Fibroblastlike primary cells from human colon adenocarcinoma explants: Collagen biosynthesis

Summary Fibroblastlike primary cells have been obtained from human colon adenocarcinoma explants. Such cells disappear during cell culture and thus have not been previously studied. These cells have a number of altered phenotypic characteristics: a) morphology; b) growth behavior and adherence to culture substrate (they required 3 h for 90% attachment and only presented a flattened morphology 40 h after platting); and c) collagen metabolism. Increased protein biosynthesis (about double than control colon-derived fibroblasts) and maintained ability for collagen biosynthesis have been observed for the tumor-associated fibroblastlike cells. Thus, the collagen to noncollagenous proteins ratio was decreased for these cells. They exhibited an altered type I:type III collagen (5:1 instead of 3:1 in colon fibroblasts) and procollagen (2:1 against 5:1 in colon fibroblasts) ratios as well as a decreased secretion of collagen with an abnormal deposition of procollagens in the cell layer. These studies show a permanent phenotypic alteration in the tumor-associated fibroblastlike cells.     

Phagocytosis of collagen fibrils by periosteal fibroblasts in long bone explants. Effect of concanavalin A

In an attempt to determine whether phagocytosis of collagen by fibroblasts involves binding of the fibril to the plasma membrane, the effect of the lectin concanavalin A (Con A) was studied in an in vitro model system. Metacarpal bone rudiments from 19-day-old mouse fetuses were incubated with varying concentrations of the lectin. Quantitative electron microscopic analysis indicated that Con A caused a dose-related increase in the amount of phagocytosed collagen fibrils in periosteal fibroblasts, suggesting either an enhanced uptake or a decreased intracellular breakdown of fibrils. Since a Con A-inducible increase was not seen in the combined presence of both the lectin and the proteinase inhibitor leupeptin, which is known to inhibit the intracellular digestion of phagocytosed fibrillar collagen, it is unlikely that Con A stimulated phagocytosis. Based on the finding that Con A interfered with the digestion of a synthetic substrate by the collagenolytic lysosomal enzyme cathepsin B it is suggested that the augmentation of intracellular fibrillar collagen under the influence of the lectin was due to a decreased intracellular digestion. Since Con A did not inhibit the uptake of collagen fibrils by the fibroblasts it is concluded that Con A-inhibitable binding sites for collagen molecules are unlikely to be involved in phagocytosis of collagen fibrils by fibroblasts.     

Factors controlling the rhythmic contraction of collagen gels by neonatal heart cells

Summary A floating collagen matrix culture of neonatal rat heart myocardial cells shows rhythmic contractions which are dependent on localization of cells, cell density, and collagen concentration. The rhythmic contractions of the collagen matrix can be registered by a device scanning the optical density at the edge of the gel and have been observed over a temperature range from 9° to 40° C. The results of the present study underline the usefulness of myocardial cell populated collagen matrixes for studies on coherent contractions of heart cell cultures.     

Fibroblast heterogeneity in glucocorticoid regulation of collagen metabolism: Genetic or epigenetic?

Summary Cultured fibroblasts derived from normal human dermis show a consistent 62% inhibition of collagen synthesis by hydrocortisone, whereas cultures derived from keloids average only 30% inhibition and show a much larger strain to strain variation ranging from 75% inhibition to 49% stimulation. Examination of fibroblast clones indicates that this high variation among keloid strains is not due to differences in the proportion of normal and keloid cells in the mass culture populations. Small but significant differences in the effect of hydrocortisone on collagen deposition are also seen among these clonal populations, but are not related to the type of tissue from which cultures were derived. Two to three-fold differences among clones derived from a single individual were observed, possibly suggesting functional heterogeneity of dermal fibroblasts with regard to collagen metabolism under control conditions and in response to hydrocortisone. However, this variation among clones may simply reflect differences in clonal growth, inasmuch as both collagen synthesis and deposition, and the effect of hydrocortisone on these processes, are strongly affected by population density. This work was supported in part by PHS grants, CA-17229 from the National Cancer Institute and AG-02046 from the National Institute on Aging, DHHS; and by Grant RIM 78-17313 from the National Science Foundation.     

An axial periodic fibrillar arrangement of antigenic determinants for fibronectin and procollagen on ascorbate treated human fibroblasts

Fibronectin and collagens are major constituents of the cell matrix of fibroblasts. Fibronectin is a 220,000 dalton glycoprotein that mediates a variety of adhesive functions of cells examined in vitro. Fibronectin is secreted in a soluble form and interacts with collagen to form extracellular filaments. Fibronectin and procollage type I were localized using the peroxidase anti-peroxidase method. Under standard culture conditions, fibronectin and procollagen were localized to non-periodic 10 nm extracellular fibrils, the cell membrane and plasma membrane vesicles. Ascorbate treatment of cells leads to a new larger fibril with a diameter of approximately 40 nm. Antibodies to fibronectin and procollagen I react to these native collagen fibrils with an axial periodicity of approximately 70 nm. Fibronectin is clearly associated with native collagen fibrils produced by ascorbate treated cells and there is an asymetric distribution or segregation of fibronectin on these collagen fibrils with a 70 nm axial repeat.     

