Purification and characterization of placental heparanase and its expression by cultured cytotrophoblasts

The role of different extracellular matrix (ECM)-degrading enzymesin the normal functioning of the placenta is well documented.Heparan sulphate proteoglycan (HSPG) is an integral constituentof the placental and decidual ECM. Because this proteoglycanspecifically interacts with various macromolecules in the ECM,its degradation may disassemble the matrix. Hence, in the caseof the placenta, this may facilitate normal placentation andtrophoblast invasion. Crude placental specimens were collectedfrom first and third trimester placentas. Heparanase (endo-P-glucuronidase)was isolated and purified by ammonium sulphate precipitationfollowed by sequential chromatographies on carboxymethyl-, heparin-and ConA-Sepharose columns. The placental enzyme was furthercharacterized for its molecular weight and specific inhibitionby heparin, and was shown to resemble heparanase expressed byhighly metastatic tumour cells and activated cells of the immunesystem. In order to locate the source of heparanase activityin the placenta, primary cytotrophoblast cultures were established.Intact cells, as well as conditioned medium and cell lysates,were analysed for heparanase activity using metabolically sulphate-labelledECM as a natural substrate. Heparanase was highly active inlysates of cytotrophoblasts. This activity was also expressedby intact cytotrophoblasts seeded on ECM, but no activity couldbe detected in the culture medium. Incubation of the cytotrophoblastsin contact with ECM resulted in release of ECM-bound basic fibroblastgrowth factor (bFGF). We propose that the cytotrophoblasticheparanase facilitates placentation, through cytotrophoblastextravasation and localized neovascularization. cytotrophoblast/extracellular matrix/heparanase/heparan sulphate proteoglycan/placenta     

<Emphasis Type="Italic">TCF7L2</Emphasis>variant genotypes and type 2 diabetes risk in Brazil: significant association,but not a significant tool for risk stratification in the general population

Background  

Genetic polymorphisms of the TCF7L2 gene are strongly associated with large increments in type 2 diabetes risk in different populations worldwide. In this study, we aimed to confirm the effect of the TCF7L2 polymorphism rs7903146 on diabetes risk in a Brazilian population and to assess the use of this genetic marker in improving diabetes risk prediction in the general population.     

Age, gender and litter-related variation in T-lymphocyte cytokine production in young pigs

The capacity of farm animals to produce cytokines could be an important determinant of robustness and health. From research in rodents and humans it appears that the production and the balance of T helper 1 (Th1) and T helper 2 (Th2)-type cytokines influences susceptibility to autoimmune and infectious diseases. It is known that pigs show a large variation in many immune response parameters. So far the extent of individual variation in the production of Th1- and Th2-type cytokines in commercial outbred pigs has not been reported. In the current experiment we determined mRNA expression, as well as protein production of cytokines in 32 pigs from eight litters. From each litter two male and two female pigs were tested at 2, 5 and 8 weeks of age. Two Th1-type cytokines, interleukin (IL)-2 and interferon (IFN)-gamma, and two Th2-type cytokines, IL-4 and IL-10, were measured after phytohaemagglutinin (PHA)-stimulation of blood mononuclear cells. Cytokine production and the Th1/Th2-ratio were highly variable. The variation in cytokine protein production was moderately consistent across ages, i.e. pigs that produced high levels of cytokine at 2 weeks of age tended to do so as well at 5 and 8 weeks of age. Cytokine production tended to increase with age, and gilts and boars differed in their IL-2/IL-4 ratio. Unexpectedly, age, gender and litter effects often differed for mRNA and protein production data. We hypothesize that cytokine production is a consistent trait in pigs, especially at the protein production level. Future investigations in more animals and across a wider age range are necessary.     

Nkx2.5‐negative myocardium of the posterior heart field and its correlation with podoplanin expression in cells from the developing cardiac pacemaking and conduction system

