SV40 transformed fibroblasts recognize the same 140 kD fibronectin chemotactic fragment as non-transformed cells

SV40-virus-transformed human embryonal fibroblasts show an enhanced chemotactic response to the glycoprotein fibronectin. However, they recognize the same chemotactic active region as non-transformed fibroblasts. The result suggests that an enhancement of chemotaxis by fibroblasts which have been transformed with Simian Virus 40 is due not to the utilization of further chemotactic domains in the molecule, but to an increased sensitivity of the cells to the chemoattractant.     

Bioassay for hamster macrophage chemotaxis: application to study particle-lung interactions

Attraction of lung macrophages to particle deposition sites has been demonstrated in different animal species. We reported a threefold increase of the number of macrophages to occur within 40 min after polystyrene particle deposition in hamster airways [Geiser et al. (1994) Am. J. Respir. Cell Mol. Biol. 160: 594–603]. Complement-derived chemotactic activity is one of the mechanisms postulated for macrophage recruitment. It was the aim of this study to test whether complement-derived chemotactic activity is involved in the rapid recruitment of macrophages to the site of deposited polystyrene particles in hamster airways. We first developed an in vitro cell migration assay for hamster macrophages to assess complement-derived chemotaxis. Second, the bronchoalveolar lavage fluids (BALF) of four hamsters that had inhaled aerosols of polystyrene microspheres were tested for chemotactic activity by this bioassay and compared with BALF of four sham-exposed hamsters. Chemotactic response of macrophages was found toward complement-activated hamster serum, whereas macrophage migration was not increased toward BALF of particle and sham-exposed hamsters. In contrast, macrophage migration to BALF of both groups was reduced by 1.6-fold. Thus, the stimulus for macrophage recruitment to the site of deposited polystyrene particles in hamster airways could not be demonstrated using this bioassay. Received 10 September 1997; received after revision 24 November 1997; accepted 10 December 1997     

Chemotactic and random movement of cord-blood granulocytes

Summary Chemotactic responsiveness and random movement of cord-blood granulocytes were studied with a modified Boyden's method. Cord-blood granulocytes were less active chemotactically than granulocytes from healthy children and adults, whereas the random filter movement of the cells from all three sources was about the same. In cord sera, concentrations of cell directed chemotaxis inhibitors were equal to those in sera from other age groups. Compared with the situation in healthy children and adults, the generation of chemotactic factors in cord-blood sera was impaired. This impairment was not related to an increased activity of chemotactic factor inactivators. Measurement of the cyclic nucleotide levels in granulocytes from cord-blood and from children belonging to various age groups revealed that the cord granulocytes have significantly lower concentrations of cAMP and cGMP, which could have been responsible for the decreased chemotactic responsiveness.     

Stimulation of the synthesis and non-competitive inhibition of alkaline phosphatase by theophylline in normal hamster fibroblasts and absence of response in transformed fibroblasts]

Theophylline increases the synthesis of alkaline phosphatase in normal Hamster fibroblasts (activity X6 after 24 hours with theophylline 10-3 M). The enzymatic activity of these cells is inhibited by theophylline in vitro (80% inhibition in the presence of theophylline 10-3 M). The inhibition seems to be non competitive, since the apparent Km of the enzyme is not modified. This stimulator and inhibitor effect of theophylline is absent in Hamster fibroblasts transformed by SV 40 virus.     

Chemotactic and random movement of cord-blood granulocytes

Chemotactic responsiveness and random movement of cord-blood granulocytes were studied with a modified Boyden's method. Cord-blood granulocytes were less active chemotactically than granulocytes from healthy children and adults, whereas the random filter movement of the cells from all three sources was about the same. In cord sera, concentrations of cell directed chemotaxis inhibitors were equal to those in sera from other age groups. Compared with the situation in healthy children and adults, the generation of chemotactic factors in cord-blood sera was impaired. This impairment was not related to an increased activity of chemotactic factor inactivators. Measurement of the cyclic nucleotide levels in granulocytes from cord-blood and from children belonging to various age groups revealed that the cord granulocytes have significantly lower concentrations of cAMP and cGMP, which could have been responsible for the decreased chemotactic responsiveness.     

Kinetics of chemo-attraction of polymorphonuclear leukocytes towards N-formyl peptide studied with a novel polycarbonate (Nucleopore) membrane in the Boyden chamber

The motile responses of human polymorphonuclear leukocytes (PMN) to N-formyl-methionyl-leucyl-phenylalanine (FMLP) in the Boyden chamber using a new sparse-pore polycarbonate membrane (pores 3 micron in diameter and occupying 0.1% of surface area) were compared with those demonstrated by using a standard polycarbonate (Nuclepore) filtration membrane (pores 3 micron in diameter and occupying 5% of surface area). Motility of PMN in gradients of FMLP using the new membrane was not influenced by chemokinetic effects of the factor, and the 'background' migration of the cells was minimal. However, motility of PMN in gradients of FMLP using the standard membrane was found to be influenced by chemokinetic effects of the chemotactic factor, and the 'background' or 'control' migration (in the absence of chemotactic factor) of the cells was substantial. Greater directional migration of PMN according to steepness of the gradient of chemotactic factor was demonstrated with the use of the new membrane. The new membrane may be of considerable value in the further study of the chemotactic responses of PMN.     

