheit von O2 zu erklären. Ein weiterer denkbarer Grund könnte auch die allgemeine Tendenz sein, unter Hypoxie aus Energiespargründen den Metabolismus herunter zu regulieren. Cytoglobin, das bei normalen Sauerstoffbedingungen nur im Gehirn von Spalax (nicht jedoch in Herz und Leber) ebenfalls um Faktor 2 bis 3 stärker exprimiert wird als in der Ratte, ist mit einiger Sicherheit ebenfalls von adaptivem Nutzen für die Anpassung von Spalax an niedrige Sauerstoffbedingungen, wenngleich seine Funktion unklar bleibt. Unter Hypoxie wird die Cytoglobin-mRNA sowohl in Spalax als auch in der Ratte hochreguliert. Es konnte in der vorliegenden Arbeit dargelegt werden, dass die Expression von Cygb höchstwahrscheinlich durch den Transkriptionsfaktor Hif-1 gesteuert wird, der die molekulare Hypoxieantwort vieler Tierarten zentral steuert. In der vorliegenden Arbeit wurde ebenfalls die Expression von Ngb und Cygb im Gehirn des Hausschweins (Sus scrofa) untersucht. Diese Spezies diente in der Arbeit als weiterer hypoxiesensitiver Organismus sowie als biomedizinisch relevantes Modell für eine Operation an Säuglingen mit angeborenen Herzkrankheiten. Die Versuche haben gezeigt, dass die Gabe bestimmter Medikamente wie dem Immunsuppressivum FK506 zu einer erhöhten Ngb-Konzentration auf mRNA-Ebene führen kann, was potenziell im Zusammenhang mit beobachteten protektiven Effekten der Medikamentengabe während und nach der Herzoperation steht.

Entwicklung eines pharmakologischen Modells zur Prüfung immuntherapeutischer Antikörper mit dreidimensionalen Tumorzellkulturen in vitro

