Ex vivo expansion and clonality of CD34+ selected cells from bone marrow and cord blood in a serum-free media

Although umbilical cord blood is increasingly being used in allogeneic marrow transplantation, delayed platelet engraftment is often a concern for cord blood transplant recipients. We evaluated the potential of ex vivo expansion and clonality in CD34+ cells separated from a bone marrow source, and cord blood, in a serum-free Media. The CD34+ cells, selected from bone marrow (BM) and umbilical cord blood (CB), were expanded with hematopoietic growth factors. They were then cultured for burst-forming units of erythrocytes (BFU-E), colony-forming units of granulocytes and monocytes (CFU-GM) and colony-forming units of megakaryocytes (CFU-Mk) at days 0, 4, 7, and 14 under the combination of growth factors, with cell counts. The cytokines included the recombinant human megakaryocyte growth and development (100 ng/ml), interleukin-3 (10 ng/ml), stem cell factor (100 ng/ml), flt-3 ligand (50 ng/ml) and interleukin-11 (200 ng/ml). The CB-selected CD34+ cells showed significantly higher total cell expansion than those from the BM at day 7 (3.0 fold increase than BM), day 14 (2.4 fold), and day 17 (2.6 fold). The colony count of the BFU-E/CFU-E per CD34+ cell at day 0 was 0.14 +/- 0.023 in the CB, which was significantly higher than 0.071 +/- 0.015 in the BM. The CB-selected CD34+ cells produced more BFU-E colonies than the BM on culture days 4, 7, and 14. The BFU-E colonies from the CB cells increased markedly on culture days 4 and 7, with a 4-fold increase at day 14. The colony count of the CFU-Mk per CD34+ cell at day 0 was 0.047 +/- 0.011 in the CB-selected CD34+ cells cultures, which was higher than the 0.026 +/- 0.014 in the BM. The CB-selected CD34+ cells produced more CFU-Mk colonies than the BM on culture days 4, 7 and 14. In conclusion, the ex vivo expansion of the CB cells may be very promising in producing total cellular expansion, CFU-Mk and BFU-E compared with BM, especially at day 7. The ex vivo expansion of the CB may have rationale in making an ex vivo culture for 7 to 14 d.     

Influence of plating density on individual cell growth, cell division and differentiation of neonatal rat heart primary cultures

The influence of plating cell density of an originally enriched myocardial cell population has been studied in neonatal rat heart cells in culture. Low density (LDM) is defined as a density (24 h after plating) of 209 +/- 44 cells/mm2 (mean +/- SEM) and is compared with high density (HDM), 419 +/- 67 cells/mm2. Cell growth is evaluated by the total cell number, the percentage of myocardial cells (M) in culture (PAS method) and the protein content per cell. Some differentiation parameters such as beating rates, glycogen concentration, enzymatic activities (cytochrome C oxidase and glycogen phosphorylase) are studied with time in culture (48, 96 and 192 hr). High density was designed to yield a complete confluency of the cells within 24 hr after plating and to minimize cell division of the non-muscle cells (F). At high density, cell division of F cells is effectively limited, thus leading to a more stable model regarding the cell density per plate and the percentage of M cells: 85.7 +/- 4% and 33.4 +/- 6% in LDM cultures compared with 86.5 +/- 4.7% and 51.7 +/- 9.8% in HDM cultures at 24 and 192 hr (mean +/- SEM). Heart cells increase similarly in size with age in culture in both groups. In HDM cultures the spontaneous contractions begin sooner (24 hr) than in LDM cultures and are more rapidly synchronized. The beating rate is higher in HDM cultures between 48 and 96 hr; however, after this time it falls in HDM and does not fall in LDM. Thus the overgrowth of muscle cells by non-muscle cells is not responsible for loss of beating with time in culture but more likely high density could be a limiting factor for isotonic contraction. There is more glycogen per myocyte in LDM than in HDM cultures. The cell density influences the enzymatic activities of cytochrome C oxidase and glycogen phosphorylase. The cytochrome oxidase activity is higher in HDM cultures than in LDM cultures at 96 hr whereas glycogen phosphorylase activity is higher in LDM cultures at time 96 and 192 hr. In LDM cultures, the ratio cytochrome C oxidase/glycogen phosphorylase decreases with time in culture from 1.685 +/- 0.680 at 48 hr to 0.780 +/- 0.290 at 192 hr but not in HDM cultures (2.13 +/- 0.36 and 1.64 +/- 0.34 respectively). Thus plating density influences properties of heart cell cultures with regard to the overgrowth of the F-cell population and the differentiated state of M cells.     

