M. Michael Sigel

Studies with Cells From Marine Fish in Tissue Culture.

Summary Cells of tissues obtained from marine fish were shown to grow by the explant method at 20 °C in a medium designed for mammalian cells. Preparation of monolayers from cells dispersed by primary trypsinization was accomplished when additional sodium chloride was added to mammalian cell media. Cells in continuous culture were found to multiply more efficiently (as regards time required for sheet formation and number of cells required for satisfactory growth) than did cells in primary culture.

IgM antibodies in fish mucus.

Discussion and Summary As early as 1935, Nigrelli (4) observed that the mucus of certain fishes was toxic to parasites and suggested that this activity might be due to antibody or that the mucus itself might be toxic to parasites. Our studies have indicated that normal gar mucus contains detectable antibody to several erythrocytes. Other work in this laboratory (Bradshaw, C. M., and Sigel, M. M., unpublished work) with mucus of normal snapper, Lutjaneus griseus, and normal bowfin, Amia calva, also demonstrated detectable antibody activity in surface mucus. After primary immunization of gar with SHE their surface mucus antibody levels increases considerably. Lower titers of hemagglutinin were recently reported by Fletcher and Grant in the mucus of plaice (5). We have previously reported that normal gar serum contains cold agglutinins with maximum reactivity at 4°. Interestingly, the normal and immune mucus antibody titers were increased by downshifting the temperature from 35 to 20° but no further enhancement was noted at 4°. The hemagglutinin in gar mucus has several properties of macroglobulin antibody. It is located in the excluded fraction from Sephadex G-200, its activity is removed by absorption with RAGIgM and it is totally susceptible to reduction. The absence of 7S immunoglobulin is consistent with our findings (Bradshaw, C. M. and Sigel, M. M., to be published) on the absence of 7S IgM in gar serum. Fletcher and Grant (5) found that the antibody present in plaice serum and mucus was not retained by Sephadex G-200, and had similar carbohydrate and amino acid compositions. Although they did not determine the S value of the mucus antibody, the serum antibody had an S20,w of 12.4. The provisional conclusion based on our investigation and on the findings of Fletcher and Grant (5) is that IgM of fish occurs as a macroglobulin secretory protein.

Differential effects of recombinant interleukin-1α and β on Leydig cell function

Abstract Previously we have reported that human chorionic gonadotropin(hCG)-stimulated testosterone biosynthesis was markedly inhibited by purified natural human interleukin-1(IL-1). In the present study we evaluated the effects of human and murine recombinant IL-1 (rIL-1) on Leydig cell steroidogenesis in primary culture. Human rIL-1β caused a dose-dependent inhibition of hCG-, 8-bromo cyclic AMP-, and forskolin-induced testosterone formation. In contrast, human rIL-1α was considerably less potent. When the effects of the cytokines were corrected for their biological potencies, human rIL-1 β and murine rIL-1α were still more effective than human rIL-1α in inhibiting testosterone production (at least 100-fold more potent). Thus, even though IL-1α and IL-1β bind to the same receptors on T cells, Leydig cells exhibit differential sensitivity in response to rIL-1α and rIL-1β which is partly species dependent.

Phylogenetic studies on T cells. I. Lymphocytes of the shark with differential response to phytohemagglutinin and concanavalin A

Abstract Peripheral blood lymphocytes of the nurse shark ( Ginglymostoma cirratum ) respond to stimulation by concanavalin A (Con A) as evidenced by increased incorporation of tritiated thymidine. Separation by means of Ficoll-Isopaque yields two or more bands and a sediment, all of which contain lymphocytes responsive to Con A. Only the bottom cells react to phytohemagglutinin (PHA). This reaction cannot be detected in the unseparated lymphocyte population. Thus, only a unique subset of lymphocytes appears to be responsive to PHA and is probably blocked in its response by other cells. The findings suggest that differentiation toward Con A responsiveness may have preceded phylogenetically the responsiveness to PHA. Judging by the requirement for high concentrations of both mitogens the receptor sites on shark lymphocytes appear to be present in lower densities than on lymphocytes of higher vertebrates.

Tumor necrosis factor-α enhances inhibitory effects of interleukin-1β on Leydig cell steroidogenesis

Abstract Human recombinant tumor necrosis factor-α (rTNFα) alone (up to 1000 units/ml) did not alter either basal or human chorionic gonadotropin (hCG)-Induced testosterone formation in primary culture of rat Leydig cells. However, concomitant addition of rTNFα with human recombinant interleukin-1β (rIL-1β) enhanced the inhibitory effects of rIL-1β. The rIL-1β dose response curve was shifted to the left (IC50 changed from 1 ng/ml to 0.3 ng/ml). Even though rTNFα had no effect on testosterone formation, hCG-stimulated cyclic AMP formation was inhibited by rTNFα in a dose dependent manner. In the presence of both rTNFα and rIL-1β, hCG-induced cyclic AMP formation and binding of [ 125 I]-hCG to Leydig cells were further inhibited. Testicular macrophages represent about 20% of the interstitial cells. TNFα and IL-1 may by produced locally by interstitial macrophages and have paracrine effects on Leydig cell function.

Antibody response of fish to viral antigens.

INTRODUCTION It was previously reported from this laboratory that marine fishes can make antibodies to PR8 influenza virus.I , 2 The initial studies have now been extended to other viruses. These, along with further observations on the nature of the immunologic response, and on quantitative and qualitative differences in the response of fishes belonging to four families of two classes, are the subject of the present paper.