In den letzten Jahren hat die Tumorbehandlung mit immunologischen Präparaten an Bedeutung gewonnen. Der allgemeine Ablauf der Testung eines Arzneimittelkandidaten sieht vor, zunächst in Zellkulturversuchen und Tierversuchen Wirkweise und Sicherheit, sowie voraussichtliche Abbauwege und mögliche Gefahren so beurteilen zu können, dass sie für einen Einsatz im Menschen in Frage kommen. Zur präklinischen in vitro-Testung werden dabei in der Regel Monolayer-Zellkulturen oder Einzelzellsuspensionen eingesetzt. Der Einsatz von 3D-Zellkulturmodellen, welche den Aufbau von Mikrometastasen oder intervaskuläre Areale in Tumoren exakter widerspiegeln, führt zu wesentlich besseren Voraussagen bezüglich der klinischen Wirksamkeit neuer Präparate. Das Ziel dieser Arbeit war daher die Entwicklung und Anwendung eines neuen 3D-Zellkulturbasierten Systems zur Testung trifunktionaler bispezifischer Antikörper für die Tumorbehandlung, welches sich auch auf andere vergleichbare Präparate übertragen lässt.rnIn meiner Arbeit konnte ich mehrere humane Tumorzelllinien definieren, mit denen es gelang, stabile Co-Kulturen von Multi Cellular Tumour Spheroids (MCTS) mit Peripheral Blood Mononuclear Cells (PBMC) in miniaturisierten Spinner-Flaschen zu etablieren. Spinner-Flaschen, in denen die im Kulturmedium befindlichen Immunzellen, MCTS und Therapeutika ständig frei zirkulieren, sind besonders für eine wirklichkeitsnahe Nachbildung der in vivo-Simulation mit disseminierten Tumorzellen oder mit malignem Aszites geeignet. Diese Art der Kultivierung erlaubte Beobachtungszeiten von ≥20 Tagen für eine große Bandbreite Analysemethoden. Zu den mit dem erstellten Protokoll standardmäßig durchführbaren Analysemethoden zählen unter anderem immunhistochemische Färbungen an Sphäroid-Gefrierschnitten, Vitalitätstest, Untersuchung der Plattierungs-Effizienz, Bestimmung der Sphäroidvolumina, Zytokinbestimmungen aus dem Medienüberstand mit Cytokine Bead Arrays, PCR-Analysen immunzellspezifischer Antigene, sowie durchflusszytometrische Analysen. Diese Methodenkombination erlaubt einen sehr detaillierten Einblick in die Wirkweise und Effizienz neuer Immuntherapeutika aus verschiedensten Blickwinkeln und stellt ein reproduzierbares Testsystem zur präklinischen Testung von Immuntherapeutika dar, das zukünftig als Bindeglied zwischen Monolayer-Zellkulturen und klinischen Prüfungen einen festen Platz einnehmen könnte.rnMit dem beschriebenen 3D-Zellkultur-System wurden in der vorliegenden Arbeit die trifunktionalen bispezifischen Antikörper catumaxomab (unter dem Handelsnamen Removab® für die Behandlung maligner Ascites zugelassen) und ertumaxomab (derzeit in klinischen Prüfungen) hinsichtlich ihrer Wirkweise untersucht. Die Antikörper besitzen im Gegensatz zu herkömmlichen monoklonalen Antikörpern zwei verschiedene Bindungsarme, einer gegen CD3 auf T-Zellen, der zweite gegen EpCAM respektive Her2/neu - beides weit verbreitete Tumorantigene - gerichtet. An ihrem Fc-Teil besitzen sie eine dritte Bindungskapazität, über welche sie an Fcγ RI, -IIa und -III positive akzessorische Zellen binden. Diese Kombination ermöglicht theoretisch die Ausbildung eines Tri-Zell-Komplexes aus T-Zelle, Tumorzelle und akzessorischer Zelle. Dies stellt eine wirkungsvolle Therapieoption unter Ausnutzung der körpereigenen, immunologischen Abwehr dar. rnIm Rahmen dieser Arbeit wurde gezeigt, dass beide Antikörper eine Größenreduktion der Sphäroide mit den entsprechenden Tumorantigenen in gleichem Maße bewirkten und die Plattierungseffizienz durch ertumaxomab dosisabhängig reduziert wurde. Mit dem erstellten Testsystem konnte der Wirkmechanismus von catumaxomab auf Sphäroide der Zelllinie FaDu (Kopf-Hals-Plattenepithelkarzinom) detaillierter gezeigt werden: catumaxomab wirkte dosisabhängig auf die Reduktion der Sphäroidvolumina und die zunehmende Infiltration von CD45+ Zellen, die als T-, NK- und/oder dendritische Zellen identifiziert wurden. Des Weiteren rief die catumaxomab-Gabe eine verstärkte Ausschüttung der Zytokine IL-2, IFN-γ und TNF-α hervor. Diese Ergebnisse sprechen dafür, dass catumaxomab die zelluläre Immunantwort aktiviert.rnDie Standard-Tumorbehandlung beinhaltet die Gabe von Chemotherapeutika. Oft werden dafür Zytostatika mit dem unerwünschten Nebeneffekt auch gesunde proliferierende Zellen anzugreifen verwendet. Dies kann prinzipiell auch die Wirksamkeit der Antikörper-Therapie beeinflussen. Aus diesem Grund wurden in dieser Arbeit zusätzlich vergleichende Kombinations-Versuche mit catumaxomab und einem gängigen Zytostatikum - Cisplatin - durchgeführt. Mit Untersuchungen der Sphäroidvolumina, Vitalitätstests und Plattierungseffizienz konnte gezeigt werden, dass die Wirkung von catumaxomab bei gleichzeitiger Anwendung beider Therapeutika aufrecht erhalten bleibt und diese sogar additiv verstärkt wird. Eine Kombinationstherapie im Menschen ist daher denkbar.rnrn

Platelet released growth factors boost expansion of bone marrow derived CD34(+) and CD133(+) endothelial progenitor cells for autologous grafting

Stem cell based autologous grafting has recently gained mayor interest in various surgical fields for the treatment of extensive tissue defects. CD34(+) and CD133(+) cells that can be isolated from the pool of bone marrow mononuclear cells (BMC) are capable of differentiating into mature endothelial cells in vivo. These endothelial progenitor cells (EPC) are believed to represent a major portion of the angiogenic regenerative cells that are released from bone marrow when tissue injury has occurred. In recent years tissue engineers increasingly looked at the process of vessel neoformation because of its major importance for successful cell grafting to replace damaged tissue. Up to now one of the greatest problems preventing a clinical application is the large scale of expansion that is required for such purpose. We established a method to effectively enhance the expansion of CD34(+) and CD133(+) cells by the use of platelet-released growth factors (PRGF) as a media supplement. PRGF were prepared from thrombocyte concentrates and used as a media supplement to iscove's modified dulbecco's media (IMDM). EPC were immunomagnetically separated from human bone morrow monocyte cells and cultured in IMDM + 10% fetal calf serum (FCS), IMDM + 5%, FCS + 5% PRGF and IMDM + 10% PRGF. We clearly demonstrate a statistically significant higher and faster cell proliferation rate at 7, 14, 21, and 28 days of culture when both PRGF and FCS were added to the medium as opposed to 10% FCS or 10% PRGF alone. The addition of 10% PRGF to IMDM in the absence of FCS leads to a growth arrest from day 14 on. In histochemical, immunocytochemical, and gene-expression analysis we showed that angiogenic and precursor markers of CD34(+) and CD133(+) cells are maintained during long-term culture. In summary, we established a protocol to boost the expansion of CD34(+) and CD133(+) cells. Thereby we provide a technical step towards the clinical application of autologous stem cell transplantation.