Protein content in rabbit periodontal ligament fibroblasts during growth in culture

The protein content of an individual periodontal ligament fibroblast decreases with increasing cell density during growth in culture. A mean total protein concentration of 3.3 +/- 0.4 micrograms per 10(3) cells was calculated. This reference value can be used in studies evaluating enzyme activities, transport rates or metabolic functions.     

Regulation of prostaglandin biosynthesis in dispersed choriodecidual cells in culture

We have evaluated the prostaglandin (PG) production and PG biosynthetic gene expression in a choriodecidual dispersed cell culture system. Cells dispersed from human choriodecidual membranes by dispase and trypsin digestion were evaluated after 1,3,5 and 7 days of culture for basal and tumour necrosis factor alpha (F-alpha) stimulated PGE2 production. The highest rates of production (P < 0.05) were obtained with cells treated after 3 days of culture, (3.7 +/- 1) x 10(2) pg PGE2 per 16 h per microg total cellular protein (mean +/- SEM), which was 3.9 times basal rate after 3 days culture. In choriodecidual cells treated after 3 days in culture, expression of prostaglandin endoperoxide H synthase-2 (PGHS-2) mRNAwas similarly responsive toTNF-alpha (3.9 times basal within 3 h of 30 ng/ml TNF-alpha) while there was little effect on PGHS-1 or cytosolic phospholipase A2 expression. Hence, the dispersed choriodecidual cell culture system described retainsTNF-alpha responsive PG biosynthetic capacity which is at least in part upregulated via increased expression of PGHS-2 mRNA.     

Lipid metabolism in cultured cells. XVI. Lipoprotein binding and HMG CoA reductase levels in normal and tumor virus-transformed human fibroblasts.

The loss in feedback control of cholesterol biosynthesis in tumor cells was examined in tissue culture. Human fibroblasts from normal subjects, SV40 tumor virus-transformed cell lines, and homozygous familial hypercholesterolemic cells as reference, were grown in tissue culture. Experiments were conducted to relate the regulatory enzyme for cholesterol biosynthesis, HMG CoA reductase, and the membrane-located binding receptors for low density lipoproteins (LDL) that mediate feedback control in normal cells. Monolayers of virus-transformed tumor cells exhibited specific (125)I-labeled LDL binding of 152 +/- 21 ng/mg cell protein, which was essentially the same as that of normal fibroblasts (135 +/- 20 ng/mg). Binding of LDL by familial hypercholesterolemic cells used as controls was only 8 +/- 3 ng/mg under the same test conditions. Basal levels of HMG CoA reductase in tumor cells of 45.2 +/- 6.5 units/mg cell protein were about twice those of normal cells. However, in contrast to the lack of feedback control of this enzyme observed with tumors in vivo, in both the normal and the transformed cells in vitro, activity of the enzyme decreased about fourfold when serum lipids were added. These findings demonstrate that tumor cells growing in vitro contain a normal complement of the membrane-located binding receptors for low density lipoproteins and, although the basal levels are higher than normal, an effective feedback regulation of the enzyme HMG CoA reductase is retained.     

