Increase In Total Cell Number Research Articles

Marrow stromal osteoblast function on a poly(propylene fumarate)/ β-tricalcium phosphate biodegradable orthopaedic composite

The objective of this study was to assess the osteoconductivity of a poly(propylene fumarate)/ β-tricalcium phosphate (PPF/ β-TCP) composite in vitro. We examined whether primary rat marrow stromal cells would attach, proliferate, and express differentiated osteoblastic function when seeded on PPF/ β-TCP substrates. Attachment studies showed that a confluent monolayer of cells had adhered to the substrates within an 8 h time frame for marrow stromal cells seeded at confluent numbers. Proliferation and differentiated function of the cells were then investigated for a period of 4 weeks for an initial seeding density of 42 000 cells/cm 2. Rapid proliferation during the first 24 h as determined by 3 H -thymidine incorporation was mirrored by an initial rapid increase in total cell number by DNA assay. A lower proliferation rate and a gradual increase in cell number persisted for the remainder of the study, resulting in a final cell number of 128 000 cells/cm 2. Differentiated cell function was assessed by measuring alkaline phosphatase (ALP) activity and osteocalcin (OC) production throughout the time course. Both markers of osteoblastic differentiation increased significantly over a 4-week period. By day 28, cells grown on PPF/ β-TCP reached a maximal ALP activity of 11 (±1)×10 −7 μmol/min/cell, while the OC production reached 40 (±1)×10 −6 ng/cell. These data show that a PPF/ β-TCP composite exhibits in vitro osteoconductivity similar to or better than that of control tissue culture polystyrene.

Inhibitory effects of a lecithinized superoxide dismutase on bleomycin-induced pulmonary fibrosis in mice.

Oxidant/antioxidant imbalance is thought to be involved in the pathogenesis of idiopathic pulmonary fibrosis (IPF). Therefore, antioxidants, such as superoxide dismutase (SOD), are expected to have an inhibitory potential against IPF. To elucidate whether a lecithinized SOD (phosphatidylcholine [PC]-SOD) has the potential to be a new therapeutic agent for IPF, we investigated the inhibitory effects of PC-SOD at doses of 1 mg/kg/d (low dose) and 10 mg/kg/d (high dose) and of methylprednisolone (mPSL) on bleomycin (BLM)-induced pulmonary fibrosis in mice. Histopathologic evaluation and lung hydroxyproline content revealed that the severity of fibrosis was attenuated in mice treated with low-dose PC-SOD, whereas no significant effect was observed in other mice. In bronchoalveolar lavage fluid on Day 1 after treatment with BLM, BLM-induced increases in total cell number, populations of lymphocytes and neutrophils, and expression of messenger RNA for interleukin-1beta and platelet-derived growth factor (PDGF)-A were significantly suppressed in PC-SOD-treated mice. The suppression of PDGF-A expression was significantly greater in mice treated with low-dose PC-SOD than in mice treated with high-dose PC-SOD or mPSL. In summary, this study demonstrated the inhibitory effects of low-dose PC-SOD on the development of pulmonary fibrosis, which indicates the potential usefulness of PC-SOD as a new treatment agent for IPF or at least for BLM-induced pulmonary fibrosis in humans.

FGF-2-induced imbalance in early embryonic heart cell proliferation: a potential cause of late cardiovascular anomalies.

This laboratory previously demonstrated that placement of fibroblast growth factor-2 (FGF-2)-soaked beads adjacent to the developing ventricle at stage 24 caused cardiovascular anomalies by embryonic day 15. We sought to characterize early cellular changes that may suggest mechanisms for the abnormalities observed at day 15. Because levels of both myocyte proliferation and immunohistochemically detectable endogenous FGF-2 begin to decline before stage 24 in untreated embryos, it was of interest to determine whether exogenous FGF-2 might maintain cardiac myocyte proliferation at or near peak levels. Chick embryos were incubated to stage 18 (2.8 days), at which time beads soaked in phosphate-buffered saline (PBS) or 100 microg/ml FGF-2 were placed adjacent to the developing ventricle and development was allowed to continue. After 3 days (stage 29), bromodeoxyuridine (BrdU) was applied to mark dividing cells, followed by double fluorescent assessments to detect relative numbers of dividing and nondividing cells. Quantitative image analysis, using Metamorph software, showed that exogenous FGF-2 caused a 62% increase in the overall number of dividing cells (P < 0.01), concomitant with a 25% increase in total cell number (cell density: P < 0.05). Expressed in relative terms, these changes corresponded to a 25% increase in the proliferation labeling index: 30% of all cells were proliferating in FGF-treated hearts, in contrast with only 24% in control hearts. Taken together, these data suggest that an FGF-induced imbalance in myocardial cell proliferation at early developmental stages of heart development causes cardiovascular anomalies during late embryogenesis.

