The complete primary structure of the spermadhesin AWN, a zona pellucida-binding protein isolated from boar spermatozoa

AbstractAWN is a boar protein which originates in secretions of the male accessory glands and which becomes sperm surface-associated upon ejaculation. It is one of the components thought to mediate sperm adhesion to the egg's zona pellucida through a carbohydrate-recognition mechanism. AWN may, thus, participate in the initial events of fertilization in the pig. In this report we describe its complete primary structure by combination of protein-chemical and mass spectrometric methods. AWN exists as two isoforms, AWN-1 and AWN-2, which differ in that AWN-2 is N-terminally acetylated. The amino acid sequence of AWN contains 133 amino acid residues and two disulphide bridges between nearest-neighbour cysteine residues. Analysis of the amino acid sequence of the AWN proteins showed significant similarity only to AQN-1 and AQN-3, two other boar spermadhesins

Luteal angiogenesis and its control

Angiogenesis, the formation of new blood vessels from pre-existing ones, is critical to luteal structure and function; In addition, it is a complex and tightly regulated process. Not only does rapid and extensive angiogenesis occur to provide the corpus luteum (CL) with an unusually high blood flow and support its high metabolic rate, but in the absence of pregnancy the luteal vasculature must rapidly regress to enable the next cycle of ovarian activity. This review describes a number of the key endogenous stimulatory and inhibitory factors, which act in a delicate balance to regulate luteal angiogenesis and ultimately luteal function. In vitro luteal angiogenesis cultures have demonstrated critical roles for fibroblast growth factor 2 (FGF2) in endothelial cell proliferation and sprouting, whilst other factors such as vascular endothelial growth factor (VEGFA) and platelet derived growth factor (PDGF) were important modulators in the control of luteal angiogenesis. Post-transcriptional regulation by small non-coding micro-RNAs, is also likely to play a central role in the regulation of luteal angiogenesis. Appropriate luteal angiogenesis requires the coordinated activity of numerous factors expressed by several cell types at different times and this review will also describe the role of perivascular pericytes and the importance of vascular maturation and stability. It is hoped that a better understanding of the critical processes underlying the transition from follicle to CL, and subsequent luteal development will benefit the management of luteal function in the future

Perivascular-like cells contribute to the stability of the vascular network of osteogenic tissue formed from cell sheet-based constructs

In recent years several studies have been supporting the existence of a close relationship in terms of function and progeny between Mesenchymal Stem Cells (MSCs) and Pericytes. This concept has opened new perspectives for the application of MSCs in Tissue Engineering (TE), with special interest for the pre-vascularization of cell dense constructs. In this work, cell sheet technology was used to create a scaffold-free construct composed of osteogenic, endothelial and perivascular-like (CD146+) cells for improved in vivo vessel formation, maturation and stability. The CD146 pericyte-associated phenotype was induced from human bone marrow mesenchymal stem cells (hBMSCs) by the supplementation of standard culture medium with TGF-b1. Co-cultured cell sheets were obtained by culturing perivascular-like (CD146+) cells and human umbilical vein endothelial cells (HUVECs) on an hBMSCs monolayer maintained in osteogenic medium for 7 days. The perivascular-like (CD146+) cells and the HUVECs migrated and organized over the collagen-rich osteogenic cell sheet, suggesting the existence of cross-talk involving the co-cultured cell types. Furthermore the presence of that particular ECM produced by the osteoblastic cells was shown to be the key regulator for the singular observed organization. The osteogenic and angiogenic character of the proposed constructs was assessed in vivo. Immunohistochemistry analysis of the explants revealed the integration of HUVECs with the host vasculature as well as the osteogenic potential of the created construct, by the expression of osteocalcin. Additionally, the analysis of the diameter of human CD146 positive blood vessels showed a higher mean vessel diameter for the co-cultured cell sheet condition, reinforcing the advantage of the proposed model regarding blood vessels maturation and stability and for the in vitro pre-vascularization of TE constructs.Funding provided by Fundacao para a Ciencia e a Tecnologia project Skingineering (PTDC/SAU-OSM/099422/2008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Nanoparticles as carrier systems for transcutaneous vaccination - Antigen- delivery across the skin barrier