Differential regulation of pig theca cell steroidogenesis by LH, insulin-like growth factor I and granulosa cells in serum-free culture

The regulation of pig theca cell steroidogenesis was studied by the development of a physiological serum-free culture system, which was subsequently extended to investigate potential theca-granulosa cell interactions. Theca cells were isolated from antral follicles 6-9 mm in diameter and the effects of plating density (50-150x10(3) viable cells per well), LH (0.01-1.0 ng ml(-1)), Long R3 insulin-like growth factor I (IGF-I) (10, 100 ng ml(-1)) and insulin (1, 10 ng ml(-1)) on the number of cells and steroidogenesis were examined. The purity of the theca cell preparation was verified biochemically and histologically. Co-cultures contained 50x10(3) viable cells per well in granulosa to theca cell ratio of 4:1. Wells containing granulosa cells only were supplemented with 'physiological' doses of androstenedione or 100 ng ml(-1). Oestradiol production by co-cultures was compared with the sum of the oestradiol synthesized by granulosa and theca cells cultured separately. Oestradiol and androstenedione production continued throughout culture. High plating density decreased steroid production (P < 0.01). LH increased androstenedione (P < 0.001) and oestradiol (P < 0.05) synthesis and the sensitivity of the cells increased with time in culture. Oestradiol production was increased by 10 ng IGF-I ml(-1) (P < 0.001) but androstenedione required 100 ng ml(-1) (P < 0.001). Co-cultures produced more oestradiol than the sum of oestradiol synthesized by theca and granulosa cells cultured separately (P < 0. 001), irrespective of the androstenedione dose. This serum-free culture system for pig theca cells maintained in vivo steroidogenesis and gonadotrophin responsiveness. Thecal androstenedione and oestradiol production were differentially regulated and were primarily stimulated by LH and IGF-I, respectively. Theca-granulosa cell interactions stimulated oestradiol synthesis and this interaction was mediated by factors additional to the provision of thecal androgen substrate to granulosa cells.     

Kinase pathways in dominant and subordinate ovarian follicles during the first wave of follicular development in sheep

The mechanism by which one or more dominant ovarian follicles continue development while other subordinate follicles regress is not known. The mitogen activated protein kinases (MAPKs) are a group of kinases that are activated by hormonal factors and form a cascade of processes that regulate cell growth, division and differentiation. The aim of the present experiment was to characterise the presence of the MAPKs, Erk 1/Erk 2 and Akt in healthy dominant follicles and regressing subordinate follicles. Following in vivo monitoring of ovarian follicle development, three ewes were ovariectomised and the follicular fluid and follicle wall (theca and granulosa cells) saved from the dominant and largest subordinate follicle. The dissected diameter and follicular fluid oestradiol concentration of the dominant follicle was larger (P<0.01) than the largest subordinate follicle (6.5+/-0.0mm and 41.3+/-4.9ng/ml versus 4.7+/-0.3mm and 0.6+/-0.4ng/ml). Western blot analyses showed that there was more Akt (202.7+/-6.4 versus 59.6+/-32.7 units; P<0.05) and Erk 1/Erk 2 (104.5+/-10.6 versus 0.3+/-0.2 units; P<0.01) present in follicle wall samples from the dominant compared to the largest subordinate follicles. Phosphorylated forms of Akt and Erk 1/Erk 2 were detected in samples from dominant but not subordinate follicles. We suggest that signal transduction pathways involving Akt and Erk 1/Erk 2 may play an important role in determining the outcome of ovarian follicle growth and development in sheep.     

The effect of hypoxia on uptake and degradation of low density lipoproteins by cultured human arterial smooth muscle cells.