Chromosome fragility and susceptibility of Bloom's syndrome fibroblasts to SV40 transformation

A comparison of the frequencies of chromosomal aberrations and the rates of SV40 transformation was made using fibroblasts obtained from 2 patients with Bloom's syndrome (BS) and from a normal individual. BS cells were found to be more susceptible to chromosome damage, in confirmation of earlier reports, but surprisingly, BS cells were distinctly less prone to transformation.     

The effect of acidic polysaccharides and prostaglandin-like substances isolated fromPropionibacterium acnes on granulocyte chemotaxis

Summary Three acidic polysaccharide (AP) fractions and the prostaglandin-like substances (PLS) isolated fromP. acnes were investigated regarding their chemotactic activities on polymorphonuclear leukocytes. Both AP's and PLS induced a significant chemotactic response, which suggests their involvement in inflammatory acne vulgaris.     

Chromosome fragility and susceptibility of Bloom's syndrome fibroblasts to SV40 transformation

Summary A comparison of the frequencies of chromosomal aberrations and the rates of SV40 transformation was made using fibroblasts obtained from 2 patients with Bloom's syndrome (BS) and from a normal individual. BS cells were found to be more susceptible to chromosome damage, in confirmation of earlier reports, but surprisingly, BS cells were distinctly less prone to transformation.Supported in part by the Michael J. Connell Foundation Medical Genetics Fund, and Health, Education and Welfare, MCH project 422.     

The effect of mouse erythrocyte rosette forming lymphocytes on lymphokine production in T-cell cultures

An enhancement effect of mouse erythrocyte rosette forming (MERF) cells on the production of migration inhibitory factor, chemotactic factor for neutrophils and skin reactive factor in T-lymphocyte cultures stimulated with the purified protein derivative of tuberculin was observed. We consider it likely that the MERF cells, possessing the appropriate cell surface constituents to construct an immunogenic moiety, present antigen on their surfaces to elicit lymphokine production.     

The CD40/CD154 receptor/ligand dyadRID="†"ID="†" Review

Until recently, the expression and primary function of the cell surface receptor CD40 and its ligand CD154 were considered restricted to B and T lymphocytes, and their interactions required for the thymus-dependent humoral response. However, current work from several groups challenges this view of the CD40/CD154 dyad as a mere mediator of lymphocyte communication. A variety of non-lymphocytic cell types express both receptor and ligand, including hematopoetic and non-hematopoetic cells, such as monocytes, basophils, eosinophils, dendritic cells, fibroblasts, smooth muscle, and endothelial cells. Accordingly, ligation of CD40 mediates a broad variety of immune and inflammatory responses, such as the expression of adhesion molecules, cytokines, matrix-degrading enzymes, prothrombotic activities, and apoptotic mediators. Consequently, CD40 signaling has been associated with pathogenic processes of chronic inflammatory diseases, such as autoimmune diseases, neurodegenerative disorders, graft-versus-host disease, cancer, and atherosclerosis. This review focuses on the synthesis and structure of CD40 and outlines CD154/CD40 signaling pathways, and emphasizes the previously unexpected importance of the CD40/CD154 receptor/ligand dyad in a spectrum of immunoregulatory processes and prevalent human diseases. Received 10 January 2000; revised 16 June 2000; accepted 5 July 2000 RID="†" ID="†" Review RID="*" ID="*" Corresponding author.     

The effect of mouse erythrocyte rosette forming lymphocytes on lymphokine production in T-cell cultures

Summary An enhancement effect of mouse erythrocyte rosette forming (MERF) cells on the production of migration inhibitory factor, chemotactic factor for neutrophils and skin reactive factor in T-lymphocyte cultures stimulated with the purified protein derivative of tuberculin was observed. We consider it likely that the MERF cells, possessing the appropriate cell surface constituents to construct an immunogenic moiety, present antigen on their surfaces to elicit lymphokine production.This work was supported by the Scientific Research Council, Ministry of Health, Hungary, Code No. 421030401 1/s     

Highly efficient cell-mediated gene transfer using non-viral vectors and FuGene6: in vitro and in vivo studies