Primitive immunoglobulins and other proteins with binding functions in the shark.

The nurse shark (Cinglymostonta cirraturn) has proved to be a valuable animal in studies on the development of specific and nonspecific immunity. This species occupies an important phylogenetic niche; its origin dates back to the Mesozoic era, more than 100 million years ago. These animals can be maintained in captivity in good health for long periods of time. This paper collates the findings of investigations (some published, others not yet in print) conducted in my laboratory, with the participation of Drs. L. W. Clem, S. Rudikoff, D. M. Lopez, R. J. McDonough, and Miss F. de Boutaud; in Dr. Clem’s laboratory at the University of Florida, with the collaboration of Drs. P. A. Small, G. A. Leslie, and D. G. Klapper; in Dr. E. W. Voss’s laboratory at the University of Illinois, and in Dr. E. A. Kabat’s laboratory at the College of Physicians and Surgeons, Columbia University, with Dr. V. Harisdangkul. For the past several years we have been studying the serum proteins of this animal, placing the major emphasis on immunoglobulins. Initial immunoelectrophoretic analyses suggested that the shark serum contained an immunoglobulin of the IgG type. This, however, proved to be a cathodic moving transferrin.1 Exhaustive investigations have failed to disclose the presence of IgG in the blood of either nurse sharks or the more recently evolved lemon sharks (Negaprion brevirostris) . In both species the immunoglobulins are restricted to the IgM class.’. This immunoglobulin was found to occur as a 19s pentamer and a 7s monomer,t however. In this presentation I will distinguish between IgM molecules that have specific antigen-binding activity (conventional antibody) and IgM molecules that appear to possess binding sites capable of binding antigens of diverse specificities (primitive binding). Another group of proteins with ligand-binding capabilities is represented by the fructosanspecific protein, which is not an immunoglobulin. In some respects these proteins resemble lectins. Thus the nurse shark appears to represent a link in the development of molecules with specialized binding sites, in that it is capable of forming proteins with the immunological functions of the true antibody of higher vertebrates, while also retaining the vestigial capacity to form lectinlike proteins analogous to those found in invertebrates and plants.

A differential effect of IgM and IgG antibodies on the blastogenic response of lymphocytes to rubella virus.

Abstract Peripheral blood lymphocytes of rabbits immunized with live rubella vaccine respond to rubella virus antigens in tissue culture with increased DNA synthesis as measured by incorporation of 3 H-thymidine. This reaction can be inhibited by rubella antibody. A dose dependent effect was observed when antibodies in whole serum were mixed with virus prior to addition to lymphocyte cultures. When antisera were fractionated and their individual immunoglobulins tested, a paradoxical effect was obtained. Immune IgG although it was highly effective in neutralizing the virus was incapable of inhibiting the lymphocyte response and at times caused an increased response. In contrast, immune IgM which was less efficient in neutralizing virus caused significant suppression of the blastogenic reaction. By themselves these results might have signified that IgG and IgM antibodies have different specificities or different binding properties with respect to viral surface antigens. However, immune complexes consisting of virus and IgM reduced response of both rubella immune and normal rabbit lymphocytes to PHA. This nonspecific inhibitory action required a specific step of antigen and IgM antibody interaction and normal IgM-virus mixtures or mixtures of anti-rubella IgM and poliovirus or influenza virus did not suppress lymphocyte response to PHA. Anti-rubella IgG complexed with rubella virus did not suppress the PHA response. The IgG function was apparently limited to neutralization of the infectivity of rubella virus whereas the major role of IgM was manifested through its suppressive effect on lymphocyte reactions.

Quantitative Methods for Measuring Cell Growth and Death

Publisher Summary This chapter examines the quantitative methods for measuring cell growth and death. Cell growth and death can be investigated by microscopic observations of cells in monolayers. More precise measurements are achieved by cell counts performed by the means of a hemocytometer or by automatic electronic cell counters. Visual counting is usually accompanied by vital stains which distinguish viable cells from damaged or dead ones. The measurement of cell growth by the determination of cell protein concentration is a rapid and reliable method which can be employed in large-scale testing. The amount of cell growth can be determined with cultures grown in suspension and also with cultures adhering to a surface. The ease of determination of the initial protein concentration and of the subsequent concentrations during the time course of the experiment makes this method very attractive. With appropriate conversion factors, the values may be changed to dry weight, protein nitrogen, or cell count. This method has found great utility for assessing cytocidal and cytostatic effects of extrinsic substances and is also useful for monitoring the dynamics of cell populations during the logarithmic phase of growth.

An interferon antagonist: Its effect on interferon action in mengo-infected ehrlich ascites tumor cells☆

Abstract Ehrlich ascites tumor (EAT) cells were pretreated with crude or purified interferon made in L cells with live NDV. After 14 hours of infection with mengovirus, in the presence of actinomycin D and orthophosphate- 32 P, the cells were extracted for RNA with a mixture of hot phenol-Dupanol. The extracts were chromatographed on MAK columns and assayed for radioactivity and infectivity. Crude interferon, active in L cells, had no inhibitory effect on the synthesis of viral RNA in EAT cells, and in fact caused a three- to fourfold stimulation. In contrast, interferon purified on a CM-Sephadex (C25) column completely suppressed the production of viral RNA. A substance eluting just ahead of interferon was found to prevent activity of interferon in EAT cells. Enzyme inactivation studies revealed the antagonist to be a protein. It was concluded that the apparent intraspecific selectivity of crude interferon was due to an effect of the antagonist which is manifest in EAT, but not in L cells.