Atheroma have been produced in experimental animal by systemic hypoxia. This study assessed the effects of hypoxia on binding, uptake and degradation of human low density lipoprotein (LDL) by human arterial smooth muscle cells, the cell involved in atherogenesis. The LDL content of the smooth muscle cell grown in the usual conditions (95% air [20% O2], 5% CO2) increased with the incubation time of LDL in the medium (7.5 mug protein/ml of medium); the trypsin releasable LDL "binding" reached a plateau by 24 h (2.2 +/- 1.3 [x +/- S.D.]) ng/mug LDL protein added per 10(6) cells whereas the LDL in the cell after trypsinization ("net uptake") continued to increase up to 48 h (6.5 +/- 4.6 ng/mug LDL protein added per 10(6) cells at 48 h). LDL protein degradation increases rapidly between 7 and 48 h (10.4 ng/mug LDL protein added per 10(6) cells at 24 h) after an initial delay of approximately 7 h. Smooth muscle cells grown under hypoxic conditions (5%02) had similar LDL "binding " but showed increased "net uptake" (10.7 +/- 4.8 ng/mug LDL protein added per 10(6) cells) and a 36 +/- 13% decrease in degradation (p less than 0.05; n =8). The impaired degradation of lipoprotein by smooth muscle cells may, in part, explain the role of hypoxia in atherogenesis.     

Replication of the broad-host-range plasmid RK2: direct measurement of intracellular concentrations of the essential TrfA replication proteins and their effect on plasmid copy number.

The trfA gene of the broad-host-range plasmid RK2 is essential for initiation of plasmid replication. Two related TrfA proteins of 43 and 32 kilodaltons (kDa) are produced by independent translation initiation at two start codons within the trfA open reading frame. These proteins were o overproduced in Escherichia coli and partially purified. Rabbit antisera raised against the 32-kDa TrfA protein (TrfA-32) and cross-reacting with the 43-kDa protein (TrfA-43) were used in Western blotting (immunoblotting) assays to measure intracellular TrfA levels. In logarithmically growing E. coli HB101, RK2 produced 4.6 +/- 0.6 ng of TrfA-32 and 1.8 +/- 0.2 ng of TrfA-43 per unit of optical density at 600 nm (mean +/- standard deviation). On the basis of determinations of the number of cells per unit of optical density at 600 nm, this corresponds to about 220 molecules of TrfA-32 and 80 molecules of TrfA-43 per cell. Dot blot hybridizations showed that plasmid RK2 is present in about 15 copies per E. coli cell under these conditions. Using plasmid constructs that produce different levels of TrfA proteins, the effect of excess TrfA on RK2 replication was tested. A two- to threefold excess of total TrfA increased the copy number of RK2 by about 30%. Additional increases in TrfA protein concentration had no further effect on copy number, even at levels 170-fold above normal. An RK2 minimal origin plasmid showed a similar response to intracellular TrfA concentration. These results demonstrate that TrfA protein concentration is not strictly rate limiting for RK2 replication and that a mechanism that is independent of TrfA concentration functions to limit RK2 copy number in the presence of excess TrfA.     

Permeability of epidural somatostatin and morphine into the intrathecal space of dogs

In an in vivo saline perfusate of the intrathecal space of 6 dogs, the concentration of somatostatin was determined by radioimmunoassay before and over 2 h after epidural administration of 3 mg somatostatin. The total recovered amount of somatostatin was negligible, about 0.02%. However, within 50 min after the bolus epidural injection of somatostatin, the concentration per ml perfusate increased from 0.1 +/- 0.02 ng/ml to 138 +/- 102 ng/ml (P less than 0.001) and declined to 4 +/- 1.7 ng/ml after 120 min. This increase of the somatostatin concentration by 3 orders of magnitude might explain why epidurally administered somatostatin is effective in treatment of acute and chronic pain. In a control investigation with epidural morphine in another 6 dogs to prove the feasibility of the method, the total recovered amount of morphine in the intrathecal perfusate over 2 h was about 12%.     

Distribution of chymase-containing mast cells in human bronchi.