Recent advances in the study of cardiac development have shown the relevance of addition of myocardium to the primary myocardial heart tube. In wild‐type mouse embryos (E9.5–15.5), we have studied the myocardium at the venous pole of the heart using immunohistochemistry and 3D reconstructions of expression patterns of MLC‐2a, Nkx2.5, and podoplanin, a novel coelomic and myocardial marker. Podoplanin‐positive coelomic epithelium was continuous with adjacent podoplanin‐ and MLC‐2a‐positive myocardium that formed a conspicuous band along the left cardinal vein extending through the base of the atrial septum to the posterior myocardium of the atrioventricular canal, the atrioventricular nodal region, and the His‐Purkinje system. Later on, podoplanin expression was also found in the myocardium surrounding the pulmonary vein. On the right side, podoplanin‐positive cells were seen along the right cardinal vein, which during development persisted in the sinoatrial node and part of the venous valves. In the MLC‐2a‐ and podoplanin‐positive myocardium, Nkx2.5 expression was absent in the sinoatrial node and the wall of the cardinal veins. There was a mosaic positivity in the wall of the common pulmonary vein and the atrioventricular conduction system as opposed to the overall Nkx2.5 expression seen in the chamber myocardium. We conclude that we have found podoplanin as a marker that links a novel Nkx2.5‐negative sinus venosus myocardial area, which we refer to as the posterior heart field, with the cardiac conduction system. Anat Rec, 290:115–122, 2007. © 2006 Wiley‐Liss, Inc.     

Hereditary Cerebral Hemorrhage with Amyloidosis-Dutch Type (HCHWA-D): I - A Review of Clinical, Radiologic and Genetic Aspects

Hereditary cerebral hemorrhage with amyloidosis - Dutch type (HCHWA-D) is an autosomal dominant disease caused by deposition of β-amyloid in the leptomeningeal arteries and cortical arterioles, in addition to preamyloid deposits and amyloid plaques in the brain parenchyma.
The disease is due to a point mutation at codon 693 of the amyloid precursor protein (βPP) gene at chromosome 21. Since this point mutation is diagnostic for HCHWA-D, presymptomatic testing is feasible and offered, together with genetic counselling and psychological support, to subjects at risk. HCHWA-D is clinically characterized by recurrent strokes, in addition to dementia, which can occur after the first stroke but also preceding it. Radiological studies revealed focal lesions (hemorrhages, hemorrhagic and non-hemorrhagic infarctions) and diffuse white matter damage. Diffuse white matter hyperintensities on MRI are an early symptom of HCHWA-D since they have been found on MRI scans of subjects who had not suffered a stroke.
The presence of the diagnostic point mutation makes HCHWA-D a useful model to study the effects of cerebral amyloid angiopathy in vivo. The characteristic pathological abnormalities and its implications for Alzheimer's disease will be discussed in Part II of this article     

Variations of the origin of the artery of the sinoatrial node in normal human hearts

Summary The artery of the sino-atrial node was studied in 100 normal human hearts after injection of each coronary artery with coloured gelatine containing a radiopaque substance. The hearts belonged to 69 males and 31 females, being 64 Caucasians and 36 non-Caucasians (Negroes and Mulattoes) whose age ranged from 7 to 80 years. Since the individuals had committed suicide or were victims of accidents, their hearts, after pathologists' evaluation, were considered normal. The sinoatrial node of the normal human heart is supplied by the right coronary artery more frequently (58%±4.9% of the cases) than by the left (42%±4.9). The right anterior medial atrial artery, originating from the right coronary at the level of the medial third of the right anterior quadrant of the atrial dome, is most frequently (50%±5) responsible for the blood supply of the sinoatrial node. Among the branches of the left coronary artery, the left anterior medial atrial artery, originating at the level of the medial third of the left. anterior quadrant of the atrial cupola, was the most frequent blood supplier (25%±4.3) of the sinoatrial node. The origin of the artery of the sinoatrial node from the proximal portion or trunk of the left coronary artery was less frequent (12%±3.2) than the origin from the circumflex artery (30%±4.5). Neither sex nor race influenced the variations of the origin of the sino-atrial node.

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Variations d'origine de l'artère du noeud sinu-atrial du coeur humain normal

Résumé L'a. du noeud sinu-atrial a été étudiée sur 100 coeurs humains normaux après injection de chaque a. coronaire à la gélatine colorée additionnée d'une substance radio-opaque. Les coeurs provenaient de 69 hommes et 31 femmes, 64 caucasiens et 36 non caucasiens (nègres et mulâtres) âgés de 7 à 80 ans. Ces sujets étant décédés par suicide ou des suites d'accidents, leurs coeurs ont été considérés comme normaux après examen anatomo-pathologique. Le noeud sinu-atrial du coeur humain est vascularisé par l'a. coronaire droite plus fréquemment (58 %±4,9) que par l'a. coronaire gauche (42 %±4). L'a. atriale antéro-médiale droite, issue de l'a. coronaire droite au niveau du tiers médial du quadrant antérieur droit du dôme atrial est l'artère la plus fréquemment en cause (50 %±5) dans la vascularisation du noeud sinuatrial. Parmi les branches de l'a. coronaire gauche, l'a. atriale antéro-médiale gauche, née au niveau du tiers médial du quadrant antérieur gauche du dôme atrial, était la branche la plus fréquemment en cause (25 %±4,3) dans la vascularisation du noeud sinu-atrial. La naissance de l'a. du noeud sinu-atrial à partir de la partie proximale ou du tronc de l'a. coronaire gauche était moins fréquente (12 %±3,2) que son origine à partir du rameau circonflexe (30 %±4,5). Les variations d'origine de l'a. du noeud sinu-atrial n'apparaissaient pas influencées par le sexe ou la race.

