Galectin fingerprinting detects differences in expression profiles between bovine endometrium and placentomes as well as early and late gestational stages.

The concept of a network within the family of adhesion/growth regulatory galectins implies distinct spatio-temporal expression profiles. To test this assumption immunohistochemically for bovine placenta, placentomal (P) and interplacentomal tissues (IP) were collected at a slaughterhouse from the three stages of pregnancy (early gestation = day 30-130; mid gestation = day 130-220; late gestation = day 220-275). The specimens were snap-frozen or fixed in Bouin's solution, then embedded in paraffin. Gene expression for galectins-1, -3, -4 and -9 in P and IP of late gestational stages was monitored by RT-PCR. Galectin-type-specific antibodies were used for immunohistochemical localization. In IP, galectin-1 was present in stroma cells and early gestational trophoblast giant cells (TGC), whereas galectin-3 was confined to uterine epithelial cells. In contrast, both galectins were found in epithelia of P tissue. Uterine epithelial cells and blood vessel walls were positive for galectin-4, while galectin-9 was detected predominantly in uterine epithelial cells and late gestational TGC. Our study thus reveals individual profiles among the galectins tested, an indication for specific functions exerted by each protein in the bovine endometrium and placenta.

Expression of matrix metalloproteinase (MMP)-2, MMP-14 and tissue inhibitor of matrix metalloproteinase (TIMP)-2 during bovine placentation and at term with or without placental retention.

Matrix metalloproteinases (MMPs) and counteracting tissue inhibitors of metalloproteinases (TIMPs) are balancing extracellular matrix (ECM) formation and degradation. The latter is believed to be an important aspect for the detachment of fetal membranes postpartum when loosening the feto-maternal connection which is a prerequisite to avoid placental retention a common disease in cows leading to considerable economic loss. Membrane-type (MT) MMPs have been suggested as potential activators controlling ECM remodelling. In particular, MT1-MMP (MMP-14) is able to degrade ECM substrates and activate MMP-2 through binding TIMP-2 at the cell surface. Since the connection between the trophoblast and the maternal caruncular epithelium is supported by integrin receptors bound to ECM, we hypothesize that impaired modulation of the ECM by TIMPs/MMPs participates in the aetiology of bovine retained fetal membranes. To analyse this involvement, placentomes were collected from cows after term parturition and timely release of fetal membranes (n = 4) and cows with retained fetal membranes after various treatments for the induction of parturition using progesterone antagonist (aglepristone), PGF(2α) analogue, glucocorticoid, and after elective caesarean sections (each group n = 3). The expression of MMP-14, MMP-2 and of TIMP-2 was examined by real-time-PCR, immunohistochemistry, Western blot and zymography. The relative mRNA expression levels of MMP-14 remained unchanged, while the expression levels of TIMP-2 and MMP-2 partly increased in animals with induced parturition and retention of fetal membranes compared to animals without placental retention. MMP-14 protein was expressed in cells of the uninucleated trophoblast, the fetal mesenchyme and maternal stroma. TIMP-2 was present exclusively in trophoblast giant cells, while MMP-2 could be detected in uninucleated trophoblast cells and the fetal mesenchyme. The presence of the activated enzyme was confirmed by zymography. In conclusion, MMP-14, MMP-2 and TIMP-2 are co-localized in the fetal compartment and therefore could influence the timely release of fetal membranes in cattle.

Formation of bovine placental trophoblast spheroids.

INTRODUCTION: In this study, we aimed to form spheroids with the bovine placental trophoblast cell line F3. Spheroids are 3-dimensional culture models which can be used to conduct versatile in vitro and in vivo experiments. MATERIALS AND METHODS: The spheroids were generated using the hanging drop technique, 25% methocel and matrigel. The F3 spheroids were characterized morphologically by light microscopy and transmission (TEM) and scanning electron microscopy (SEM) and immunohistochemistry (ezrin, vimentin, cytokeratin, placental lactogen). The fluorescent dyes calcein and ethidium homodimer were used to determine the viability of the spheroidal F3 cells by immunofluorescence microscopy. RESULTS: The cell line F3 only formed spheroids by the hanging drop technique when matrigel was added. The trophoblast spheroids were delimited and fully covered by extracellular matrix (light microscopy/TEM/SEM). Cells contributing to spheroids could not be discriminated from each other (light microscopy). The outer spheroidal layer consisted of cells which possessed an apical pole with microvilli that were directed to the outside (light microscopy/TEM). All of the spheroidal F3 cells expressed ezrin, vimentin and cytokeratin, but not placental lactogen. The spheroid core contained degenerating cells whilst the F3 cells of the outer rim were viable (TEM/immunofluorescence microscopy). DISCUSSION: We have established a 3-dimensional spheroid model for the bovine placental trophoblast cell line F3. The developed culture model might prove valuable for future in vitro studies on the differentiation of bovine trophoblast cells.

