The tumorigenic phenotype of a mutated form of Fc gamma RIIB1, lacking the ability to generate soluble receptor and allowing a low-level of ligand binding.

Non immunohematopoietic murine tumor cells ectopically expressing Fc gamma RIIB1 (B1) were recently shown to express a higher tumorigenicity phenotype than cells not expressing this receptor. Utilizing a genetic approach we studied the possible contribution of a soluble form of B1 to tumor enhancement. A mutated form of the B1, lacking the cleavage site responsible for the generation of soluble B1 was produced using gene splicing by overlap extension PCR. A deletion confirmed by sequence analysis from 172 to 178 residues was generated. Stable transfectants expressed the B1 deleted form (B1 Delta) both as specific RNA and as a membrane protein receptor allowing a low level of ligand binding. The soluble form of B1 was undetectable in tissue culture supernatants of Bib transfected cells while it was present in supernatants of wild type B1-transfectants. Stable B1 Delta transfectants were significantly more tumorigenic than negative control transfectants. Tumor incidence was almost as high as that of intact B1 and lagged in the latency period before the appearance of palpable tumors. It is suggested that the soluble B1 has a minimal contribution to tumor enhancement.

TNFalpha and anti-Fas antibodies regulate Ly-6E.1 expression by tumor cells: a possible link between angiogenesis and Ly-6E.1.

Angiogenic and poorly angiogenic tumor variants were obtained by an intraperitoneal inoculation of cells from clones of polyoma-virus transformed BALB/c 3T3 cells into syngeneic mice. The angiogenic tumor cells expressed a higher tumorigenicity phenotype and a higher capacity to produce artificial pulmonary metastases than cells from the poorly angiogenic tumors. The former cells expressed also significantly higher levels of the lymphocyte activation protein Ly-6E.1 than the former cells. The two types of cells did not differ in expression levels of CD44 and of a polyoma-virus specific membrane antigen. These results raise the possibility that the angiogenic phenotype is coregulated with Ly-6. The effect on Ly-6 expression of signal transduction through TNF receptors, functioning as pivotal regulators of angiogenesis was therefore studied. It was found that TNFalpha and more so antibodies against Fas down-regulate expression levels of Ly-6. This down-regulation seemed to be selective as expression levels of CD44 were not affected by this treatment.

Endothelial albumin binding proteins are membrane-associated components exposed on the cell surface.

The heterobifunctional, photoactivatable, thiol-cleavable cross-linker sulfosuccinimidyl 2-(p-azido-salicylamido)ethyl-1,3'-dithiopropionate (SASD) was radioiodinated and used to determine whether endothelial albumin binding proteins (ABP) recently identified (Ghinea, N., Fixman, A., Alexandru, D., Popov, D., Hasu, M., Ghitescu, L., Eskenasy, M., Simionescu, M., and Simionescu, N. (1988) J. Cell Biol. 107, 231-239) are plasma membrane-associated components exposed on the cell surface. Microvascular endothelial cells (MEC) freshly isolated from rat epididymal fat were incubated with 125I-2-(p-azidosalicylamido)ethyl-1,3'-dithiopropionate (ASD)-albumin conjugate which upon photolysis by UV light was cross-linked to the receptor proteins. By cleaving the disulfide linkages of the cross-linker with 5% beta-mercaptoethanol and the ligand-receptor interactions with 0.1% sodium dodecyl sulfate, the radioiodinated ASD moiety remained attached to the receptor peptides which were further detected by 5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by autoradiography. In parallel, samples were examined by ligand blotting with albumin-gold complex. The results showed that in these experimental conditions ABP are represented by two major peptides of 31 and 18 kDa and two minor bands of 73 and 56 kDa. Densitometric scanning showed that the two major bands constitute more than 70% of the total ABP. The four peptides were not apparent if the samples were not UV-irradiated. The binding of the radioiodinated ligand to ABPs was reduced by approximately 82% in the presence of excess competitive unlabeled albumin. When MEC were incubated with unlabeled SASD and exposed to UV light, the autoradiographic banding pattern obtained was similar to that of either radioiodinated receptor proteins or MEC not treated with SASD. This indicated that the four albumin binding peptides are distinct proteins of the endothelial cell plasmalemma.

Culture of pericytes isolated from rat adipose microvasculature and characterization of their prostanoid production.

In order to investigate the role of pericytes (PCs) at microvascular level, a PC line from rat epididymal fat pads was isolated and its prostanoid production in culture was further examined. PC cultures were characterized morphologically by phase contrast and electron microscopy. PC prostanoid production was compared with that of a smooth muscle cell (SMC) line isolated from bovine aorta. The same PGI2 production magnitude was assayed in PC and SMC cultures, but TXA2 and PGF2 alpha synthesis was 8-10 times higher in the PC line. At 4-day postconfluence, when PC layers started retraction, prostanoid synthesis was significantly lower than at confluence. Histamine and bradykinin (both 100 nM) acted similarly, increasing the PGI2 basal production of PC cultures. The results argue for a possible contractile role of PCs at microvascular level.

Identification of albumin-binding proteins in capillary endothelial cells.