CD34 + cells from mobilized peripheral blood retain fetal bone marrow repopulating capacity within the Thy-1 + subset following cell division ex vivo

Ex vivo cell cycling of hematopoietic stem cells (HSC), a subset of primitive hematopoietic progenitors (PHP) with engrafting capacity, is required for transduction with retroviral vectors and to increase transplantable HSC numbers. However, induction of division of HSC ex vivo also may lead to differentiation and loss of in vivo marrow repopulating potential. We evaluated mobilized peripheral blood (MPB) PHP for maintenance of stem cell function after ex vivo culture under conditions that we show can induce cycling of a majority of PHP with minimal differentiation. The following methods were combined: cell labeling with the division tracking dye carboxyfluorescein-diacetate succinimidylester (CFSE), analysis of primitive cell surface marker expression, an ex vivo PHP assay, and an in vivo marrow repopulating assay. MPB-purified CD34 + Thy-1 + cells were labeled with CFSE dye and cultured for 112 hours in serum-deprived medium in the presence of the cytokine combinations of thrombopoietin (TPO), flt3 ligand (FL), and c-kit ligand (KL), or TPO, FL, and interleukin 6 (IL-6). Both cytokine combinations supported division of greater than 95% of cells within 112 hours with an average 2.1-fold (TPO, FL, KL) or 1.3-fold (TPO, FL, IL-6) increase in total cell numbers. An average of 21.6% (TPO, FL, KL) and 27.4% (TPO, FL, IL-6) of the divided cells still expressed the Thy-1 marker after 112 hours. Functional assays were performed to compare cultured and uncultured cells. CD34 + Thy-1 + CFSE lo (post division) cells showed maintenance of cobblestone area-forming cell (CAFC) frequency (a mean of 1/9.0) relative to the starting population of uncultured CD34 + Thy-1 + cells (a mean of 1/8.4). In contrast, CD34 + cells that had lost Thy-1 expression during culture (CD34 + Thy-1 − CFSE lo) showed a mean 5.8-fold reduction in CAFC frequency (a mean of 1/52.5). Only the Thy-1–expressing fraction of cells post culture could engraft in vivo in the SCID-hu bone assay. Because the majority of HSC functional activity post culture was found in the CD34 + Thy-1 + fraction, we focused on this fraction for subsequent analysis. CFSE labeling allows segregation and purification by flow cytometry of cells having undergone discrete numbers of divisions during culture. Very few cells that divided more than four times in culture still expressed Thy-1. Cells that retained expression of Thy-1 during culture retained CAFC activity relative to fresh CD34 + Thy-1 + cells, after undergoing at least two divisions. CAFC frequency decreased after four divisions in culture with TPO, FL, and KL or after three divisions in TPO, FL, and IL-6. We then compared populations of Thy-1 + cells that had undergone sequential numbers of divisions in culture for their ability to engraft in the SCID-hu bone assay. Engrafting ability was retained throughout four divisions in both cytokine combinations. These data demonstrate that primitive MPB CD34 + cells maintain HSC function coincident with Thy-1 expression while undergoing two to four divisions under these culture conditions. Essentially all CD34 + Thy-1 + cells divided under the conditions tested, promoting susceptibility to retroviral transduction.

