Eamonn Fitzpatrick

Glycosidase activity in the excretory-secretory products of the liver fluke, Fasciola hepatica

Fasciola hepatica secretes proteolytic enzymes and other molecules that are essential for host penetration and migration. This mixture may include enzymes required for the degradation of supramucosal gels, which defend epithelial surfaces against pathogen entry. These contain hydrated mucins that are heavily glycosylated. Excretory-secretory products (ES) from F. hepatica were examined for a range of glycosidase activities, using synthetic 4-methylumbelliferyl glycosides as substrates. The ES product contained at least 8 different glycosidase activities, the most abundant of which were beta-N-acetylhexosaminidase, beta-galactosidase and beta-glucosidase. Alpha-fucosidase, beta-glucuronidase, alpha-galactosidase, alpha-mannosidase and neuraminidase were also present. Beta-N-acetylhexosaminidase and beta-galactosidase were present in multiple isoforms (at least 4), whereas beta-glucosidase appeared to exist as one isoenzyme with a pI < 3.8. All three enzymes had acidic pH optima (4.5-5.0). Ovine small intestinal mucin was degraded by ES at pH 4.5 or 7.0, with or without active cathepsin L, the major protease found in F. hepatica ES. The ability of F. hepatica ES to degrade mucin in the presence or absence of active cathepsin L suggests that cathepsin L is not essential for mucin degradation. The abundance of beta-galactosidase and beta-hexosaminidase in ES supports a role for these enzymes in mucin degradation.

Glycoproteins and glycosidases of the cervix during the periestrous period in cattle.

The cervix and its secretions undergo biochemical and physical changes under the differential influences of estrogen and progesterone. These include changes in the glycoprotein profile of the endocervix and its secretions. A comprehensive survey of such changes in cervical epithelium and cervical secretions was performed on bovine samples throughout the periestrous period. Cervical tissue samples and swabs were collected from synchronized beef heifers that were slaughtered 1) 12 h after controlled intravaginal progesterone-releasing device (CIDR) removal, 2) 24 h after CIDR removal, 3) at the onset of estrus, 4) 12 h after the onset of estrus, 5) 48 h after the onset of estrus, and 6) 7 d after the onset of estrus. Histological staining with hematoxylin and eosin, periodic acid Schiff, Alcian blue, and high-iron diamine was carried out to map overall patterns of stored glycoproteins and tissue structure. Biotinylated lectins were also used to detect the presence and distribution of a range of saccharide structures. The activities of β-galactosidase, α-L-fucosidase, β-N-acetyl-hexosaminidase, and sialidase were measured in cervical swabs using specific substrates. The epithelial layer of the cervix exhibited dynamic changes in cellular hypertrophy and amounts of stored glycoprotein. The greatest content of neutral and acidic mucins was observed 48 h after onset of estrus (P < 0.05). Sialylated mucins predominated at the bases of cervical folds, whereas sulfated mucins were more abundant (P < 0.05) at their apices. The stained area of core mucin glycans changed (P < 0.05) in association with follicular versus luteal phases, whereas terminal glycans changed (P < 0.05) mainly at the time of estrus and shortly thereafter. The greatest activity of β-galactosidase and sialidase was observed 12 h after onset of estrus, whereas β-hexosaminidase and α-fucosidase peaked at the luteal time point (P < 0.05). Taken together, we suggest that the well-known changes in the endocervix and its secretions that are associated with the physiological modulation of sperm transport and function of the cervical barrier are, in part, driven by glycosylation changes.

Eosinophil-nerve interactions and neuronal plasticity in rat gut associated lymphoid tissue (GALT) in response to enteric parasitism.

Intestinal lymphoid tissues and Peyers patches (PP) are innervated sites of immune surveillance in the gastrointestinal tract. Following infection with F. hepatica, neuronal hyperplasia and significantly increased eosinophil and mast cell trafficking to colonic PP sites were evident in rat tissues. Nerve-eosinophil associations were significantly elevated in infected colon and colonic PP, as were colonic tissue levels of the circulatory recruitment factors IL-5 and eotaxin. Increased immunoreactivity for neuronal plasticity markers GAP-43 and neural cell adhesion molecule (NCAM) was also found in infected tissues. Such neuronal alterations in the PP during enteric parasitism may have functional consequences on particular or pathogen uptake.

Ascending placentitis in the mare: A review.