Primary culture of mouse mammary tumor epithelial cells embedded in collagen gels

Summary Mammary tumor epithelial cells from BALB/cfC3H mice were dispersely embedded inside the collagen gels in Ham's F-12 medium containing horse serum. A sustained cell growth leading to a 5- to 10-fold increase in cell number over initial level was observed in less than 2 weeks. The extent of this growth was found to be dependent on serum concentration. However, addition of various protein and steroid hormones, both singly and in combination, to low-serum-containing medium failed to achieve a comparable level of growth to that promoted by higher serum concentration. Mammary tumor cells can now be consistently propagated in primary culture. This investigation was supported by Grants CA05388 and CA09041 awarded by the National Cancer Institute, Department of Health, Education and Welfare, and by cancer research funds of the University of California.     

Histochemical and ultrastructural studies on the metacercarial cyst of Echinostoma revolutum (Trematoda).

Histochemical and ultrastructural studies were made on the metacercarial cyst of Echinostoma revolutum obtained from the kidney of experimentally infected Physa and Lymnaea snails. Ultrastructural studies revealed three cyst walls, an outer, middle and inner. The outer wall was more electron-dense than the middle, and contained coarser granules than those found in the middle layer. The inner wall was lamellated and contained membranous whorls. Collagenous fibers presumably of host origin surrounded the outer cyst wall. The outer and middle cyst walls stained identically with all histochemical procedures used. These walls contained acid mucopolysaccharides and glycoprotein, whereas the inner cyst wall contained glycoprotein. All cyst walls stained positively with a variety of protein stains.     

Growth characteristics of human epidermal kerationcytes from newborn foreskin in primary and serial cultures

Summary Using gels of acid-soluble, collagen as a culture surface, trypsin-released keartinocytes from 0.1-mm, split-thickness sections of newborn foreskin may be plated with high efficiency and subcultured at a 1∶5 split a 2- to 3-week intervals for three subpassages. When plated at a density of 3.2×10⁴ cells per cm², keratinocytes attach to the gel with an efficiency of over 70%; after a lag phase of 3 days, the cells multiply exponentially with a doubling time of 60 hr. Cultures reach a growth-plateau phase at a density of 47.7×10⁴ cells per cm². Both hydrocortisone and epidermal growth factor (EGF) stimulate slightly the growth of primary cultures; both factors are required for proliferation of the 2nd and further passage of keratinocytes. As the cultures reach, confluence multilayers, of stratified cells are formed and cells of squamous morphology are spontaneously released from the surface. When the released cells and the attached cells are pulsed with [³H]-histidine and [¹⁴C]-leucine, a higher ratio of histidine to leucine is observed in the released cells indicating the biochemical onset of maturation. Orange G-Aniline Blue staining of the released cells show some of the cells to be completely keratinized. Fibrous proteins extracted from the cultured cells and analyzed by sodium dodecyl sulfate (SDS) gel electrophoresis display the characteristic stratum corneum proteins of 60,000 and 66,000 daltons. Supported in part by Grants AM 14121 and AM 19595 of the U.S. Public Health Service.     

The synthesis of cartilage collagen by rabbit and human chondrocytes in primary cell culture

Summary This report describes a method for preparing primary cell cultures of differentiated rabbit sternal and human vertebral cartilage cells. These cell cultures were shown to synthesize primarily α1 chains, which is taken to mean that at least 82% of the collagen produced is cartilage specific collagen (type II). This work was supported in part by grant HD-05505 from NIH.     

The effect of streptococcus on the persistence of Brugia malayi and on the production of elephantiasis in cats

Cats were exposed to 200 Brugia malayi larvae on one hind foot over a 3 week period. Six weeks after the initial exposure to B. malayi, 10 of the cats were challenged on both hind legs with a Group G streptococcus. The remaining 10 cats were not exposed to the streptococcus. Following bacterial challenge, the B. malayi-infected leg of 9 of 10 cats displayed sequelae including erysipelas and abscesses. In addition, 5 of the affected legs had an elephantoid appearance, both by gross observation and as seen at necropsy 10 weeks after the initial B. malayi infection. The contralateral, uninfected leg of each cat remained normal in appearance. Histologic processing and examination of the elephantoid tissue showed it to be collagen; eosinophils and mast cells were plentiful in the collagen matrix. In the controls, only 1 animal displayed erysipelas and no abscesses were seen. Lymphedema seen in the B. malayi-infected leg of 5 control cats was less extensive than in uninfected cats challenged with streptococci and at necropsy no significant collagen matrix was evident. The location and number of worms in the lymphatics were noted. This study demonstrated that secondary microbial infections can contribute to the causation of elephantiasis under certain circumstances and that developing B. malayi were in some way adversely affected by the streptococcal involvement of the filaria-infected lymphatics.