Bone morphogenetic proteins‐2 and ‐4 attenuate apoptosis in a cerebellar primitive neuroectodermal tumor cell line

Similarities between primitive neuroectodermal tumors and central nervous system (CNS) progenitor cells have evoked interest in the response of these tumors to endogenous growth factors. The bone morphogenetic proteins (BMPs) have recently been found to regulate survival and differentiation of CNS progenitor cell populations. In this study, we investigated the effects of BMP-2, BMP-4, and BMP-6 on the undifferentiated cerebellar primitive neuroectodermal tumor or medulloblastoma cell line DAOY. Analysis by reverse transcriptase-polymerase chain reaction showed that mRNAs for type IA and type II BMP receptors were present in control cultures. In cultures treated with BMP-2, mRNAs for BMP receptor type IB and the activin R-I receptor became evident. Cultures were analyzed for total cell counts, proliferating cell nuclear antigen (PCNA), and apoptotic DNA fragmentation. There was a significant increase in total cell number in the BMP-2 and BMP-4 treatment groups, without any change in PCNA reactivity, and a dramatic decrease in the proportion of apoptotic nuclei at concentrations of BMP-2 and BMP-4 above 5 ng/ml (P < 0.001). These effects were not observed with BMP-6, TGF-β1 or GDNF. These results suggest that the increase in total cell number is due to the attenuation of apoptosis by BMP-2 and BMP-4. The anti-apoptotic effect of BMP-2 and BMP-4 on this neuroectodermal cell line has potential clinical implications for neuroectodermal tumors. J. Neurosci. Res. 56:248–258, 1999. © 1999 Wiley-Liss, Inc.

Long-Term Culture of Human CD34+ Progenitors With FLT3-Ligand, Thrombopoietin, and Stem Cell Factor Induces Extensive Amplification of a CD34−CD14− and a CD34−CD14+ Dendritic Cell Precursor

Current in vitro culture systems allow the generation of human dendritic cells (DCs), but the output of mature cells remains modest. This contrasts with the extensive amplification of hematopoietic progenitors achieved when culturing CD34+ cells with FLT3-ligand and thrombopoietin. To test whether such cultures contained DC precursors, CD34+ cord blood cells were incubated with the above cytokines, inducing on the mean a 250-fold and a 16,600-fold increase in total cell number after 4 and 8 weeks, respectively. The addition of stem cell factor induced a further fivefold increase in proliferation. The majority of the cells produced were CD34−CD1a− CD14+(p14+) and CD34−CD1a−CD14−(p14−) and did not display the morphology, surface markers, or allostimulatory capacity of DC. When cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), both subsets differentiated without further proliferation into immature (CD1a+, CD14−, CD83−) macropinocytic DC. Mature (CD1a+, CD14−, CD83+) DCs with high allostimulatory activity were generated if such cultures were supplemented with tumor necrosis factor- (TNF). In addition, p14− cells generated CD14+ cells with GM-CSF and TNF, which in turn, differentiated into DC when exposed to GM-CSF and IL-4. Similar results were obtained with frozen DC precursors and also when using pooled human serum AB+ instead of bovine serum, emphasizing that this system using CD34+ cells may improve future prospects for immunotherapy.

Long-Term Culture of Human CD34+ Progenitors With FLT3-Ligand, Thrombopoietin, and Stem Cell Factor Induces Extensive Amplification of a CD34−CD14− and a CD34−CD14+ Dendritic Cell Precursor

AbstractCurrent in vitro culture systems allow the generation of human dendritic cells (DCs), but the output of mature cells remains modest. This contrasts with the extensive amplification of hematopoietic progenitors achieved when culturing CD34+ cells with FLT3-ligand and thrombopoietin. To test whether such cultures contained DC precursors, CD34+ cord blood cells were incubated with the above cytokines, inducing on the mean a 250-fold and a 16,600-fold increase in total cell number after 4 and 8 weeks, respectively. The addition of stem cell factor induced a further fivefold increase in proliferation. The majority of the cells produced were CD34−CD1a− CD14+(p14+) and CD34−CD1a−CD14−(p14−) and did not display the morphology, surface markers, or allostimulatory capacity of DC. When cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin-4 (IL-4), both subsets differentiated without further proliferation into immature (CD1a+, CD14−, CD83−) macropinocytic DC. Mature (CD1a+, CD14−, CD83+) DCs with high allostimulatory activity were generated if such cultures were supplemented with tumor necrosis factor-α (TNF). In addition, p14− cells generated CD14+ cells with GM-CSF and TNF, which in turn, differentiated into DC when exposed to GM-CSF and IL-4. Similar results were obtained with frozen DC precursors and also when using pooled human serum AB+ instead of bovine serum, emphasizing that this system using CD34+ cells may improve future prospects for immunotherapy.

