ADRENOCORTICOID-DEPENDENT α-LACTALBUMIN SYNTHESIS IN RAT MAMMARY GLAND EXPLANTS: ANTAGONIST STUDIES

Though adrenal steroids are required for the production of alpha-lactalbumin by mammary gland explants, the physiological class of steroid activity (mineralocorticoid, glucocorticoid) remains to be established. alpha-Lactalbumin production by mammary gland explants from mid-pregnant rats has been shown in previous studies to be increased by high doses of aldosterone, but not by deoxycorticosterone; in six of 11 experiments corticosterone, the physiological glucocorticoid in the rat, elevated alpha-lactalbumin; in five other studies corticosterone had no effect. In the present studies the mineralocorticoid antagonist, spirolactone, at very high doses (3-10 mumol/l) blocked the stimulatory effect on alpha-lactalbumin levels of both 30 nmol/l corticosterone and 3 nmol/l RU 26988, a pure synthetic glucocorticoid (Type II) receptor agonist. Receptor studies, however, indicated that this antagonism is consistent with Type II, glucocorticoid receptor occupancy by spirolactone. Since deoxycorticosterone is without agonist effect, and only very high doses of spirolactone affect alpha-lactalbumin synthesis, we conclude that the effect of adrenal steroids on alpha-lactalbumin production is a manifestation of glucocorticoid and not mineralocorticoid activity.     

Chorionic villus sampling as a source of trophoblasts

Unlike trophoblasts obtained from pregnancy termination material, trophoblasts grown from explanted chorionic villus samples (CVS) from 11-14 weeks of gestation potentially enable investigation of pre-eclampsia and other pregnancy disorders as the pregnancy outcome will later be known. CVS surplus to diagnostic needs were cultured as explants on either Matrigel or gelatin and the outgrowing cells characterised. Cell morphology was examined and the cells were stained for cytokeratin-7 and HLA-G. Outgrowing trophoblasts co-stained strongly for HLA-G and cytokeratin-7. While outgrowths on Matrigel grew faster and were 100% positive for cytokeratin-7, they proved to be embedded in the matrix and difficult to passage. Outgrowths on gelatin could be released by trypsinisation and were subcultured and further characterised before and after freezing. These cells should prove a valuable resource for the examination of disorders of pregnancy.     

Evidence for neuron-survival and neurite-promoting factors from skeletal muscle: Their effects on embryonic spinal cord

Explants of 8- and 9-day embryonic chick spinal cord were cultured for 4 days in medium which had been conditioned by skeletal muscle cells. When the conditioned medium was preincubated over polylysine substrata, it lost the ability to induce extensive neuritic outgrowth from the spinal cord. This ability was restored when the spinal cord was exposed to preincubated conditioned medium on preincubated polylysine substrata. Skeletal muscle thus appears to contain two separable components; one is a soluble factor which supports neuronal survival, and the other is an adsorbable factor which binds to appropriate substrata and promotes neuritic outgrowth.     

Infection of cultivated CNS tissue with herpes virus,HSVII a reappraisal

Summary Organized cultures of newborn rat and hamster cerebellum were infected with herpes virus type II, after 7 and 14 days in vitro. 48 h after the infection, electron microscopic examination of the cultures showed that astrocytes contained numerous intranuclear and intracytoplasmic viral particles, while neurons remained apparently intact. The specificity of the infection for a given cell type is discussed.     

Milk Thistle Extract and Silymarin Inhibit Lipopolysaccharide Induced Lamellar Separation of Hoof Explants in Vitro

The pathogenesis of laminitis is not completely identified and the role of endotoxins (lipopolysaccharides, LPS) in this process remains unclear. Phytogenic substances, like milk thistle (MT) and silymarin, are known for their anti-inflammatory and antioxidant properties and might therefore have the potential to counteract endotoxin induced effects on the hoof lamellar tissue. The aim of our study was to investigate the influence of endotoxins on lamellar tissue integrity and to test if MT and silymarin are capable of inhibiting LPS-induced effects in an in vitro/ex vivo model. In preliminary tests, LPS neutralization efficiency of these phytogenics was determined in an in vitro neutralization assay. Furthermore, tissue explants gained from hooves of slaughter horses were tested for lamellar separation after incubation with different concentrations of LPS. By combined incubation of explants with LPS and either Polymyxin B (PMB; positive control), MT or silymarin, the influence of these substances on LPS-induced effects was assessed. In the in vitro neutralization assay, MT and silymarin reduced LPS concentrations by 64% and 75%, respectively, in comparison PMB reduced 98% of the LPS concentration. In hoof explants, LPS led to a concentration dependent separation. Accordantly, separation force was significantly decreased by 10 µg/mL LPS. PMB, MT and silymarin could significantly improve tissue integrity of explants incubated with 10 µg/mL LPS. This study showed that LPS had a negative influence on the structure of hoof explants in vitro. MT and silymarin reduced endotoxin activity and inhibited LPS-induced effects on the lamellar tissue. Hence, MT and silymarin might be used to support the prevention of laminitis and should be further evaluated for this application.     

