Frederik B. Bang

Long-term growth of chicken fibroblasts on a collagen substrate☆

Abstract In 3 separate experiments, cells derived from chick embryo muscle explants have been grown in either Waymouths medium or Lings (AN.54) medium with 20% human placental cord serum. Continuous transfer and new outgrowth from a succession of 2 × 2 mm fragments has continued for periods of 24 to 44 months. Continuous growth was achieved only on a collagen substrate, and no continuous growth was obtained when cells were transferred to glass. When incubator temperature was raised to 43 °C over a period of 1 month, new cell types developed and had the capacity to both survive and grow directly on glass for several months.

RESPONSES OF A VARIETY OF NORMAL AND MALIGNANT CELLS TO CONTINUOUS CULTIVATION, AND SOME PRACTICAL APPLICATIONS OF THESE RESPONSES TO PROBLEMS IN THE BIOLOGY OF DISEASE

For about half a century now, published reports show that investigators have progressively widened their interests in, and have more seriously examined the behavior of, explanted living tissues and their constituent cells, and that these investigators have tried in a number of ways to influence and control cellular activity. These biopsy studies of primary explants and continuous cell cultures have described work on tissues, especially from avian and mammalian hosts, in various stages of development up to senescence and death, and from experimentally altered and diseased hosts. Especially during the latter half of this period, there has developed an expanding interest in improving the techniques of tissue culture and a more serious application and planned projection of these methods to certain problems in the biology and control of disease. We have tried to contribute to these areas during this period.‘, 2 , 3 4 . The technical achievements to date, and their potential applications, demonstrate the feasibility of comparing the derived normal or controlled state with a diseased state of the tissues or cells examined freshly during several days or more following biopsy, or as a more or less stabilized cell population during continuous cultivation. The tissue and cell responses in vilro, however, should be expected to vary somewhat from the natural state following varying periods of maintenance in vitro. In fact, whatever may have been the original state of the tissue prior to explantation, and however dependent, of course, it may have been upon the conditions imposed, one should be prepared to expect a variety of responses following explantation, including considerable shifts in cell composition. The responses of certain cells to continuous cultivation might be: a continuation of a previous rapid growth, as in a tumor; a continuation of an original dormant state (an apparently difficult state to maintain in vilro) ; a relative reversal of a previous state of increased physiological activity; a new transformation or adaptation;6. or, perhaps, whether wanted or unwanted, varying degrees of cell degeneration and death. In this latter category, we must hold ourselves responsible for the degenerative changes that have set in and attempt to account for them in a true spirit of scientific endeavor. Socalled ‘Lnonspecific” degeneration, for example, often plagues those wishing to keep cells in a good s t d e of health during continuous cultivation. Are the

Cell recognition: Reactions of the sea star (Asterias vulgaris) to the injection of amebocytes of sea urchin (Arbacia punctulata)

Abstract Recognition of self and non-self by invertebrates apparently varies from species to species and as yet there is no general understanding of this variation within the phylum. Injection of Arbacia (sea urchin) cells, about half of which are deeply pigmented, into Asterias (sea star) in vivo was followed by an abrupt drop in the number of circulating amebocytes of the host. The Arbacia cells adhered to and were phagocytosed by the host cells; the latter consistently clumped within the papulae and later ruptured to the exterior. The interaction of Asterias and Arbacia amebocytes was not prevented by either the treatment of the host Asterias , or by the treatment in vitro of Arbacia cells, wth N -ethyl maleimide (NEM). Injection of Asterias cells into Asterias did not elicite cell clumping, nor was it followed by a drop in the circulating amebocytes.

Genetics of resistance of animals to viruses. I. Introduction and studies in mice.

Publisher Summary Inherited resistance to animal viruses may be conveniently classified into three types: monogenetic, following simple mendelian ratios; polygenetic; and cytoplasmic. A virus is a unique cellular parasite, dependent upon the host for reproduction and nourishment in a variety of different ways. Since, as with the other types of parasites, the host and the parasite have necessarily evolved together. It is a distortion to consider the resistance of the host, without considering the evolutionary steps in the development of this extreme form of parasitism; therefore, this chapter reviews some of the ideas put forward about host-agent interactions in plants as well as in animals. The importance of genes in regulating the resistance to disease, including parasites and parasitoids, is apparent if the disease is considered to be an important evolutionary force. The selective effects of viruses have not yet been adequately studied. Continued attempts to find a correlation between the different blood groups and differing severity of smallpox infection clearly searched for selective forces, but the results were inconclusive. Most of the knowledge of genetic resistance to virus disease rests on the study of resistance to selected agents in various inbred strains of mice and chickens, rather than on any knowledge of the effects of genetic resistance in a natural heterozygous population. The increasing frequency, however, with which genetic resistance is found, is in itself an evidence that these genes are important in natural outbred populations. In addition, there are increasing numbers of virus diseases, in which the viral agent seems to be inherited in a mendelian fashion.

The effect of vitamin A deficiency and Newcastle disease on lymphoid cell systems in chickens.