Mast cell chymase stimulates secretion from cultured airway gland serous cells and hydrolyzes bronchoactive peptides in vitro. To explore the likelihood of these interactions occurring in situ, we examined the distribution and concentration of chymase-containing mast cells near glands and smooth muscle of major human bronchi from eight individuals without known airway disease. Total airway mast cells and the subset of mast cells containing chymase were detected by staining for methylene blue metachromasia and chloroacetate esterase activity, respectively. The percentage of chymase-containing mast cells was found to differ strikingly among bronchial tissue compartments. Near glands, for example, the concentration of chymase-positive mast cells (640 +/- 120 cells/mm3) was 73 +/- 9% that of total mast cells (910 +/- 130 cells/mm3), whereas in smooth muscle the concentration of chymase-positive mast cells (450 +/- 200 cells/mm3) was only 14 +/- 4% that of total mast cells (2920 +/- 620 cells/mm3). Of all chymase-containing mast cells in the airway subepithelium, 30 +/- 4% were located within 20 microns of submucosal glands. Although the percentage of chymase-containing cells varied, the absolute concentration of chymase-containing mast cells was similar in all compartments. These results reveal a differential distribution of mast cell subpopulations in human airway and suggest that mast cells containing chymase are near gland and smooth muscle targets.     

Disruption of seminiferous epithelial function in the rat by ovarian protein

The granulosa cell produces an inhibitor of aromatase activity, which recently was purified to homogeneity (follicle-regulatory protein: FRP). Since extracts of testicular homogenates also contain factor(s) with biological properties similar to FRP, including inhibition of granulosa cell aromatase, we examined the effects of ovarian FRP on testicular function. Forty-five-day-old rats received daily FRP injections (100 micrograms or 300 micrograms). After 15, 30, 45, and 70 days of therapy, (n = 5 each group), trunk serum was measured for testosterone, androstenedione, estradiol, and FSH levels by established radioimmunoassays (RIA). One testis from each rat was homogenized, centrifuged, and evaluated for sperm head counts; the other testis was dissected by transillumination, and the length of seminiferous epithelial stages determined. After 15 (control: 4.8 +/- 0.2 mm; 100 micrograms: 6.0 +/- 0.3 mm; 300 micrograms: 6.6 +/- 0.3 mm) and 30 days (control: 4.6 +/- 0.2 mm; 100 micrograms: 6.3 +/- 0.2 mm; 300 micrograms: 5.9 +/- 0.2 mm) of treatment the length of the "strong" seminiferous tubule segment in FRP-treated rats was greater than in control rats (p less than 0.05). Serum levels of steroids and FSH were similar in all groups. After 30, 45, and 70 days of treatment, the sperm head counts for the 100-micrograms and 300-micrograms dosages were 26%, 29%, 30% and 20%, 34%, and 24% of control values, respectively. By 70 days of treatment, cycle Stage VII was markedly reduced or absent in FRP-treated rats, and their round spermatids contained ring chromatin; both conditions indicate degeneration. FRP (50 micrograms/ml) was added to rat Sertoli cell cultures for 4 days after which transferrin and androgen-binding protein (ABP) were measured. FRP enhanced Sertoli cell secretion of ABP (58 vs. 138 +/- 7 microliters eq/culture) and transferrin (2.1 vs. 4.7 +/- 0.6 microgram/culture). In conclusion, systemic injection of FRP alters seminiferous epithelial function by reducing maturation of mature sperm forms. Adding FRP to Sertoli cells in culture enhances secretion of transferrin and ABP; this suggests that maturation of the germinal elements may be linked to the secretory function of seminiferous tubules.     

Co-culture of human lung-derived mast cells with mouse 3T3 fibroblasts: morphology and IgE-mediated release of histamine, prostaglandin D2, and leukotrienes