Insulin‐Like Growth Factor‐1 (IGF‐1) has a Costimulatory Effect on Proliferation of Committed Progenitors Derived from Human Umbilical Cord CD34 + Cells

The effects of insulin-like growth factor-1 (IGF-1) on highly enriched human umbilical cord CD34+ cells were investigated in vitro. CD34+ cells were cultured in serum-free medium containing stem cell factor (SCF), GM-CSF, and interleukin-3 (IL-3). Culture of CD34+ cells for one week in the presence of these cytokines resulted in a dose-dependent increase in total cell number. Addition of G-CSF together with SCF+IL-3+GM-CSF increased the proliferation of myelopoietic cells as determined by the number of cells expressing the myelomonocytic marker CD64 and the granulocytic marker CD15 without significantly altering the number of CD34+ cells in the cultures. In the presence of G-CSF, IGF-1 induced a dose-dependent increase in the total cell number and a moderate but significant increase in the percentages of CD15+, CD64+ cells with sustained CD34+ cell proliferation. We conclude that IGF-1 can enhance the in vitro proliferation of committed progenitor cells derived from umbilical cord CD34+ cells.

The Effect of α4β1-Integrin Binding Sequences of Fibronectin on Growth of Cells From Human Hematopoietic Progenitors

Highly regulated interactions between adhesion receptors on progenitor cells and their extracellular matrix ligands are essential for the control of hematopoiesis in bone marrow stroma. We have examined the relationship between alpha4beta1-integrin-mediated adhesion and growth of CD34(+) cells by assessing their adhesive and migratory patterns of proliferation in a mixture of hematopoietic growth factors in the presence of different recombinant fragments of the HepII/IIICS region of fibronectin. CD34(+) cells were isolated from cord blood and placed in culture wells containing serum-free medium and growth factors. Wells were precoated with either the H120 fragment of fibronectin, which contains three alpha4beta1-integrin binding sites, or the H0 fragment, which lacks the two highest affinity alpha4beta1 binding sequences. Proliferation of single cells of CD34(+)38(+)DR+ and CD34(+)38(-)DR+ phenotypes occurred in contact with the H120 substrate and was associated with migration. Larger numbers of cells were used to quantitate proliferative responses. Cells growing in wells coated with H120 formed attachments to the base of the wells throughout the culture period. Higher total cell counts were consistently found in wells coated with H120 compared with H0 and bovine serum albumin controls. The difference was first apparent at day 8 of culture and reached a maximum at days 11 through 13, when expansion with H120 was a mean of 1.8-fold higher than that seen with H0 (P</= .0001). The greatest expansion (2.25-fold) with H120 compared with H0 was seen when the growth factor concentrations were reduced to 1/16 of the standard levels (P </= .001). The increase in total cell numbers was not at the expense of CD34(+) cells as numbers of these were similar in H120 and control cultures. These results provide evidence for synergy between growth factors and integrins that may be relevant to understanding hematopoiesis in marrow stroma.

The Effect of α4β1-Integrin Binding Sequences of Fibronectin on Growth of Cells From Human Hematopoietic Progenitors

AbstractHighly regulated interactions between adhesion receptors on progenitor cells and their extracellular matrix ligands are essential for the control of hematopoiesis in bone marrow stroma. We have examined the relationship between α4β1-integrin–mediated adhesion and growth of CD34+cells by assessing their adhesive and migratory patterns of proliferation in a mixture of hematopoietic growth factors in the presence of different recombinant fragments of theHepII/IIICS region of fibronectin. CD34+cells were isolated from cord blood and placed in culture wells containing serum-free medium and growth factors. Wells were precoated with either the H120 fragment of fibronectin, which contains three α4β1-integrin binding sites, or the H0 fragment, which lacks the two highest affinity α4β1binding sequences. Proliferation of single cells of CD34+38+DR+and CD34+38−DR+phenotypes occurred in contact with the H120 substrate and was associated with migration. Larger numbers of cells were used to quantitate proliferative responses. Cells growing in wells coated with H120 formed attachments to the base of the wells throughout the culture period. Higher total cell counts were consistently found in wells coated with H120 compared with H0 and bovine serum albumin controls. The difference was first apparent at day 8 of culture and reached a maximum at days 11 through 13, when expansion with H120 was a mean of 1.8-fold higher than that seen with H0 (P≤ .0001). The greatest expansion (2.25-fold) with H120 compared with H0 was seen when the growth factor concentrations were reduced to 1/16 of the standard levels (P≤ .001). The increase in total cell numbers was not at the expense of CD34+cells as numbers of these were similar in H120 and control cultures. These results provide evidence for synergy between growth factors and integrins that may be relevant to understanding hematopoiesis in marrow stroma.