Glomerulonephropathy in Aged Captive Key Largo Woodrats (Neotoma floridana smalli)

A retrospective review of mortality records of Key Largo woodrats (Neotoma floridana smalli) in a captive breeding program revealed chronic renal disease in 5 of 6 woodrats older than 4 years of age. Two of the 5 woodrats with chronic renal disease also had clinical evidence of diabetes mellitus. Kidneys from all 5 woodrats were examined via light microscopy, histochemical staining, immunohistochemical staining, and transmission electron microscopy. The dietary histories of the affected animals were examined as well. The most striking histopathologic abnormality in the affected kidneys was the presence of large protein casts within cortical and medullary tubules in combination with lesions of membranous glomerulopathy and glomerulosclerosis. Transmission electron microscopy revealed thickening and undulation of the tubular and glomerular mesangial basement membranes with the variable presence of electron-dense deposits within the capillary endothelial basement membrane. Patchy glomerular immunoreactivity for IgG was noted in 2 cases, but IgA and IgM immunoreactivity were not present. The pathologic changes in the kidneys of the Key Largo woodrats mirrored many of the features of chronic progressive nephropathy commonly diagnosed in laboratory rats. Woodrats in the captive population were fed an ad libitum high-protein diet similar to diets that have been shown in laboratory rats to exacerbate the development and progression of chronic progressive nephropathy. It is concluded that Key Largo woodrats develop glomerulonephropathy with features similar to chronic progressive nephropathy described in laboratory rats. Age, concomitant disease, and dietary factors may contribute to the development and severity of this potentially age-limiting disease in Key Largo woodrats.

Vascular endothelial growth factor induces contralesional corticobulbar plasticity and functional neurological recovery in the ischemic brain

Vascular endothelial growth factor (VEGF) is a potent angiogenic factor, which also has neuroprotective activity. In view of these dual actions on vessels and neurons, we were interested whether VEGF promotes long distance axonal plasticity in the ischemic brain. Herein, we show that VEGF promotes neurological stroke recovery in mice when delivered in a delayed way starting 3 days after middle cerebral artery occlusion. Using anterograde tract-tracing experiments that we combined with histochemical and molecular biological studies, we demonstrate that although VEGF promoted angiogenesis predominantly in the ischemic hemisphere, pronounced axonal sprouting was induced by VEGF in the contralesional, but not the ipsilesional corticobulbar system. Corticobulbar plasticity was accompanied by the deactivation of the matrix metalloproteinase MMP9 in the lesioned hemisphere and the transient downregulation of the axonal growth inhibitors NG2 proteoglycan and brevican and the guidance molecules ephrin B1/2 in the contralesional hemisphere. The regulation of matrix proteinases, growth inhibitors, and guidance molecules offers insights how brain plasticity is controlled in the ischemic brain.

Protein synthesis in jejunum and liver of neonatal calves fed vitamin A and lactoferrin

Rates of protein synthesis (PS) and turnover are more rapid during the neonatal period than during any other stage of postnatal life. Vitamin A and lactoferrin (Lf) can stimulate PS in neonates. However, newborn calves are vitamin A deficient and have a low Lf status, but plasma vitamin A and Lf levels increase rapidly after ingestion of colostrum. Neonatal calves (n = 6 per group) were fed colostrum or a milk-based formula without or with vitamin A, Lf, or vitamin A plus Lf to study PS in the jejunum and liver. l-[(13)C]Valine was intravenously administered to determine isotopic enrichment of free (nonprotein-bound) Val (AP(Free)) in the protein precursor pool, atom percentage excess (APE) of protein-bound Val, fractional protein synthesis rate (FSR) in the jejunum and liver, and isotopic enrichment of Val in plasma (APE(Pla)) and in the CO(2) of exhaled air (APE(Ex)). The APE, AP(Free), and FSR in the jejunum and liver did not differ significantly among groups. The APE(Ex) increased, whereas APE(Pla) decreased over time, but there were no group differences. Correlations were calculated between FSR(Jej) and histomorphometrical and histochemical data of the jejunum, and between FSR(Liv) and blood metabolites. There were negative correlations between FSR(Liv) and plasma albumin concentrations and between FSR(Jej) and the ratio of villus height:crypt depth, and there was a positive correlation between FSR(Jej) and small intestinal cell proliferation in crypts. Hence, there were no effects of vitamin A and Lf and no interactions between vitamin A and Lf on intestinal and hepatic PS. However, FSR(Jej) was correlated with histomorphometrical traits of the jejunum and FSR(Liv) was correlated with plasma albumin concentrations.