Human mast cells, dispersed from lung tissue by proteolytic treatment and enriched to a purity of 23 to 68% by density-gradient centrifugation, were maintained ex vivo for up to 13 days when co-cultured with mouse skin-derived 3T3 fibroblasts in RPMI 1640 containing 10% fetal calf serum. The human mast cells were adherent to the fibroblast cultures within 2 to 4 hr after seeding, and after 7 days of co-culture were localized between the layers of fibroblasts. The cell surfaces of the mast cells and the fibroblasts did not form tight junctions, but rather approached within 20 nm of each other. The co-cultured mast cells did not divide; they maintained their cellular content of histamine and TAMe esterase and resembled in vivo mast cells in that their secretory granules exhibited scroll patterns and their nuclei were oval. Both the freshly isolated and the co-cultured mast cells responded to activation with anti-human IgE by exocytosing histamine and generating and releasing arachidonic acid metabolites. When freshly isolated mast cells were activated immunologically, they exocytosed 38 +/- 8% of their total histamine content and released 28 +/- 1.9 ng (mean +/- range, n = 2) of immunoreactive equivalents of prostaglandin D2 (PGD2) per microgram of total cellular histamine, but did not generate significant amounts of either leukotriene C4 (LTC4) or leukotriene B4 (LTB4). The 1-wk co-cultured mast cells, on the other hand, exocytosed 43 +/- 2.4% of their total histamine content, and released 86 +/- 10, 43 +/- 20, and 5.2 +/- 5.2 ng (mean +/- SD, n = 4) of immunoreactive equivalents of PGD2, LTC4, and LTB4, respectively, per microgram of histamine. Thus, human lung-derived mast cells can be maintained ex vivo when co-cultured with fibroblasts, and, when treated with anti-IgE, they metabolize arachidonic acid via both the cyclooxygenase and the 5-lipoxygenase pathways.     

Nighttime immunoreactive somatostatin content of the median eminence in hypo- and hyperthyroid rats

The median eminence content of immunoreactive somatostatin (IRS) was measured by radioimmunoassay in 107 male albino rats, who were either hypothyroid after surgical thyroidectomy (N = 38), hyperthyroid following a subcutaneous implant of 5 mg of L-thyroxine (N = 36), or otherwise untreated (N = 33). Thyroid function was assessed by determining plasma levels of T4 and TSH from trunk blood obtained at the time of decapitation. Subgroups of animals from the 3 groups were killed either before (1800 hr), during (2200, 0200, 0400 hr), or after the dark portion of their 14:10 LD photoperiod. Although no changes in median eminence IRS content were found throughout the period of study within any of the 3 groups, hypothyroid animals (297.23 +/- 13.47 ng per ME; 620.41 +/- 58 ng IRS/mg protein) had a significantly lower median eminence IRS concentration than untreated rats (355.86 +/- 16.55 ng of IRS per ME, P less than 0.01; 906.86 +/- 96.38 ng IRS/mg protein, P less than 0.05) and hyperthyroid animals (384.12 +/- 14.67 ng per ME, P less than 0.001; 874.1 +/- 104.5 ng IRS@mg protein, P less than 0.05). It is concluded, that the feedback of thyroid hormones on the hypothalamic-pituitary axis during thyroid hormone excess in vivo, contrary to what occurs in hypothyroidism, is probably independent of hypothalamic somatostatin.     

Ex vivo expansion of hematopoietic stem cells derived from umbilical cord blood in rotating wall vessel

Expansion of umbilical cord blood mononuclear cells (UCB MNCs) was carried out in a rotating wall vessel (RWV) bioreactor and tissue culture flasks (T-flasks) in serum-containing medium supplemented with relatively low doses of purified recombinant human cytokines (5.33 ng/ml IL-3, 16 ng/ml SCF, 3.33 ng/ml G-CSF, 2.13 ng/ml GM-CSF, 7.47 ng/ml FL and 7.47 ng/ml TPO) for 8 days. The cell density, pH and osmolality of the culture medium in the two culture systems were measured every 24h. Flow cytometric assay for CD34+ cells was carried out at 0, 144 and 197 h and methylcellulose colony assays were performed at 0, 72, 144 and 197 h. The pH and osmolality of the medium in the two culture systems were maintained in the proper ranges for hematopoietic stem cells (HSCs) and progenitors culture. The RWV bioreactor, combined with a cell-dilution feeding protocol, was efficient to expand UCB MNCs. At the end of 200 h culture, the total cell number was multiplied by 435.5+/-87.6 times, and CD34+ cells 32.7+/-15.6 times, and colony-forming units of granulocyte-macrophage (CFU-GM) 21.7+/-4.9 times. While in T-flasks, however, total cells density changed mildly, CD34+ cells and CFU-GM decreased in number. It is demonstrated that the RWV bioreactor can provide a better environment for UCB MNCs expansion, enhance the contact between HSCs and accessory cells and make the utilization of cytokines more effective than T-flask.     