Ziel dieser experimentellen Arbeit waren Untersuchungen zum Targeting epidermaler Langerhanszellen sowie dem Antigentransport mittels Nanopartikel gekoppeltem p24-Antigen im Hinblick auf eine transepidermale Immunisierung. Zu diesem Zweck wurden die follikuläre Penetration von Partikeln in menschlicher Haut, das Aufnahmeverhalten durch epidermale Langerhanszellen und die Aktivierung derer durch p24-Antigen beladene Partikel untersucht. Es zeigte sich, dass nach einem CSSS und dem Auftragen unterschiedlich großer und beschichteter Partikeltypen (PLA/PS-Partikel, >/< 200nm, +/-p24-Antigen) auf exzidierte humane Haut eine Penetration entlang des Haarfollikels erfolgte. Mittels der Fluoreszenzmikroskopie ließen sich in mehr als der Hälfte (50-70 %) der untersuchten Haarfollikel PLA- sowie PS-Partikel nachweisen. Bei PLA- Partikeln einer Größe > 200 nm zeigte sich in etwa 20 % der HF eine Penetration bis zum tiefen Infundibulum. Somit stellt sich der follikuläre Weg als geeignet für transepidermale Vakzinierungsstrategien dar, da über ihn die immunkompetenten Zielzellen der Haut, die im Bereich des distalen Infundibulums resident sind, erreicht werden können (Targeting). Das zweite Ziel dieser experimentellen Arbeit war, aufbauend auf die vorangegangenen Penetrationsuntersuchungen, eine Aufnahme der p24-gekoppelten Partikel durch die LZ nach transfollikulärer Penetration nachzuweisen. Die epidermalen LZ gelten als potente Immunzellpopulation und können durch Internalisierung der Antigen-beladenen Nanopartikel möglicherweise zusätzlich über MHC-I-Kreuzpräsentation des Antigens eine effektive zelluläre Immunantwort initiieren; diese ist gerade im Hinblick auf transepidermale Immunisierungsstrategien gegen HIV von großer Bedeutung. Alle im Rahmen dieser experimentellen Arbeit untersuchten PS-Partikel (>/< 200 nm, +/- p24-Antigen) konnten nach topischer Applikation durch die Hautbarriere in die epidermalen Zellschichten gelangen und von den dort residenten Langerhanszellen aufgenommen werden. Es zeigte sich in der Durchflusszytometrie eine größenabhängige Aufnahme der Partikel durch epidermale LZ, die Zahl der „Partikel-positiven“ Zellen schwankte zwischen 1,5-3,5 %. Somit wird sichtbar, dass die intrazelluläre Aufnahme der Partikel größenabhängig erfolgt und auch für die Penetrationseigenschaften wesentlich ist. Eine Aufnahme von PLA- Partikeln konnte, wie auch in früheren Untersuchungen gezeigt, nicht nachgewiesen werden und liegt möglicherweise an der Instabilität der Partikel mit nachfolgender Freisetzung des Farbstoffes sowie Konglomeratbildung. Neue unpublizierte Forschungsergebnisse der AG Dr. A. Vogt zeigen jedoch, dass sich HIV-1-p24-Antigen, welches an PLA-Partikel gekoppelt ist, nach transepidermaler Applikation in epidermalen LZ nachweisen lässt und somit prinzipiell für eine Translokation des Antigens über die Hautbarriere spricht. Nach der Untersuchung der follikulären Penetration sowie Aufnahme der Partikel durch epidermale LZ bestand der letzte Teil dieser experimentellen Arbeit in der Erforschung der Aktivierung der LZ durch p24-Antigen beschichtete Partikel. Es zeigte sich eine Stimulation der Immunzellen nach einem CSSS und transkutaner Applikation vor allem mit PLA 215_p24 durch Hochregulierung der Aktivierungsmarker CD80 sowie CD83. Da speziell PLA-Partikel bioabbaubar sind, gelten sie als attraktive Trägersysteme für transkutane Immunisierungsstrategien. In der hier vorliegenden Dissertation konnte bestätigt werden, dass p24-PLA-Partikel über den Haarfollikel penetrieren und die LZ aktivieren, was für die Initiierung einer effektiven Immunantwort von Bedeutung ist. Auch ist eine Aufnahme transepidermal applizierter Partikel mit p24-Antigen durch LZ möglich und im Hinblick auf ein Targeting der epidermalen Immunzellpopulation wünschenswert. Diese Arbeit zeigt, dass Nanopartikel mit p24-Antigen als Träger für eine transepidermale Immunisierung viel versprechend sind, da sie sowohl über den follikulären Kanal penetrieren, über die Hautbarriere translozieren, dort von epidermalen Langerhanszellen aufgenommen werden und diese zusätzlich stimulieren.The objective of this experimental work was to investigate the complex interaction of nanoparticles and epidermal Langerhans cells with regard to the possibilities of transepidermal immunisation. For that purpose we looked into the process of follicular penetration of nanoparticles into human skin, the reaction of absorption of antigen-coated nanoparticles by LCs and their activation by various particle types. It was shown that penetration via the hair follicle duct took place after a CSSS and application of p24-antigen coated particle types of different sizes and material classes. By means of fluorescence microscopy proof of PLA and PS-particles could be established in more than half of the examined hair follicles. With PLA Particles >200nm penetration into the deep infundibulum could be detected. Because of this the follicular pathway seems to be suitable for transcutaneous vaccination strategies as by that method the immune competent target cells of the skin which are resident in the area of the distal infundibulum can be reached. He second aim of this experimental work was to give proof of internalisation of the particles by the LCs after transfollicular penetration. The epidemal LCs are regarded as potent immune cell population and might initiate a strong cellular immune response by internalisation of antigen-coated nanoparticles, additionally via MHC-1 cross-presentation of antigen. In view of transepidermal immunisation strategies, especially against the HI-Virus this fact is of utmost importance. For this reason the internalisation of PS- particles +/- p24 antigen by Langerhans cells was investigated after topic application. All PS-particles investigated within the bounce of this work were taken up after topic application by the Langerhans cells. In flow cytometry absorption by Langerhans cells took place corresponding to the size of particles. Therefore it is apparent that the intracellular internalisation of particles works dependent on size and is also essentiell for the quality of penetration,. The last part of these experimental studies was to look more closely into the activation of Langerhans cells by p24 antigen-covered particles. Stimulation of immune cells after transepidermal incubation was discovered, especially with PLA 215 p24 particles due to higher regulation of the activation markers CD 80 and CD83. As especially PLA-particles are biodegradable they are considered as attractive carrier systems for transcutaneous vaccination strategies. Fundamental absorption of transepidermally applied particles coated with antigen by LCs is not only possible, it is also highly desirable concerning the targeting of epidermal immune cell population. The studies show that nanoparticles as antigen-carrier systems are most promising as they penetrate via the follicluar duct as well as translocate through the skin barrier where they are absorbed by LCs and furthermore stimulate them

