Optimization of 3-D hepatocyte culture by controlling the physical and chemical properties of the extra-cellular matrices

Hepatocytes are anchorage-dependent cells sensitive to microenvironment; the control of the physicochemical properties of the extra-cellular matrices may be useful to the maintenance of hepatocyte functions in vitro for various applications. In a microcapsule-based 3-D hepatocyte culture microenvironment, we could control the physical properties of the collagen nano-fibres by fine-tuning the complex-coacervation reaction between methylated collagen and terpolymer of hydroxylethyl methacrylate-methyl methacrylate-methylacrylic acid. The physical properties of the nano-fibres were quantitatively characterized using back-scattering confocal microscopy to help optimize the physical support for hepatocyte functions. We further enhanced the chemical properties of the collagen nano-fibres by incorporating galactose onto collagen, which can specifically interact with the asialoglycoprotein receptor on hepatocytes. By correlating a range of collagen nano-fibres of different physicochemical properties with hepatocyte functions, we have identified a specific combination of methylated and galactosylated collagen nano-fibres optimal for maintaining hepatocyte functions in vitro. A model of how the physical and chemical supports interplay to maintain hepatocyte functions is discussed.     

H2O2 enhances Ca2+ release from osteoblast internal stores

The physiological activity of osteoblasts is known to be closely related to increased intracellular Ca2+ activity ([Ca2+]i) in osteoblasts. The cellular regulation of [Ca2+]i in osteoblasts is mediated by Ca2+ movements associated with Ca2+ release from intracellular Ca2+ stores, and transmembrane Ca2+ influx via Na+-Ca2+ exchanger, and Ca2+ ATPase. Reactive oxygen species, such as H2O2, play an important role in the regulation of cellular functions, and act as signaling molecules or toxins in cells. In this study, we investigated the effects of H2O2 on cellular Ca2+ regulation in osteoblasts by measuring intracellular Ca2+ activities using cellular calcium imaging techniques. Osteoblasts were isolated from the femurs and tibias of neonatal rats, and cultured for 7 days. The cultured osteoblasts were loaded with a Ca2+-sensitive fluorescent dye, Fura-2, and fluorescence images were monitored using a cooled CCD camera, and subsequently analyzed using image analyzing software. The results obtained are as follows: (1) The osteoblasts with lower basal Ca2+ activities yielded a transient Ca2+ increase, a Ca2+ spike, while osteoblasts with higher basal Ca2+ activities showed a continuous increase in [Ca2+]i leading to cell death. (2) Ca2+ spikes, generated after removing Na+ from superfusing solutions, were blocked by H2O2 and this was followed by a sustained increase in Ca2+ activity. (3) ATP- induced Ca2+ spikes were inhibited by pretreating with H2O2 and this was followed by a continuous increase of [Ca2+]i. When cells were pretreated with the exogenous nitric oxide (NO) donor S-Nitroso-N-acetylpenicilance (SNAP, 50 microM), treatments of ATP (1 mM) induced a Ca2+ spike-like increase, but [Ca2+]i did not return to the basal level. (4) The expression of inositol- 1,4,5-triphosphate receptor (IP3R) was enhanced by H2O2. Our results suggest that H2O2 modulates intracellular Ca2+ activity in osteoblasts by increasing Ca2+ release from the intracellular Ca2+ stores.     

Comparative in vitro activity of the new erythromycin derivative dirithromycin against gram-positive bacteria isolated from cancer patients

The in vitro activity of dirithromycin (LY-237216), a new macrolide erythromycin derivative, was compared to that of four other agents (clarithromycin, erythromycin, roxithromycin, clindamycin) against 334 gram-positive isolates obtained from cancer patients. Dirithromycin was similar in potency and antimicrobial spectrum to the other agents tested. It was very active against beta-haemolytic streptococci andStreptococcus pneumoniae, and moderately active against penicillin and methicillin susceptibleStaphylococcus aureus, Bacillus spp.,Listeria monocytogenes andCorynebacterium jeikeium. Erythromycin resistant organisms were also resistant to dirithromycin.     

A randomized, double blind, placebo-controlled study to investigate the use of conscious sedation in conjunction with paracervical block for reducing pain in termination of first trimester pregnancy by suction evacuation

BACKGROUND: This study evaluated the role of conscious sedation in pain relief during termination of first trimester pregnancy by suction evacuation (SE) under local anaesthesia. METHODS: A hundred women undergoing SE before 12 weeks gestation were randomized by computer using the sealed envelope method to receive placebo (saline) or conscious sedation (2 mg midazolam and 25 microg fentanyl) i.v. 5 min before cervical dilatation. Paracervical block was given to all patients, 2 min later. Pain scores during and after SE, post-operative side-effects and satisfaction level were compared. RESULTS: No statistically significant differences in pain scores were found between the two groups. Post-operative side-effects such as dizziness (P = 0.015) and drowsiness (P < 0.001) were significantly more severe in the conscious sedation group. However, patients in the conscious sedation group reported better satisfaction levels than the control group (P = 0.003). CONCLUSION: The use of conscious sedation significantly improved patient satisfaction during termination of first trimester pregnancy by SE under local anaesthesia, despite a lack of improvement in pain relief and the presence of increased severe dizziness/drowsiness in the post-operative period.     