The present study was undertaken to develop an efficient non-viral gene delivery system for cardiovascular gene therapy. We investigated transfection efficiency and toxic properties of the new transfection reagent, FuGene6, and compared it with two other transfection reagents, Tfx-50 and LipoTaxi. For in vivo experiments, the plasmid was delivered intramuscularly via transplantation of fibroblasts transfected with plasmid and FuGene6. Conditions for efficient gene delivery were initially studied in vitro. Human and rabbit fibroblasts were isolated from skin, cultured and transfected with phVEGF165 or pCMVbeta gal plasmids, coding for vascular endothelial growth factor (VEGF) or beta-galactosidase, respectively. The effect of the DNA amount and the DNA:transfection reagent ratio on plasmid uptake were studied. Of the transfection reagents tested, only FuGene6 provided high-efficiency and dose-dependent plasmid transfer both for cell-localised (beta-galactosidase) and secreted (VEGF) gene products. When analysed with an MTT assay, FuGene6 showed no toxicity at low doses. Optimised conditions were applied for in vivo reporter gene delivery. Rabbits were injected intramuscularly with ex vivo-transfected fibroblasts. As in in vitro studies, ex vivo-transfected fibroblasts showed highly efficient gene expression in vivo. Tissue sections were analysed with macrophage-specific immunostaining. No signs of inflammation were seen in the region of fibroblast injection. This study demonstrates that FuGene6 is a highly efficient transfection reagent that may be useful for in vitro non-viral transfection of primary human and rabbit fibroblasts and for in vivo therapeutic non-viral gene delivery.     

Lysis of Rous sarcoma virus in the presence of anti-chicken fibroblast serum and complement]

Rous sarcoma virus produced by Chick embryo fibroblasts is inactivated by an antiserum prepared against uninfected fibroblasts in the presence, but not in the absence, of complement. This inactivation which demonstrates the presence of one or more antigens of the surface of the producer cell on the viral envelope, is due to virolysis. This is demonstrated by the release of the viral internal proteins and by the fact that the viral RNA becomes entirely degradable by RNase.     

Relation between stimulus intensity and neutrophil chemotactic response

Summary The effect of chemotactic peptides which lack chemokinetic activity has been investigated. The neutrophil response is proportional to the logarithm of the stimulus intensity, or alternatively a power function with an exponent of 0.3. Equal responses are obtained for equal ratios between the peptide concentration in the lower compartment and the threshold concentration. The significance of Weber-Fechner's law in leucocyte chemotaxis is discussed.We thank Miss B. Zanolari, Mrs M. Marti, Mrs Hauck and Mr Dietz for excellent technical assistance. This work was supported by the Swiss National Foundation for Scientific Research.     

Side effects of Ricinus lectin (RCA 120) on nucleic acid synthesis in chick embryo fibroblasts

When fibroblasts from chick embryos were treated with Ricinus lectin, the effects observed depended on the stage of development of the embryo from which the cells were prepared. Thus, in 16-day fibroblasts, which have a weak proliferative capacity, nucleic acid synthesis was less sensitive to the effect of this lectin than that in 8-day fibroblasts, whose proliferative capacity is high.     

Dehydroepiandrosterone sulfate-binding sites in plasma membrane from human uterine cervical fibroblasts.

Dehydroepiandrosterone sulfate (DHA-S) plays a critical role in cervical dilatation at labor. Incubation of cervical fibroblasts with [3H]DHA-S caused a rapid and saturable increase in cellular radioactivity: an apparent equilibrium was reached by 2 min. There was no detectable conversion of DHA-S into DHA or oestradiol. When the fibroblasts loaded with [3H]DHA-S were homogenized and fractionated, the specific radioactivity in the plasma membrane fraction was enriched approximately 8- to 9-fold compared with the whole homogenate; only low amounts of radioactivity were observed in the other subcellular fractions. The binding of DHA-S to plasma membrane preparations showed saturation kinetics with an apparent equilibrium dissociation constant (Kd) of 12 nM, and the binding capacity (Bmax) was calculated to be 1.25 fmol/mg protein. Neither DHA nor oestrone sulfate affected [3H]DHA-S binding to the plasma membrane. The plasma membranes of skin fibroblasts did not show specific binding sites for DHA-S. These findings demonstrate the presence of specific binding sites for DHA-S in the plasma membrane of cervical stroma cells. The fetal adrenal steroid may exert its action on cervical ripening at least in part through membrane-associated binding sites, or receptors.     

The effect of acidic polysaccharides and prostaglandin-like substances isolated from Propionibacterium acnes on granulocyte chemotaxis.

Three acidic polysaccharide (AP) fractions and the prostaglandin-like substances (PLS) isolated from P. acnes were investigated regarding their chemotactic activities on polymorphonuclear leukocytes. Both AP's and PLS induced a significant chemotatic response, which suggests their involvement in inflammatory acne vulgaris.     

The effect of halothane on in vitro human neutrophil chemotaxis

Summary Increasing concentrations of halothane were shown to sequentially inhibit directed (chemotactic) and random movement of human peripheral blood neutrophils. No influence on neutrophil movement was apparent at clinically important concentrations, halothane may effect neutrophil microtubule and actomyosin microfilament systems.Acknowledgments. We thank Mr Lous Matej for his skillful technical assistance.