Regulation of woody plant development by auxin and polar auxin transport

Auxin is a key regulator in plant growth and development. This dissertation examines the role of auxin and polar auxin transport in woody growth and development. Strategies of promoter reporter system, microarray expression analysis, transgenic modification, physiological assays, anatomical analysis, and histochemical/biochemical assays were employed to improve our understanding of auxin study in Populus. The results demonstrate various aspects of auxin regulation on shoot growth, root development, wood formation, and gravitropism in woody tissues. We describe the behavior of the DR5 reporter system for measuring auxin concentrations and response in stably transformed Populus trees. Our study shows that DR5 reporter system can be efficiently used in Populus to study auxin biology at a cellular resolution. We investigated the global gene expression in responding to auxin in Populus root. The results revealed groups of IBA up- and down- regulated genes involved in various biological processes including cell wall modification, root growth and lateral root formation, transporter activity and hormone crosstalk. We also verify two of the identified genes' function by transgenic modification in Populus, which encode auxin efflux carrier PtPIN9 and transcription factor PtERF72. We investigated the role of PtPIN9 in woody growth and development, especially in wood formation and gravitropic response in woody stem. We found that overexpressing PtPIN9 enhanced several growth parameters while suppression of PtPIN9 has inhibited tension wood formation. Our results show that PIN9 and other members from PIN family could be possible useful tools for increasing biomass productivity, wood quality, or in modifying plant form.

Effects of Ethylene on Secondary Xylem Formation in Arabidopsis thaliana

Ethylene has myriad roles as a plant hormone, ranging from senescence and defending against pathogen attacks to fruit ripening and interactions with other hormones. It has been shown to increase cambial activity in poplar, but the effect on wood formation in Arabidopsis hypocotyl has not previously been studied. The Auxin-Regulated Gene involved in Organ Size (ARGOS), which increases organ size by lengthening the time for cell division, was found to be upregulated by ethylene. We tested the effect of ethylene treatment at 10 and 100 µM ACC on three genotypes of Arabidopsis, Col0 (wild-type), an ARGOS deficient mutant (argos), and ein3-1, an ethylene insensitive mutant. ARGOS expression analysis with qPCR indicated that ACC does induce ARGOS and ARGOS-LIKE (ARL) in the hypocotyl. As seen in poplar, ethylene also decreases stem elongation.Histochemical staining, showed that ethylene changes the way secondary xylem lignifies, causing gaps in lignification around the outer edge of secondary xylem. Our results also implied that ethylene treatment changes the proportion of secondary to total xylem, resulting in less secondary, whereas in poplar, ethylene treatment caused an increase.

BMP-2 liberated from biomimetic implant coatings induces and sustains direct ossification in an ectopic rat model

INTRODUCTION: Using a rat model, we evaluated the kinetics and histomorphometry of ectopic bone formation in association with biomimetic implant coatings containing BMP-2. MATERIALS AND METHODS: One experimental and three control groups were set up: titanium-alloy discs coated with a biomimetically co-precipitated layer of calcium phosphate and BMP-2 [1.7 microg per disc (incorporated-BMP group)]; uncoated discs (control); discs biomimetically coated with a layer of calcium phosphate alone (control); and discs biomimetically coated with a layer of calcium phosphate bearing superficially adsorbed BMP-2 [0.98 microg per disc (control)]. Discs (n = 6 per group) were implanted subcutaneously in rats and retrieved at 7-day intervals over a period of 5 weeks for kinetic, histomorphometrical, morphological and histochemical analyses. RESULTS: In the incorporated-BMP-2 group, osteogenic activity was first observed 2 weeks after implantation and thereafter continued unabated until the end of the monitoring period. The net weekly rates of bone formation per disc were 5.8 mm3 at 2 weeks and 3.64 mm3 at 5 weeks. The total volumes of bone formed per disc at these junctures were 5.8 mm3 and 10.3 mm3, respectively. Bone tissue, which was formed by a direct ossification mechanism, was deposited at distances of up to 340 microm from the implant surfaces. The biomimetic coatings were degraded gradually, initially by foreign body giant cells alone and then also by osteoclasts. Forty percent of the coating material (and thus presumably of the incorporated BMP-2) remained at the end of the monitoring period. Hence, 60% of the incorporated BMP-2 had been released. At this 5-week juncture, no bone tissue was associated with any of the control implants. CONCLUSION: BMP-2 incorporated into biomimetic calcium phosphate coatings is capable not only of inducing bone formation at an ectopic site in vivo but also of doing so with a very high potency at a low pharmacological level, and of sustaining this activity for a considerable period of time. The sustainment of osteogenic activity is of great clinical importance for the osseointegration of dental and orthopedic implants.