Somatostatin sst5 inhibition of receptor mediated regeneration of rat aortic vascular smooth muscle cells.

1. The aim of the present study was to determine the effect of somatostatin (SRIF) on mitogen-induced regeneration of rat aortic vascular smooth muscle cells (VSMC) and for comparison Chinese hamster ovary (CHO)-K1 cells expressing human recombinant sst5 receptors (CHOsst5), following partial denudation of a confluent cell monolayer. Regeneration was assessed by measuring areas of recovery into the denuded area and by counting total cell numbers. 2. In VSMC, SRIF (0.1 nM - 1 microM) had no effect on the basal levels of regeneration but caused a concentration-dependent inhibition (pIC50 8.0-8.6) of the stimulated regeneration induced by submaximal concentrations of basic fibroblast growth factor (bFGF, 10 ng ml[-1]), platelet-derived growth factor-BB (PDGF, 5 ng ml[-1]) or endothelin-1 (ET-1, 100 nM). SRIF (pIC50 8.8) also inhibited bFGF-induced regeneration of CHOsst5 cells. 3. In VSMC, the inhibitory action of SRIF on the regeneration induced by bFGF (10 ng ml[-1]) was due to an anti-proliferative effect, rather than an effect on cell migration, as SRIF (0.1 nM - 1 microM) abolished bFGF-induced increases in total cell numbers. The bFGF-induced increase in cell numbers was also abolished by actinomycin D (0.1 microg ml[-1]). 4. The sst5 receptor-selective agonist, L-362,855 (pIC50 10.5), was about 100 times more potent than SRIF at inhibiting bFGF-induced regeneration of both VSMC and CHOsst5 cells whilst the sst2 receptor-selective agonist, BIM-23027 (pIC50 6.8), was approximately 20 times weaker than SRIF. 5. The sst5 receptor antagonist, BIM-23056 (100 nM), antagonized SRIF-induced inhibition of bFGF-induced regeneration in both VSMC and CHOsst5 cells (estimated pKB values 8.8 and 8.3, respectively). 6. SRIF-induced inhibition of bFGF-induced regeneration of VSMC and CHOsst5 cells was abolished by pretreating cells with pertussis toxin (100 ng ml[-1]) for 20 h. 7. These findings suggest that SRIF-induced inhibition of the proliferation of rat aortic VSMC is mediated via activation of receptors which are similar to human sst5 receptors. Furthermore this inhibitory effect is transduced via pertussis toxin-sensitive Gi/Go proteins.

A simple method for the propagation and purification of γδT cells from the peripheral blood of glioblastoma patients using solid-phase anti-CD3 antibody and soluble IL-2

Although γδT cells make up no more than 10% of the peripheral blood mononuclear (PBM) cells, they appear to play an important role in host defense against tumor growth. In order to evaluate their functional activity against tumors and their response to various cytokines, large numbers of cells are required. Here, we describe a newly-devised method for the isolation and expansion of γδT cells from the peripheral blood of cancer patients, in particular those with glioblastoma. Using this approach, a 1000–1500-fold increase in total cell numbers was achieved in two weeks, the proportion of γδT cells in the expanded population being, on average, approximately 30% after 14 days of culture. The method therefore gives a yield of approximately 10–15×10 8 γδT cells from only 5 ml of peripheral blood from glioblastoma patients and normal controls. The highly purified γδT cells of glioblastoma patients were shown to bear both a high-affinity interleukin-2 receptor (IL-2R) and a low-affinity IL-12 receptor (IL-12R). They also displayed significant cytotoxicity against autologous tumor cells, but not against autologous fresh or IL-2-treated lymphocytes, and proliferated in response to IL-2, both effects being dependent on the dose of IL-2 used for activation. In addition, overnight incubation with 700 U/ml of IL-2 or 50 ng/ml of IL-12 resulted in significant cytotoxic activity of patients' γδT cells against K562 target cells, the level of activity being almost the same as with similarly-treated γδT cells from normal controls ( P>0.05). These results demonstrate that the patients' γδT cells obtained using this method are intact in terms of cytotoxic function. Thus, this method not only makes it possible to produce large numbers of purified γδT cells but also to produce populations containing both γδT cells and NK cells, both active against tumor targets which might be suitable for clinical trials of adoptive immunotherapy, especially in cancer patients for whom no effective therapy is available.