Comparison of In-Vivo Performance Characteristics of First-Generation and Second-Generation Cross-Linked and Conventional Explants

BackgroundImplants made of ultra high molecular weight polyethylene (UHMWPE) has been used for almost 60 years in hip joint arthroplasty as articulating surface. UHMWPE implants have evolved over time from conventional to cross-linked implants. Chemical, morphologic, and micromechanical characteristics play important roles in overall in vivo performance.MethodsThis study aimed at comparing chemical, morphologic, and micromechanical in vivo performance characteristics of conventional and cross-linked explants. Optical damage scoring, Fourier transform infrared spectroscopy, differential scanning calorimetry, and depth sensing indentation techniques were used for the measurements. The measurement results were used to correlate oxidation index, degree of crystallinity, E-Modulus, and hardness.ResultsDifferent manufacturing processes directly affect implants' in vivo performance. Conventional explants are more susceptible to oxidation in the absence of thermal treatment and air ambient gamma irradiation sterilization, whereas they have higher degree of crystallinity and E-Modulus values. Introduction of a thermal treatment step to first-generation cross-linked explants has decreased the oxidation susceptibility but degraded the crystalline structure however, such explants manufactured with moderate total cross-linking irradiation dose and a combination of both remelting and annealing thermal treatment methods are the exceptions. The second-generation cross-linked explants in general have much better E-Modulus, that is, hardness values over all other types of explants and are least susceptible to oxidation.ConclusionThe results suggest that in vivo performances of explants could benefit from the hybrid UHMWPE implant manufacturing techniques, such as moderate cross-linking irradiation doses, remelting, followed by annealing and ethylene oxide or gas plasma sterilization.     

Effects of vitamin D-induced supernatant of placental explants from preeclamptic women on oxidative stress and nitric oxide bioavailability in human umbilical vein endothelial cells

The study evaluated the effect of the supernatant of placental explants from preeclamptic (PE) and normotensive (NT) pregnant women after tissue treatment with or without vitamin D (VD) on oxidative stress and nitric oxide (NO) bioavailability in human umbilical vein endothelial cells (HUVEC). Placental explants were prepared from eight NT and eight PE women, and supernatants were obtained after incubation with or without hydrogen peroxide (H₂O₂) and/or VD. HUVEC were cultured for 24 h with supernatants, and the following parameters were analyzed in HUVEC cultures: NO, nitrate (NO₃ ^-), and nitrite (NO₂ ^-) levels, lipid peroxidation, and intracellular reactive oxygen species (ROS). Results showed that the production of NO₃ ^-, NO₂ ^-, malondialdehyde (MDA), and ROS were significantly higher in HUVEC treated with explant supernatant from PE compared to NT pregnant women, while the supernatant of PE explants treated with VD led to a decrease in these parameters. A significantly high production of NO was detected in HUVEC cultured with control supernatant of NT group, and in cultures treated with supernatant of PE explants treated with VD. Taken together, these results demonstrated that cultures of placental explants from PE women with VD treatment generated a supernatant that decreased oxidative stress and increased the bioavailability of NO in endothelial cells.     

Effect of medium composition and light on root and rhinacanthin formation in Rhinacanthus nasutus cultures

Rhinacanthus nasutus (L.) Kurz (Acanthaceae) has long been used in Thai traditional medicine for treatment of tinea versicolor, ringworm, pruritic rash, and abscess. The active constituents are known as a group of naphthoquinone esters, rhinacanthins. This work focused on establishment of R. nasutus root cultures and determination of rhinacanthin production. Induction of R. nasutus root formation was accomplished on solid Gamborg’s B5 (B5) medium, supplied with 0.1?mg/L indole-3-butyric acid (IBA) and 20?g/L sucrose. The effects of explants (whole leaf explants and four-side excised leaf explants), light and medium composition on root and rhinacanthin formation were investigated. The root formation from the whole leaf explants was 10 times higher than that from the four-side excised leaf explants. In addition, light possessed an inhibitory effect on the root and rhinacanthin formation of R. nasutus. Medium manipulation found that Murashige and Skoog (MS) medium supplied with 3?mg/L IBA and 30?g/L sucrose was the most suitable for induction of the root formation. Unfortunately, the obtained root cultures produced only rhinacanthin-C in very low amount, 0.026?mg/g dry weight (DW), when they were transferred into the same MS liquid medium. With semisolid medium (4?g/L agar) of the same MS composition, however, the root cultures appeared to produce higher content of rhinacanthin-C, -D and -N (3.45, 0.07 and 0.07?mg/g DW, respectively). Our finding suggests that culturing in semisolid medium is capable of improving of rhinacanthin production in R. nasutus root cultures.