Primary active transport of organic anions on bile canalicular membrane in humans.

Biliary excretion of several anionic compounds was examined by assessing their ATP-dependent uptake in bile canalicular membrane vesicles (CMV) prepared from six human liver samples. 2, 4-Dinitrophenyl-S-glutathione (DNP-SG), leukotriene C4 (LTC4), sulfobromophthalein glutathione (BSP-SG), E3040 glucuronide (E-glu), beta-estradiol 17-(beta-D-glucuronide) (E2-17G), grepafloxacin glucuronide (GPFXG), pravastatin, BQ-123, and methotrexate, which are known to be substrates for the rat canalicular multispecific organic anion transporter, and taurocholic acid (TCA), a substrate for the bile acid transporter, were used as substrates. ATP-dependent and saturable uptake of TCA, DNP-SG, LTC4, E-glu, E2-17G, and GPFXG was observed in all human CMV preparations examined, suggesting that these compounds are excreted in the bile via a primary active transport system in humans. Primary active transport of the other substrates was also seen in some of CMV preparations but was negligible in the others. The ATP-dependent uptake of all the compounds exhibited a large inter-CMV variation, and there was a significant correlation between the uptake of glutathione conjugates (DNP-SG, LTC4, and BSP-SG) and glucuronides (E-glu, E2-17G, and GPFXG). However, there was no significant correlation between TCA and the other organic anions, implying that the transporters for TCA and for organic anions are different also in humans. When the average value for the ATP-dependent uptake by each preparation of human CMVs was compared with that of rat CMVs, the uptake of glutathione conjugates and nonconjugated anions (pravastatin, BQ-123, and methotrexate) in humans was approximately 3- to 76-fold lower than that in rats, whereas the uptake of glucuronides was similar in the two species. Thus there is a species difference in the primary active transport of organic anions across the bile canalicular membrane that is less marked for glucuronides.

Ethical dilemmas related to counseling clients living with HIV/AIDS.

This study tested an eight-factor model of client actions/decisions in terms of the extent to which professionals counseling persons with HIV/AIDS believed that those actions/decisions presented ethical dilemmas, and the frequency with which they encountered such actions. A confirmatory factor analysis lent initial support for the hypothetical eight-factor ethical-dilemma model for the ratings regarding the extent to which the participants believed those items constituted ethical dilemmas. Similar results were obtained for the frequency ratings, but in this case a second, competing model was equally plausible. Several significant predictors of participant ratings were found and are discussed.

Biliary excretion mechanism of CPT-11 and its metabolites in humans: involvement of primary active transporters.

After administration of CTP-11, a camptothecin derivative exhibiting a wide spectrum of antitumor activity, dose-limiting gastrointestinal toxicity with great interpatient variability is observed. Because the biliary excretion is a major elimination pathway for CPT-11 and its metabolites [an active metabolite, 7-ethyl-10-hydroxy-camptothecin (SN-38), and its glucuronide, SN38-Glu], several hypotheses for the toxicity involve biliary excretion. Here, we investigated whether primary active transport is involved in the biliary excretion of anionic forms of CPT-11 and its metabolites in humans using bile canalicular membrane vesicles (cMVs). Uptake of the carboxylate form of CPT-11 and the carboxylate and lactone forms of SN38-Glu by cMVs prepared from five human liver samples was ATP dependent. The concentration dependence of the ATP-dependent uptake of the carboxylate form of CPT-11 and SN38-Glu suggests the involvement of at least two saturable transport components, both with lower affinity and higher capacity than in rats. The ATP-dependent uptake of the carboxylate form of SN-38 showed a single saturable component but was detectable only in one human cMV sample. Both carboxylate and lactone forms of SN38-Glu uptake also showed a large intersample variability, although the variability was less than that observed for the carboxylate form of SN-38. On the other hand, the carboxylate form of CPT-11 exhibited much less variability. The carboxylate forms of SN38-Glu and SN-38 almost completely inhibited the ATP-dependent uptake of leukotriene C4, a well-known substrate of canalicular multispecific organic anion transporter, whereas the inhibition by the carboxylate form of CPT-11 was not as marked. Thus, multiple primary active transport systems are responsible for the biliary excretion of CPT-11 and its metabolites, and the major transport system for CPT-11 differs from that for the other two compounds. A greater degree of inter-cMV variability in the uptake of SN-38 and SN38-Glu may imply that interindividual variability in biliary excretion of these metabolites might contribute to interpatient variability in the toxicity caused by CPT-11.