Acidic pH and increasing [Ca(2+)] reduce the swelling of mucins in primary cultures of human cervical cells

BACKGROUND: Cervical mucus is a heterogeneous mixture of water, ions and mucins that form a hydrophilic polymer gel. Mucins, the main components of mucus, are condensed inside secretory granules and swell to become a hydrogel after exocytosis. Using human cervical secretory cell primary cultures, the effect of [Ca(2+)] and [H(+)] on the swelling velocity of mucin granules was investigated in vitro. METHODS and RESULTS: Immunocytochemistry demonstrated that estrogen and progesterone receptors were expressed in cultured secretory cells along with mucins type 1, 4, 5AC and 5B. Exocytosis of secretory cells, recorded by videomicroscopy, showed that during swelling, the radius of the secretory granule matrix followed first-order kinetics. An increase in extracellular [Ca(2+)] from 1 to 4 mmol/l or a reduction in pH from 7.4 to 6.5 was seen to produce a significant decrease in the velocity of swelling of the secretory granule matrix. CONCLUSIONS: The inverse relationship observed between the diffusion of the granular matrix and the extracellular [Ca(2+)] or [H(+)] suggested that changes in cation concentration might drastically affect the swelling characteristics of mucins and provide a control mechanism for the observed viscoelastic properties of mucus.     

Concentrations of endometrial protein PP 14 and CA-125 in uterine flushings performed in natural and stimulated cycles

BACKGROUND: Impaired implantation in assisted reproduction cycles with high serum estradiol (E(2)) concentrations may be attributed to abnormal endometrial development. This study compared concentrations of endometrial proteins in uterine flushings of infertile patients between natural and stimulated cycles. METHODS: Patients received a standard regimen of ovarian stimulation. Seven days after the LH surge in natural cycles or the hCG injection in stimulated cycles, uterine flushings were performed by slowly injecting and aspirating normal saline through a paediatric Foley catheter. Natural cycles were considered as group A whereas stimulated cycles with serum E(2) <20 000 pmol/l and serum E(2) >20 000 pmol/l were classified as groups B and C respectively. PP 14 and CA-125 in uterine flushings were measured and expressed per total protein content. RESULTS: Concentrations of the total protein, PP 14 and CA-125 in the uterine flushings were similar among the three groups. PP 14 per total protein in the uterine flushings was significantly correlated with serum E(2) on the day of hCG (r = 0.459; P = 0.009) in natural cycles only but not in stimulated cycles. CONCLUSION: There was no significant difference between natural and stimulated cycles in concentrations of PP 14 and CA-125 in uterine flushings performed in the mid-luteal phase.     

Cell adhesion-related proteins as specific markers of sponge cell types involved in allogeneic recognition

Sponge immunocyte identification is of interest to comparative immunologists since characterizing these cells will allow investigations into the mechanisms of non-self recognition in the oldest animal phylum. Here, we report that polyclonal antibodies raised against the core protein of a proteoglycan involved in cell adhesion in the marine sponge Microciona prolifera are specific markers for archaeocytes, the totipotent sponge cells. Archaeocytes are mobilized upon allogeneic contact and they accumulate in the contact zone. A second type of cell, the gray cells, are specifically recognized by monoclonal antibodies raised against CD44, a hyaluronan receptor. Gray cells do also accumulate in the contact area. Specific staining of a third sponge cell type, the rhabdiferous cells, shows that these do not accumulate upon allografting. These specific cell markers allow tracking of archaeocytes and gray cells, and show that they play an active role in sponge allogeneic reactions.     

Determination of Enterococcus faecalis groESL full-length sequence and application for species identification

Amplification of the partial Cpn60 (or GroEL) gene segment has been used for identification of many bacteria, including Enterococcus species. To obtain more sequence data from groESL genes of Enterococcus faecalis, the full-length sequence of the E. faecalis groESL genes containing groES (285 bp), spacer (57 bp), and groEL (1,626 bp) was determined. A database search of GenBank revealed that the deduced E. faecalis GroES and GroEL proteins show significant homology to the GroES and GroEL proteins of other bacteria. The GroEL (groEL) of E. faecalis had the highest identity with Streptococcus pneumoniae (81.8% amino acid sequence identity and 73.0% nucleotide sequence identity), followed by Lactococcus zeae, while GroES (groES) had 60.2% (64.6%) identity with Lactobacillus zeae and 58.5% (66.2%) identity with Lactococcus lactis, followed by 57.0% (65.5%) identity with Bacillus subtilis. Based on the groES sequence, an E. faecalis-specific PCR assay was developed, and this PCR assay was positive for all the E. faecalis strains tested. Dot blot hybridization using either groES or groEL as the probe distinguished E. faecalis clearly from other species, indicating that both genes can be used as suitable targets for E. faecalis identification. Moreover, broad-range PCR-restriction fragment length polymorphism of groESL was designed to differentiate eight commonly encountered Enterococcus species. The Enterococcus species of reference strains could be easily differentiated on the basis of restriction patterns produced by HaeIII and RsaI. The DNA-based assays developed in this study provide an alternative to currently used methods of identification for clinically important enterococcal species.     

Epidemic typhoid in vietnam: molecular typing of multiple-antibiotic-resistant Salmonella enterica serotype typhi from four outbreaks

Multidrug-resistant Salmonella enterica serotype Typhi isolates from four outbreaks of typhoid fever in southern Vietnam between 1993 and 1997 were compared. Pulsed-field gel electrophoresis, bacteriophage and plasmid typing, and antibiotic susceptibilities showed that independent outbreaks of multidrug-resistant typhoid fever in southern Vietnam are caused by single bacterial strains. However, different outbreaks do not derive from the clonal expansion of a single multidrug-resistant serotype Typhi strain.