Ethanol inhibits basic fibroblast growth factor-mediated proliferation of C6 astrocytoma cells.

Early ethanol exposure alters the proliferative activity of glial and neuronal precursors in the developing CNS. The present study tests the hypothesis that ethanol-induced alterations in cell proliferation result from interference with growth factors. An in vitro model of astroglia (C6 astrocytoma cells) was used to study the effects of ethanol on proliferation mediated by basic fibroblast growth factor (bFGF). bFGF stimulated the proliferation of C6 cells. This bFGF-enhanced proliferation was evident by increases in total cell number, DNA synthesis (as measured by [3H]thymidine incorporation), and the number of cells that took up bromodeoxyuridine. A synthetic peptide that specifically blocked the binding of bFGF to its high-affinity receptor completely abolished the proliferation-promoting effect of bFGF. The action of another mitogen for C6 cells, insulin-like growth factor-1, was not affected by this peptide. Therefore, the bFGF-stimulated proliferation was mediated through a specific bFGF receptor. Ethanol inhibited bFGF-mediated proliferation in a concentration-dependent manner. Ethanol concentrations of 100 and 200 mg/dl partially inhibited bFGF-mediated proliferation (by 58 and 74%, respectively), whereas concentrations of > or = 400 mg/dl completely abolished the growth-stimulating effect of bFGF. Our data show that ethanol alters proliferative activity of C6 cells by disrupting the action of bFGF. The target of ethanol neurotoxicity is a receptor-mediated activity. bFGF can affect cell proliferation by a non-receptor-mediated intracellular pathway, but ethanol does not have an impact on this pathway.

Ex vivo expansion of haemopoietic progenitor cells

Over the last few years, techniques have become available that allow the extensive proliferation of haemopoietic progenitor cells in ex vivo culture systems. The most commonly used method involves a simple liquid suspension culture system supplemented with a range of cytokines. Alternatively, more complex systems have been devised in which the formation of a stromal layer is required. Large increases in total cell numbers and committed progenitor cells can be readily obtained and, with some techniques, significant expansion of primitive haemopoietic cells has been demonstrated. Although these strategies have several potential applications, few clinical studies have been performed. It has been shown that infusion of ex vivo cultured cells is well tolerated with no associated toxicity. However, it is still unclear whether these culture systems sustain sufficient numbers of long-term repopulating cells to secure durable engraftment following myeloablative therapy. In gene therapy studies, ex vivo expansion of stem cells should improve the efficiency of gene transduction to enable the production of genetically modified cells that are capable of expressing the gene of interest for extended periods of time.

Tyrphostin inhibition of ATP-stimulated DNA synthesis, cell proliferation and fos-protein expression in vascular smooth muscle cells.

1. We and others have shown that extracellular ATP (adenosine triphosphate), released from sympathetic nerves and platelets, stimulates growth of vascular smooth muscle cells (SMC). To study the importance of tyrosine kinases for ATP-mediated proliferation in vascular smooth muscle cells we used tyrphostins, a recently developed group of highly specific inhibitors of tyrosine kinases. 2. ATP induced a powerful concentration-dependent increase in DNA synthesis measured by [3H]-thymidine incorporation in rat aorta SMC (RASMC) and an increase in total cell number after 72 h of incubation as measured by an enzymatic cell proliferation assay. Tyrphostin 25 (10(-5) M) had no effect per se on basal DNA synthesis but reduced ATP-stimulated DNA synthesis and increase in cell number in a dose-dependent manner. Higher concentrations of ATP could not reverse the inhibitory effect of tyrphostin 25. The potency of several (six) other tyrphostins was also examined and found to be slightly greater than tyrphostin 25 with equal efficacy. 3. When RASMC were incubated with 10(-5) M ATP for 2 h, nearly all of the cells (87 +/- 5%) were intensely stained with an antibody to the Fos protein while in the controls only 1 +/- 2% of the cells were weakly stained. Tyrphostin 25 greatly reduced the Fos-protein staining (14 +/- 2%). 4. ATP induced a concentration-dependent increase in 45Ca(2+)-influx and formation of inositol phosphates (IPtotal) in RASMC. These effects were not inhibited by tyrphostin 25. 5. Tyrphostin 25 did not alter ATP-induced contraction in ring segments of rat aorta. 6. In conclusion, tyrphostin 25 inhibited ATP-induced DNA synthesis, cell proliferation and Fos-protein expression, but not ATP-induced 45Ca(2+)-influx, inositolphosphate-production or vasoconstriction. This indicates that the mitogenic effect of ATP on vascular smooth muscle cells is dependent on tyrosine kinases in contrast to the contractile effect of ATP in blood vessels.