Preparation of isolated blood capillaries.

Blood capillaries have been isolated from various tissue sources yielding suspensions of capillary segments. These have provided opportunities to study the cellular properties of capillary endothelium under conditions uncomplicated by the presence of stromal tissues and in which measured parameters can be attributed to endothelial cells. Fresh capillary isolates have been used directly as experimental systems but the yield of endothelium is quite low. Amplification of endothelial biomass has been accomplished by using freshly isolated capillaries as explants for primary tissue culture. It has not been previously possible, however, to obtain large amounts of capillary endothelium from a single preparation nor have different capillary types been isolated from the same tissue. The rete mirabile of the eel swim bladder is a copious source of capillaries of two types: thick-walled, continuous capillaries heavily invested with pericytes and thin-walled, fenestrated capillaries. These can be isolated in large numbers free of large blood vessels and contaminating stromal tissue. The two types of capillaries can be isolated from each other by perfusing magnetic beads into one type prior to isolation and separating them from the other type in a magnetic field. This provides a system in which the cellular properties of the two types of endothelium can be studied in vitro and, due to a common isolation procedure, direct comparisons can be made.

Ultrastructure of capillaries in the red body (rete mirabile) of the eel swim bladder.

The retea mirabilia are paired capillary organs located on the dorsal surface of the swimbladder of the common eel. They consist of bundles of closely apposed capillary segments which function in countercurrent exchange of gases and other solutes and concentrate oxygen in the swim bladder. Ultrastructural features of the afferent arterial capillaries and efferent venous capillaries were studied by scanning EM of corrosion casts and critical-point dried retes and transmission EM of thin sections and freeze-fractured retes. A loose association between endothelial cells in the venous capillaries is indicated by penetration of casting material into interendothelial clefts and the appearance of clefts bounded by cytoplasmic flaps in exposed critical-point dried specimens. In thin sections, open gaps between venous endothelial cells are bounded by cytoplasmic processes. Sections through arterial capillaries exhibit tight occluding junctions joining endothelial cells together and these can be seen in freeze-fracture replicas to extend without interruption along the length of the arterial capillaries. These studies indicate the absence of open or hydraulically conductive pathways across the arterial capillary walls and that they probably constitute a rate-limiting barrier in countercurrent exchange of solutes.

Chlorobenzene-impaired lindane metabolism and the effect of pretreatment with chlorobenzene, lindane, or chlorobenzene plus lindane.

The storage and metabolism of lindane (gamma-HCH) was studied in the female rat after the administration of a hepatotoxic dose of chlorobenzene. Impaired lindane metabolism was observed following a challenge dose of 1.12 g chlorobenzene/kg. The data indicated that a hepatotoxic dose of chlorobenzene (CB) selectively impaired certain pathways, such as dehydrochlorination and the direct hydroxylation of lindane, to a greater extent than others, such as the dehydrogenation and dechlorination of lindane. Pretreatment with a subtoxic level of chlorobenzene produced: (1) significant increases in the dehydrogenation of lindane, (2) significant increase in the excretion of conjugated metabolites, (3) significant increases in the excretion of metabolites derived from the dehydrogenation of lindane through hexachlorocylohexene, gamma-HCCH, (4) significant improvement in the excretion of metabolites derived from CB-impaired dehydrochlorination of lindane as well as from the CB-impaired hydroxylation of lindane, and (5) significant reduction in the level of unaltered lindane stored in the adipose tissue. Repeated pretreatment with a subtoxic level of chlorobenzene offered significant protection against the reduction in lindane metabolism produced by the single hepatotoxic dose of chlorobenzene. Pretreatment with gamma-HCH alone was not as effective against the hepatotoxic effect of CB on lindane metabolism.