     

Induction of T cell proliferation with anti-CD3 switch-variant monoclonal antibodies: effects of heavy chain isotype in monocyte-dependent systems

Monoclonal antibodies (mAb) directed against the CD3 (T3), antigen are able to induce proliferation in resting human T lymphocytes. T cell proliferation only occurs in the presence of monocytes that carry the proper Fc receptor for the mAb used. To further analyze the role of the Fc portion of anti-CD3 mAb in proliferation induction, we isolated, starting from a gamma 1 anti-CD3-producing hybridoma, four heavy-chain isotype switch-variant antibody-secreting clones, producing gamma 2b, gamma 2a, epsilon and alpha, respectively. All variant antibodies recognize the CD3 antigen as determined by immunoprecipitation and cross-blocking experiments. With this series of isotype variant antibodies we were able, in proliferation induction experiments, to confirm the Fc receptor polymorphism for murine IgG2a, IgG2b and IgG1 on human monocytes. Moreover, we found that all 30 donors tested responded to the IgE anti-CD3 antibody, while no IgA responders could be identified. The induction of proliferation by the IgE variant antibody does not require the 72-kDa Fc receptor which is responsible for the interaction with mouse IgG2a. Nonresponsiveness to the IgG1 antibody, but not to the IgG2b or IgA variant antibodies, could be overcome by the addition of exogenous interleukin 2 to the cultures. When the switch-variant antibodies were used to induce IgM synthesis in peripheral blood mononuclear cells only low IgM synthesis was found, with the exception of the IgE variant, which induced excellent T cell help for IgM production.     

Viability of partially damaged human embryos after cryopreservation

In our centre, embryos are judged to have survived cryopreservation if at least half of the initial number of blastomeres remain intact. Therefore both fully intact and partially damaged embryos are transferred. The aim of this study was to investigate the viability of partially damaged human embryos after cryopreservation. We retrospectively analysed the implantation and in-vivo development of embryos which were either fully intact or had lost some blastomeres after cryopreservation. Oocytes were collected following stimulation with the gonadotrophin-releasing hormone (GnRH)-agonist Buserelin and human menopausal gonadotrophin. Supernumerary multicellular embryos with not more than 20% of their volume filled with anucleate fragments were frozen on day 2 or day 3 of the cycle using a slow cooling procedure with dimethylsulphoxide as the cryoprotectant. Following slow thawing, 431 fully intact embryos were transferred in 314 embryo transfer procedures and 488 partially damaged embryos were transferred in 327 such procedures. The percentage of gestational sacs with fetal heartbeat obtained after transfer of fully intact embryos was almost three times higher than that after transfer of partially damaged embryos (11.4 versus 3.5%). Forty-five children (birth rate 10% per embryo transfer) were born after transfer of fully intact embryos and 14 after transfer of embryos from which some blastomeres had been lost following cryopreservation. In conclusion, although children have been delivered after transfer of partially damaged embryos, the aim of a cryopreservation programme must be to obtain fully intact embryos after thawing.      

Plasma sprayed coatings of hydroxylapatite

The technique of plasma spraying has been applied to deposit a thin, dense layer of hydroxylapatite onto a titanium substrate. Bond strength of such apatite coatings with the substrate have been measured, as well as the (absence of) influence of the coating process on fatigue properties of the substrate. Animal studies showed similar histological reactions to apatite coatings as to (well documented) apatite bulk materials.     

Interaction of biodegradable β-whitlockite ceramics with bone tissue: An in vivo study

The biodegradation of different porous β-whitlockite materials are studied by in vivo experiments, radiographie follow-ups and light microscopy observations. The materials were implanted in rabbit tibiae for 16 mnth. Micropores play an important role in the biodegradation rate. The resorbing materials evoke an inflammation with plasma cells. The resorption starts in the medulla, and the phagocytosed particles are removed to the lymph nodes. Normal bone function can be restored after all the implant material is resulted.