Summary A diet without vitamin A but otherwise nutritionally complete was given to chicks from time of hatching. Control diets with adequate vitamin A were given to two other groups. At age 21 days, Newcastle disease virus (NDV) was nasally inoculated into all 3 groups; controls of each group remained uninoculated. Between Days 1 and 3 after NDV inoculation the A-depleted chicks showed significant loss of lymphocytes from the cortex of both the thymus and the bursa of Fabricius, while those on control diets did not show any loss. On Days 5 to 9, there were minimal to moderate effects on populations of lymphocytes in these organs of the group of chicks given adequate vitamin A, but consistently much more severe effects became apparent in the A-depleted group in which 3 of 7 (42%) of the cortices were essentially devoid of lymphocytes. Granulocytes were prominent in areas of thymic and bursal cortex depleted lymphocytes. Noninfected A-deprived chicks and chicks on a normal diet showed relatively modest depletion of cortical lymphocytes from both organs beginning 5 days after NDV infection. We are indebted to Dr. John G. Bieri, Laboratory of Nutrition and Endocrinology of the National Institute of Arthritis and Metabolic Diseases, Bethesda, MD, for help and advice in devising the vitamin A-deficient diets.

Congenic strains of Mice Susceptible and Resistant to Mouse Hepatitis Virus

Summary A congenic strain of C3HSS mice, which is histocompatible with C3H mice but differs from them in susceptibility to mouse hepatitis virus (MHV), has been developed by introducing the gene for susceptibility to the MHV-PRI virus from the PRI mice. This was accomplished by continual back-crossing of the hybrids to the C3H mice, but at the same time by selection of susceptibility by use of macrophage culture tests. After 20 back-crosses, a strain homozygous for susceptibility was produced by brother-sister mating of individual mice whose potential for carrying the recessive gene for resistance was tested in progeny. Since the original choice of mice for breeding was based on in vitro macrophage susceptibility, and since highly susceptible mice were developed on the same basis, it seems evident that macrophage susceptibility is an integral aspect of mouse susceptibility. The continued production of almost 50% susceptible mice in the back-crosses is further evidence of the dominant one-locus explanation of genetic susceptibility to this agent. Incomplete penetrance may also be present in 8 and 9 week old mice of the C3HSS strain since there was a sharp decrease in susceptibility of these mice even though their macrophages in culture maintained full susceptibility. We wish to thank Dr. Ilan Shif for his contribution in testing for susceptibility.

Effect of Water Deprivation on Nasal Mucous Flow.

Summary A striking depression in rate of flow of the nasal mucous blanket was observed in 4 water-deprived herring gulls, in contrast to 10 normally hydrated gulls. The observation was repeated in a series of 20 water-deprived and 10 normally hydrated chickens. In both species normal mean rate of flow was about 10 mm/minute. Two gulls and 2 chickens that were deprived then rehydrated showed a normal rate of motion of the blanket. Anchoring of the mucous sheet to the mucosal surface by fine strands of mucus was seen in whole mounts and histological sections of conchae of dehydrated baby chicks.

A fibrillar structure in rat fibroblasts as seen by electron microscopy.

During the course of comparative tissue culture studies of a strain of normal rat fibroblasts and its malignant cell derivative, 1 we obtained preparations of both of these cell types which under the electron microscope† showed an unmistakable fibrillar structure in the thinly spread cytoplasm. The fibrils, of an estimated thickness of 10 to 100 mμ, may converge and diverge in fan-like formations of great regularity (Fig. 1) but are more often seen gathered together in long bands of varying widths (Fig. 2). The composite bands are seen clearly in phase microscope movies of the cells, 3 but it is only by the increased resolution available with the electron microscope that the nature of the bands becomes clear. They are apparently composed of long thin converging fibrils which in general radiate from the dense central area. That the fibrils are not produced by a wrinkling of the formvar membrane on which the cell is stretched is indicated by their uniformity, by their sharp change in direction in certain areas of the periphery, and by their fine converging and diverging structure. Besides this, no wrinkles were seen in the membrane outside of the cellular area where clear visualization is possible. Of interest are the relations of these bands to the general structure. The mitochondria, often long and thin, seem frequently to be associated with individual fat droplets. Some of the elongated mitochondria are found in areas which show many microsomes, with the whole area bounded by parallel fibrous bands. This appears to be a static representation of the streams of material which can be seen in the movies of these cells. In other areas, the great masses of mitochondria may be seen crossing those fibrous bands.

T and B lymphocyte rosetting in undernourished children.

Summary T and B lymphocyte rosetting values were obtained for 18 children with kwashiorkor, marasmus, or nutritional edema. T cell values were subnormal in all malnutrition classes, but were lowest in children with kwashiorkor. Four of five malnourished children who were sensitized with 2,4-dinitrochlorobenzene (DNCB) before refeeding failed to respond to repeated subsequent challenges; five of six children who were sensitized after refeeding responded strongly to the first challenge.

Replacement of virus-destroyed epithelium by keratinized squamous cells in vitamin A-deprived chickens.

Summary One localized area of nasal mucous membrane in chickens was selectively susceptible to initial infection with a myxovirus (NDV) and a giant-cell-forming herpesvirus (LTV). The same quite extensive mucosal area shows earliest signs of incipient keratinizing metaplasia in vitamin A-deprived chickens, and complete conversion to keratinized epithelium following NDV infection. Specific questions of interplay of A-deficiency and respiratory infection are open to study in this whole-animal model.