Early and late phase bronchoconstrictions in conscious sensitized guinea-pigs after macro- and microshock inhalation of allergen and associated airway accumulation of leukocytes

Guinea-pigs were sensitized by i.p. injection of 10 μg OA and 100 mg aluminium hydroxide in 1 ml normal saline. Fourteen to twenty-one days after sensitization, animals were exposed to macroshock (1% OA for 2 min) or microshock (0.01% for 60 min) inhalation challenges with OA. Animals were protected against fatal anaphylaxis in the case of macroshocks with mepyramine (30mg/kg i.p.) 30 min before exposure. Specific airway conductance (sG aw) was measured in conscious animals by whole body plethysmography at intervals up to 72h after challenge. An early phase bronchoconstriction peaked significantly ( P < 0.05) at 15 min after both macroshock and microshock OA exposures, with maximum falls in sG aw of 70.8 ± 3.8 and −40.0 ± 5.9%, respectively. These had resolved after 5 h. A late phase bronchoconstriction peaked variably between 17 and 24 h: the mean peak falls in sG aw after the macro- and microshock challenges were significantly different from baseline ( P < 0.05), at −21.6 ± 3.7 and −38.0 ± 3.9%, respectively. Control exposures of OA-sensitized guinea-pigs to saline for either 2 or 60 min, in place of OA, produced no significant variation in sG aw values over the predicted early and late phases. Bronchoalveolar lavage (BAL) performed at 5 or 24h after OA challenge revealed significant increases in total cell numbers ( P < 0.05) at 5 and 24 h after the OA macroshock challenge and at 24 h after the microshock, compared with saline challenges. Differential cell counts showed a significant ( P < 0.05) increase in the proportion of neutrophils at 5 h and of neutrophils and eosinophils at 24 h after the macroshock exposure to OA, compared with saline controls. A significant ( P < 0.05) increase in the proportion of eosinophils also occurred in BAL fluid at 24 h after microshock OA challenge. Neutrophils, however, did not alter at 24 h, yet a late phase bronchoconstriction was recorded. Thus, macroshock (with mepyramine cover) and microshock (without mepyramine cover) OA challenges result in both early and late phase bronchoconstrictions. The late phase is associated with influx of eosinophils in both models but neutrophils only appear after the macroshock, indicating that late phase responses may not involve neutrophil infiltration to the airways.

The influence of extracellular matrix substrata on preadipocyte development in serum-free cultures of stromal-vascular cells.

The influence of the extracellular matrix (ECM) and ECM components on preadipocyte development was examined in primary cultures of adipose tissue stromal-vascular (S-V) cells. Extracellular matrix derived from Engelbreth-Holm-Swarm (EHS) cells or tumors enhanced several aspects of adipogenesis in vitro. In comparison to uncoated and fibronectin substrata, EHS-ECM substratum markedly increased attachment, spreading, and hypertrophy of preadipocytes while antagonizing spreading of non-preadipocytes. In addition, adipocyte number increased (P < .05) on these substrata despite no increase in total cell number: this resulted in a greater (P < .05) proportion of preadipocytes. These effects of EHS-ECM were also observed with laminin substrata per se, whereas types I and IV collagen and fibronectin had no influence. In contrast to all other substrata, adipocyte number decreased and total cell number increased 2.5-fold on ECM derived from corneal endothelial cells; this resulted in the lowest proportion of preadipocytes. Challenging cultures with adipogenic media (+serum) did not counter the inhibitory influence of corneal endothelial ECM, whereas dexamethasone partially neutralized the inhibitory influence of this ECM. These studies clearly show that source or type of the ECM dictated the influence of ECM substrata on preadipocyte development in primary S-V cultures. However, these studies indicated that the ECM and in particular laminin may play a critical role in morphological aspects of preadipocyte development.