Therapeutic Management of Subclinical Staphylococcus aureus Mastitis in Association with Iliofemoral Lymphadenopathy in a Cow

A 6.5 year old Holstein-Friesian dairy cow was initially presented with anorexia and reduced milk yield. Subclinical mastitis was diagnosed by California Mastitis Test (CMT). Bacteriological examination of a milk sample of the affected hind quarter revealed the detection of S. aureus. Additionally, rectal examination revealed very large and hard iliofemoral lymph nodes. Iliofemoral lymphadenopathy was then confirmed by ultrasound examination. This unusual case suggests that S. aureus intramammary infection (IMI) may possess the ability to invade the host organism by lymphatic route without causing visible inflammatory reactions

Expression of transforming growth factor-B3 -GF-B3- in the porcine ovary during the oestrus cycle

Transforming growth factor-ß (TGF-ß) proteins are growth factors that have been shown to be involved in regulation of ovarian follicular development. Ovarian expression, activity and functional significance of TGF-ß1 and TGF-ß2 isoforms were extensively studied in most species. However, little is known about the biological role of TGF-ß3 previously shown to be expressed independently of the other two isoforms. Therefore, expression of TGF-ß3 mRNA and protein was evaluated by RT-PCR and immunohistochemistry, respectively, in porcine ovaries collected during different phases of the oestrus cycle. Results of RT-PCR analysis showed that TGF-ß3 mRNA is expressed throughout the oestrus cycle. The level of TGF-ß3 mRNA expression was found to be higher at metoestrus and dioestrus. Weak TGF-ß3 immunoreactivity was present in follicular epithelial cells and oocytes of preantral follicles in all stages examined. TGF-ß3 protein expression was exclusively present in theca interna cell layer of antral follicles, and was particularly prominent in large antral follicles. Immediately after ovulation, almost all theca cells outside of the granulosa cell layer were intensively stained with anti-TGF-ß3. Immunostaining of TGF-ß3 in theca lutein cells rapidly decreased during corpus luteum development. It is suggested that TGF-ß3 may play an important role in modulating theca cell function of pre- and postovulatory follicles of the pig

Cellular localization of fibroblast growth factor 2 (FGF-2) in benign prostatic hyperplasia

Fibroblast growth factor 2 (FGF-2, basic fibroblast growth factor) has been reported to be elevated in tissues from benign prostatic hyperplasia (BPH), the most frequent neoplastic disease in aging men. This suggests that FGF-2 may play a significant role in the development of BPH. In this study the cellular distribution pattern of FGF-2 in tissues from BPH has been investigated by immunohistochemical and molecular biological methods. Radioimmunoassay revealed high concentrations of FGF-2, ranging between 450 and 950 ng per g tissue. Immunoblots confirmed the presence of a 18 kDa FGF-2 in tissue extracts. By immunohistochemistry done with a polyclonal antibody to recombinant FGF-2 on paraffin sections, FGF-2 was localized in fibroblasts, endothelial cells and smooth muscle cells of tissue samples of BPH. Nuclei of these cells were labelled distinctly. Moreover the cytoplasm of smooth muscle cells was labelled moderately. No immunostaining was seen in prostatic epithelium. Nonradioactive in situ hybridization with digoxygeninlabelled oligonucleotides revealed the presence of mRNA for FGF-2 in smooth muscle cells of the prostatic stroma. These results provide evidence that FGF-2 may be produced locally in the human prostate as a stromaspecific mitogen and may play a causal role in the development of BPH