Ascending placentitis is a condition that occurs late in pregnancy when bacteria enter the sterile uterus from the lower reproductive tract. It leads to abortion or the birth of premature and weakened foals. Early detection and treatment of this condition is vital for ensuring the production of a viable foal.Mares with ascending placentitis often present in late term pregnancy with signs of premature udder development and premature lactation. There may be a vulvar discharge. Early detection of placental problems is possible using trans-abdominal or trans-rectal ultrasonography. Hormones such as progesterone and relaxin may be measured as indicators of foetal stress and placental failure. Postpartum foetal membranes may be thickened and contain a fibronecrotic exudate. The region most affected is the cervical star. Definitive diagnosis of ascending placentitis is by histopathological examination of the chorioallantoic membrane.Ideal treatment strategies are aimed at curing the infection and prolonging the pregnancy to as close to term as possible and consist of anti-microbials, anti-inflammatories and hormonal support.Swabs are taken from affected mares to determine antibiotic sensitivity and to aid in treatment of foals born from these mares which are at risk of becoming septic. If detected early enough, the chances of producing a viable foal are greatly increased.

Microbial interaction with mucus and mucins

Publisher Summary Mucus represents the front-line defensive barrier between the external environment and the tissues of the host. The molecular scaffolding of mucus comprises hydrated mucins, which are very high-molecular mass, glycosylated glycoproteins presenting arrays of O-linked glycans. Mucins are integral to microbial interactions with epithelial surfaces and have potential roles in microbial trophism, the presentation of ligands to block microbial binding or stabilize colonization and the provision of feedstocks for microbial metabolism. The protective physicochemical properties of mucus are attributable to their high carbohydrate content. Microbes are capable of expressing specific lectins and mucin-degrading enzymes to engage with, utilize, and penetrate mucus gels. Normally, the dynamic nature of such gels represents an optimal adaptation through host–microbial crosstalk, enabling the maintenance of barrier function through the coregulation of mucus turnover and microbial ecology. Mucin expression and glycosylation are biologically responsive to changes in the sub- and supra-mucosal environment and are significantly influenced by inflammation and microbial colonization. Impairment of mucus gel turnover may lead to abnormal colonization, inflammatory pathology, and biofilm formation.

Intraepithelial Leucocytes in the Bovine Uterine Tube

The epithelium of the uterine tube consists of ciliated cells and secretory cells. Basal cells are a third cell type observed in tubal epithelium and they are located principally in the basal part of the epithelium. The objectives of this study were to characterize these basal cells in normal and superovulated heifers and to determine whether they participate in the replacement of the ciliated and secretory cell populations. All heifers received cloprostenol (PG) to induce oestrus (day 0). Superovulated heifers received 24 mg pFSH at doses of 4.5, 3.5, 2.5 and 1.5 mg given twice daily. Control and superovulated heifers were slaughtered on days 1, 3, 5 and 7 of the oestrous cycle. Another group of normal cycling heifers was slaughtered on days 2–3 and 11–13 of the oestrous cycle and used for immunocytochemistry. Samples from ampulla, pre-isthmus and isthmus of the uterine tube were collected and processed for light and transmission electron microscopy. Quantitative examination by light microscopy showed that there was a significant difference in the number of basal cells between the regions of the heifers’ uterine tube. On the basis of ultrastructure two populations of basal cells were observed. One (type I) had a nucleus with much condensed heterochromatin and very sparse cytoplasmic organelles. The second cell (type II) had a nucleus with heterochromatin typically clumped around the nuclear envelope. Its cytoplasm contained many organelles including a number of lysosomes. The ultrastructural features of these cells were similar in all regions and at all days of the oestrous cycle examined. Immunocytochemistry revealed that type I basal cells were lymphocytes and type II basal cells were macrophages.

Comparative Anatomy of the Human and Canine Efferent Tear Duct System – Impact of Mucin MUC5AC on Lacrimal Drainage

Purpose: To investigate the histomorphology of the canine tear drainage system and to show the distribution of mucin MUC5AC within the tissue. Methods: Conjunctiva and tear drainage systems of 19 long-nosed dogs were investigated histologically and ultrastructurally. The tissues were stained with eight different antibodies reactive against less glycosylated and highly-glycosylated MUC5AC. Results were compared with findings in human tissue received from 12 body donors. Results: Except for a distinctly longer nasolacrimal duct and several accessory openings of the duct into the nasal cavity, the morphology of the canine tear drainage system is very similar to that of humans. MUC5AC in less- and highly-glycosylated forms was present in the conjunctival tissue of dogs as well of humans. Within the tear sac and the nasolacrimal duct only less-glycosylated MUC5AC could be found in dogs and in human. Conclusions: These findings demonstrate that the canine tear drainage system is very similar to its human equivalent. In particular the distribution of MUC5AC, supposed to play an important role within the pathogenesis of dry eye syndrome (DES), is the same as in humans. Therefore the canine model seems to be an appropriate model for further DES research.