Granulomonocyte-Associated Lysosomal Protein Expression During In Vitro Expansion and Differentiation of CD34+ Hematopoietic Progenitor Cells

Using an in vitro expansion and differentiation system for human CD34+ cord blood (CB) progenitor cells, we analyzed the induction and expression kinetics of the granulomonocyte associated lysosomal proteins myeloperoxidase (MPO), lysozyme (LZ), lactoferrin (LF), and macrosialin (CD68). Freshly isolated CD34+ CB cells were negative for LZ and LF, and only small proportions expressed MPO (4% +/- 2%) or CD68 (3% +/- 1%). Culturing of CD34+ cells for 14 days with interleukin (IL)-1, IL-3, IL-6, stem cell factor, granulocyte-macrophage colony-stimulating factor (GM-CSF), and G-CSF resulted in on average a 1,750-fold amplification of cell number, of which 83% +/- 7% were MPO+. Without addition of GM-CSF and G-CSF, lower increases in total cell numbers (mean, 211-fold) and lower proportions of MPO+ cells (54% +/- 11%) were observed. The proportion of MPO+ cells slightly exceeded but clearly correlated with the proportion of cells positive for the granulomonocyte associated surface molecules CD11b (Mac-1), CD15 (LeX), CD64 (Fc gamma RI) CD66, or CD89 (Fc alpha R). At day 14 MPO+ and LZ+ cells were virtually identical. However, at earlier time points during culture (days 4 and 7), single MPO+ or LZ+ cell populations were also observed, which only later acquired LZ and MPO, respectively. Maturation of cells into the neutrophilic pathway was indicated by the acquisition of MPO, followed by LZ. In contrast, maturation of cells into the monocytic pathway was indicated by the acquisition of LZ followed by MPO and CD14. CD68 was found to be expressed at day 4 by the majority of cells and was not restricted to the granulomonocytic cells, as cells with megakariocytic (CD41+) or erythroid (CD71hi) features were CD68+. LF expression was observed only in GM- plus G-CSF-supplemented cultures, in which only 26% +/- 5% of cells expressed LF by day 14.

Effect of tumor necrosis factor receptor binding protein on cell infiltration induced by lipopolysaccharide and sephadex beads in guinea pig lung

In the present study, the effect of a tumor necrosis factor receptor binding protein (TNFbp) on the cell infiltration induced by lipopolysaccharide (LPS) and Sephadex beads in guinea pig lung was examined. The intratracheal injection of LPS (2.5 micrograms) induced a six-fold increase in total cell number recovered in bronchoalveolar lavage (BAL) fluid at 24 hr. This increase in bronchopulmonary inflammation was mainly due to a neutrophil and macrophage infiltration, representing 60% and 35% of the total cells, respectively. The intravenous or intratracheal injection of Sephadex beads to guinea pigs induced a three-fold increase in total cell number recovered in BAL at 24 h and was characterized by a prominent eosinophil, macrophage, and neutrophil infiltration representing 36%, 42%, and 16% of the total cells, respectively. In addition, bronchial tissues isolated from Sephadex-treated guinea pigs showed an increased in vitro reactivity to both histamine and acetylcholine. TNFbp (1-50 micrograms) induced a dose-dependent inhibition of cell infiltration induced by LPS. In contrast TNFbp neither attenuated the bronchopulmonary cell infiltration observed 24 h following intravenous or intratracheal administration of Sephadex beads nor inhibited the increase in bronchial reactivity. These results show that TNF plays an important role in cell infiltration induced by LPS, but not that induced by Sephadex, in the guinea pig lung.

The effect of cholinergic stimulation on cultured smooth muscle cells.

To assess the role of cholinergic angonists and antagonists on the growth control of cultured rabbit detrusor smooth muscle cells. Dosage dependent growth curve analysis of smooth muscle cells in standard media to bethanechol and atropine at concentrations from 1 x 10(-5) to 1 x 10(-9) mMol. Exogenous exposure to bethanechol results in a significant dose dependent increase in total cell number over a 10 day period. At a concentration of 1 x 10-6 and 1 x 10-5 mMol a 20% to 50% growth increase is noted. Exogenous atropine at all concentrations decreased growth by approximately five fold. The exogenous application of muscarinic cholinergic agonists and antagonists can significantly effect bladder muscle cell growth in vitro. The mechanism of growth control through these pharmacological receptors remains to be elucidated.