Lutropin-dependent protein phosphorylation and steroidogenesis in rat tumour Leydig cells

Tumour Leydig cells have been incubated in the presence or absence of lutropin (luteinizing hormone, ;LH'). Stimulation of cells with lutropin (1000ng/ml) in the presence of 1-methyl-3-isobutylxanthine (0.25mm) resulted in increased steroid production and increased protein phosphorylation. When pregnenolone metabolism was inhibited, basal pregnenolone production was 26.9+/-7.4ng/60min per 10(6) cells; stimulated production was 156.1+/-39.5ng/60min per 10(6) cells (means+/-s.d., n=4). Lutropin-dependent phosphorylated proteins of molecular mass 17000, 22000, 24000, 33000 and 57000Da were detected. A significant increase of [(32)P]P(i) incorporation into these phosphorylated proteins was observed concomitant with the increased pregnenolone production. The occurrence of the phosphoproteins in nuclei, mitochondria and postmitochondrial-supernatant was investigated. The 17000Da phosphoprotein was found in the nuclear fraction, whereas the 22000, 24000, 33000 and 57000Da phosphoproteins were localized in the postmitochondrial-supernatant fraction. Of the cholesterol-side-chain-cleavage activity, 80.3+/-6.1% (mean+/-s.d., n=5) was present in the mitochondrial fraction isolated from tumour Leydig cells, and this activity was 2.5-fold increased when cells had been preincubated with lutropin/1-methyl-3-isobutylxanthine (basal production: 194.6+/-28.6ng/30min per mg of protein; lutropinstimulated production: 498.8+/-91.5ng/30min per mg of protein; means+/-s.d., n=3). The similarities in the kinetics of the phosphorylation of proteins and the pregnenolone production after addition of lutropin/1-methyl-3-isobutylxanthine indicate that the phosphoproteins could be involved in the lutropin-dependent increase in steroidogenesis in tumour Leydig cells. It remains to be demonstrated, however, to what extent the phosphoproteins outside the mitochondria can influence the cholesterol-side-chain-cleavage activity inside the mitochondria.     

Shedding of soluble ICAM-1 into the alveolar space in murine models of acute lung injury

Intercellular adhesion molecule-1 (ICAM-1; CD54) is an adhesion molecule constitutively expressed in abundance on the cell surface of type I alveolar epithelial cells (AEC) in the normal lung and is a critical participant in pulmonary innate immunity. At many sites, ICAM-1 is shed from the cell surface as a soluble molecule (sICAM-1). Limited information is available regarding the presence, source, or significance of sICAM-1 in the alveolar lining fluid of normal or injured lungs. We found sICAM-1 in the bronchoalveolar lavage (BAL) fluid of normal mice (386 +/- 50 ng/ml). Additionally, sICAM-1 was spontaneously released by murine AEC in primary culture as type II cells spread and assumed characteristics of type I cells. Shedding of sICAM-1 increased significantly at later points in culture (5-7 days) compared with earlier time points (3-5 days). In contrast, treatment of AEC with inflammatory cytokines had limited effect on sICAM-1 shedding. BAL sICAM-1 was evaluated in in vivo models of acute lung injury. In hyperoxic lung injury, a reversible process with a major component of leak across the alveolar wall, BAL fluid sICAM-1 only increased in parallel with increased alveolar protein. However, in lung injury due to FITC, there were increased levels of sICAM-1 in BAL that were independent of changes in BAL total protein concentration. We speculate that after lung injury, changes in sICAM-1 in BAL fluid are associated with progressive injury and may be a reflection of type I cell differentiation during reepithelialization of the injured lung.     