Isolated fat tissue microvessels and lung, whose capillary endothelia express in situ specific binding sites for albumin, were homogenized and subjected to SDS-gel electrophoresis and electroblotting. The nitrocellulose strips were incubated with either albumin-gold (Alb-Au) and directly visualized, or with [125I]albumin (monomeric or polymeric) and autoradiographed. The extracts of both microvascular endothelium and the lung express albumin-binding proteins (ABPs) represented by two pairs of polypeptides with major components of molecular mass 31 and 18 kD. The ABP peptides have pIs 8.05 to 8.75. Rabbit aortic endothelium, used as control, does not express detectable amounts of ABPs. The ABPs subjected to electrophoresis bind specifically and with high affinity (Kd = approximately 60 X 10(-9)M) both monomeric and polymeric albumin: the binding is saturable at approximately 80 nM concentration and 50% inhibition is reached at 5.5 micrograms/ml albumin concentration. Sulfhydryl-reducing agents beta-mercaptoethanol and dithiothreitol do not markedly affect the ABPs electrophoretic mobility and binding properties. As indicated by cell surface iodination of isolated capillary endothelium followed by electroblotting, autoradiography, and incubation with Alb-Au, the bands specifically stained by this ligand are also labeled with radioiodine.

In vitro study of low density lipoprotein--collagen interaction.

Possibility of LDL--collagen complex formation was investigated in vitro by biochemical assay and electron microscopy. Types I and III collagen isolated from bovine thoracic aorta were incubated with human low density lipoproteins (LDL) at physiological ionic strength, pH and temperature. Biochemical quantification showed that 10-20 micrograms LDL (cholesterol) were bound per 100 micrograms collagen, binding of type III being slightly more pronounced (17%) than that of type I (11%). Binding was in inversely proportional to the extent of fibrillation. The increase of ionic strength and pH reduced the binding, indicating the electrostatic nature of the interaction. These observations suggest a possible trapping mechanism of LDL in the extracellular matrix by means of collagen, which may be relevant for the development of the atherosclerotic lesions.

Necrotropic protective effect of Ca-Mg gluconolactate on allyl alcohol-induced liver necrosis in rats.

The prevention of liver damage in rats intoxicated with allyl alcohol was attempted with 5 per cent Ca--Mg gluconolactate in reiterated doses. This preparation prevented or reduced liver necrosis only in the presence of spleen: splenectomy annihilated the effect of Ca--Mg gluconolactate.

The foetal development of the rabbit lung: a cytologic, cytochemical and histoenzymatic study.

The developmental peculiarities of the rabbit lung were analyzed in foetuses of 14 and 23 days, and in newborns having respired 30 min. and 48 hrs. Cytochemical, histoenzymatic and quantiative cytologic methods were used. The parallel evolution of epithelial and mesenchymal cells was quantified using conventional fields. The development of air spaces was morphometrically appreciated. Acid and neutral mucopolysaccarides, nucleic acids, and enzymic activities (AcPh-ase, AlkPh-ase, ATP-ase, AMP-ase, SDH, MDH, LDG, G1-6-ph-DH, proline-oxydase, hydroxyproline-2-epimerase, unspecific esterase, TwE-ase, beta-gal-ase and beta-gluc-ase, alanyl- and leucineaminopeptidase) were investigated. This complex analysis showed that in a first phase the development mainly involved the epithelial cells, while the proliferation of mesenchymal ones remained constant. In a second phase, the epithelial cell increase became slower, and the mesenchymal cells were decreasing. At the same time the air spaces were continuously increasing. During this process, neutral mucopolysaccharides were synthesized in epithelial cells and in cartilaginous nodules, and sometimes in mesenchymal cells. The RNA was continuously increasing both in epithelial and mesenchymal cells. The high enzymic activities in the 14-day foetuses appeared to be limited to AcPh-ase, AlkPh-ase, and SDH in both epithelial and mesenchymal cells, the LDH in epithelial and the ATP-ase and AMP-ase mainly in mesenchymal cells. At the same time, the G1-6-ph-DH obviously marked the epithelial cell differentiation. In the other foetal and newborn lungs, the enzymic activities appeared to be more various by limitation of AcPh-ase to epithelial elements and of AlkPh-ase to mesenchymal and vascular ones, by activation of proline-oxydase and especially of hydroxyproline-2-epimerase in pleura and peribronchovascularly, by intensification of the unspecific esterase: the other enzymes active in the 14-day foetuses were now weaker. The activity of beta-gal-ase, beta-gluc-ase, and of peptidases was missing during the entire development of the foetal rabbit lung. The corroboration of these data suggested the relation between the differentiation of enzymic activities and the development of foetal rabbit lung, the strong relations between AcPh-ase activity and the epithelial elements, and of AlkPh-ase and ATP-ase with the mesodermo-mesenchymal ones, the marking of epithelial cell differentiation by the G1-6-ph-DH activity, the presence of SDH in the basal corpuscles of differentiating cili, the increase of enzymes making inactive the hydroxyproline in zones in which connective tissue is developing, the low differentiation of hydrolases (related to the absence of air and blood transport of products) and the lack of peptidase activity corresponding to the reduced pulmonary degradation of proteins (as in adult lungs).

Kinetics of mucous cells in bronchial and bronchiolar epithelia during aerogenic elicitation with aspergillary proteins. A quantitative cytologic study.

During a prolonged exposure of rabbits to aerosols with asperigillary antigens, the content of bronchial and bronchiolar epithelia in mucus-secreting cells was quantified. In normals, the mucous cells do not exceed 3.2 per cent in bronchial epithelium and 0.44 per cent in bronchiolar one. After exposure, these proportions rapidly increase, after a primary discharge of goblet cells. The mucous cells become a majority after 7 exposures in bronchi and after 21 in bronchiolar epithelium. The maturation is more rapid in bronchioli than in bronchi. The secretion of neutral and acid mucopolysaccharides is early, concomitant in the same bronchus, and predominantly located in the impingement zones, especially the bifurcations of bronchial tree. The mitoses are extremely rare in exposed bronchi, but the epithelial and mesenchymal alveolar cells present a high mitotic index (1.14%) 24 hours after the second exposure to antigens.