The expression of mucin genes and the presence of mucin gene products in the equine endometrium

In the equine reproductive tract, little is known about mucin gene expression and the role of mucins in barrier function and host-cell interaction. The aims of the study were to identify equine orthologs of mammalian mucin genes using available equine sequence data, to profile expression of equine orthologous mucin genes in the endometrium using reverse transcriptase polymerase chain reaction (RT-PCR), to determine spatial expression patterns of mucin genes using in situ hybridisation, and to confirm the presence of mucin gene products using Western blotting and equine-specific mucin antibodies during oestrus and dioestrus. While the mucin gene expression pattern in equine endometrium is similar to that of other mammals, several mucins appear to be uniquely expressed in this tissue (eqMUC3B, 7, 18, and 20) and one is hormonally regulated (eqMUC3B).

A101–A107 Skin Substitutes and Wound Healing/Medical Applications

Skin Substitutes and Wound Healing Medical Applications

A201–A209 Gene Therapy and Growth Factors in TE/Basic Sciences/Developmental Biology and TE/Microsystem Technology in TE

Introduction: Cell-based implants for Tissue Engineering are dependent on specific bioactive factors. Transforming Growth Factor-̌ 1 (TGF-̌ 1) has be shown to promote chondrogenesis and may be useful for promoting cartilage repair when expressed from a transgene in a cell-based implant. Retroviral vectors are one of the preferred modes for transgene transduction into mesenchymal cells, however, successful transduction is dependent on active proliferating cells. This study was designed to optimize the TGFˇ1-retroviral transduction efficiency of bone marrow-derived progenitor cells and to study the effects of transgene TGFˇ1 production on chondrogenesis. Methods: Bone marrow-derived mesenchymal progenitor cells, which were obtained from human bone marrow aspirates, were cultured in DMEMC 10% FBS without (group A) or with addition of Epidermal Growth Factor(EGF) (10 ng/ml; group B) to optimize the proliferation rate. After 5 days cell counts were obtained with a MTT proliferation assay. Cells of group A and B were transduced after 24 or 72 hours in culture with retroviral vector DFG-TGF-̌1-Zeo (Polybrene: 8μg/ml; centrifugation with 1700 rpm for 1 hour). Transduced cells were selected with three selection doses of Zeocin after 4 days in culture. The amount of transduced cells was determined with a MTT proliferation assay. The TGFˇ1 protein-production was measured preand post-selection with a TGFˇ1 specific ELISA. Transduced cells of group A and B were assayed for their chondrogenic potential in anin vitro chondrogenesis model [1,2] and compared to the equivalent non-transduced controls. To assay the effect of the transgene expression, cells were incubated with/without exogenous TGF-̌1 protein. Results: An increased proliferation of bone marrow-derived mesenchymal cells could be achieved with exogenous addition of EGF (group B) (in 4 days cell count 40% above control (group A)). Without addition of EGF (group A) the different time points of transduction (transduction after 24 or 72 hours of preculture) showed no significant difference in transduction rate (approximately 10%). However, with addition of EGF (group B) douple as much cells could be transduced after 72 hours of preculture compared to 24 hours of preculture. After preculture for 72 hours the transduction rate was 20%. Furthermore, with EGF addition (group B; 100 pg/1.000 cells) a 100-fold increase of TGF-̌1 protein production could be achieved in comparison to the control (group A; 1 pg/1.000 Zellen). This ratio could also be detected after selection with Zeocin. Transduced cells, which were cultured in DMEM C 10% FBS (group A), underwentin vitro chondrogenesis with and without exogenous addition of TGFˇ1, indicating that the transgene was functional and that the cells were producing enough TGFˇ1 to promote differentiation. Cells under EGF-influence (group B) showed with and without exogenous addition of TGFˇ1 no chondrogenesis. Even in non-transduced controls, which were cultured in EGF-containing medium, no chondrogenic differentiation could be detected. Discussion: TGF-̌ 1 transduced mesenchymal progenitor cells can produce a sufficient amount of active TGFˇ1 protein, to undergo chondrogenesis. EGF-containing medium enhances cell proliferation, retroviral transduction efficiency and TGFˇ1 protein production per cell compared with DMEM and 10% FBS alone. However, mesenchymal progenitor cells seems to loose chondrogenic differentiation potential when cultured in EGF-containing medium. This study shows the potential of gene therapy to optimize cell-based implants for Tissue Engineering. Furthermore the study shows, that methods, to optimize cell-proliferation and transduction, could have also negative effects on cell differentiation.