Isolation of cloned Syrian hamster insulinoma cell lines with limited capacity for insulin production.

Cell lines derived from a Syrian hamster insulinoma have been cloned in agar and maintained continuously in culture in vitro for 1 1/2 years. The cell lines have average doubling times of 48 h, they have modal chromosome numbers between 38 and 39, and they retain the ability to form tumors when injected into Syrian hamsters. The cells do not produce immunoreactive insulin when grown in vitro (less than 0.5 ng/mg protein), but do produce immunoreactive insulin when grown in vivo as tumors (mean value from six determinations = 7.1 +/- 1.5 ng/mg protein).     

Production and secretion of retinol-binding protein by a human hepatoma cell line, HepG2

Retinol-binding protein (RBP) that is synthesized and secreted by the human hepatoma cell HepG2 has been measured using a sensitive radioimmunoassay in which RBP in media and hepatoma cell sonicates reacts identically to human serum RBP. RBP was synthesized and secreted when cells were grown in retinol-depleted as well as retinol-containing media. However, immunoreactive transthyretin (prealbumin) could not be detected in concentrated HepG2 medium. RBP secretion and accumulation per mg of cell protein could be modulated by the concentration of fetal calf serum in the growth medium: secreted RBP equaled 782 +/- 123 ng/mg of cell protein per 8 hr after preincubation with 10% fetal calf serum versus 555 +/- 86 ng/mg per 8 hr in the absence of serum, whereas RBP in cell sonicates decreased only slightly. When HepG2 cells were cultured for two or more passages in medium containing fetal calf serum depleted of retinol by ultraviolet irradiation, the amounts of RBP in the cells and released to the medium were both significantly increased. When vitamin A (90% as retinyl esters) in the form of chylomicron remnants was presented to cells, there was a significant, dose-dependent redistribution of RBP from cells to medium, both in cells grown in normal fetal calf serum and in retinol-depleted serum. These data indicate that the secretion of RBP by HepG2 can occur constitutively in the absence of retinol, but that secretion can be enhanced and regulated by retinol delivered by the chylomicron remnant.     

Hepatocyte viability and protein expression within hydrogel microstructures

Poly(ethylene) glycol (PEG) hydrogels have been successfully used to entrap mammalian cells for potential high throughput drug screening and biosensing applications. To determine the influence of PEG composition on the production of cellular protein, mammalian hepatocytes were maintained in PEG hydrogels for 7 days. Total cell viability, total protein production, and the production of two specific proteins, albumin and fibronectin, were monitored. Studies revealed that while PEG composition has no effect on cell viability, increasing amounts of PEG in the hydrogel decrease the amount of protein production by the cells after 7 days from 1.0 x 10(5) +/- 1.7 x 10(4) to 5.2 x 10(3) +/- 1.3 x 10(3) g accumulated protein/mL/million cells. Additionally, cells entrapped in PEG hydrogels produce greater amounts of protein than traditional monolayer culture (1.5 x 10(3) +/- 1.9 x 10(2) g accumulated protein/mL/million cells after 7 days). The addition of the synthetic peptide RGD to 10% PEG hydrogels altered the production of the proteins albumin and fibronectin. Hydrogels with the RGD sequence produced 287 +/- 27 ng/mL/million cells albumin after 7 days, an order of magnitude greater than monolayer cultures, whereas cells in hydrogels without the RGD sequence produced undetectable levels of albumin. Conversely, cells entrapped in 10% PEG hydrogels without the RGD sequence produced 1014 +/- 328 ng/mL/million cells fibronectin after 7 days, whereas 10% PEG hydrogels with the RGD sequence produced 200 +/- 58 ng/mL/million cells fibronectin after 7 days.