Temperature and species differences in susceptibility of kidney cell cultures to mercury toxicity

Summary The effect of temperature on inorganic mercury toxicity was investigated using kidney tissue culture systems. The relative susceptibility of rabbit (homeothermic) kidney to mercury intoxication was compared to that of Coho salmon (poikilothermic) kidney over temperature ranges consistent with the habitat of each of the two species. It was demonstrated that susceptibility to mercury toxicity is species dependent; that is, the rabbit kidney cells tolerated higher mercury concentrations in the medium than did the fish-derived cells. Within a given species, susceptibility to mercury toxicity was temperature dependent. Decreasing the temperature increased the toxicity of mercury to cultures of rabbit kidney cells, whereas decreasing temperatures decreased the effect of mercury toxicity on the salmon kidney cells. As a consequence, fish taken from arctic waters are liable to be more toxic when introduced into mammalian food chains. Albumin was shown to act as a protective agent in vitro against inorganic mercury toxicity. Research was supported in part by the University of Victoria Faculty Grant No. 08-869 and a Medical Staff Research and Education Fund Grant from Wayne County General Hospital, Eloise, Michigan.     

A primary culture of parotid acinar cells retaining capacity for agonists-induced amylase secretion and generation of new secretory granules

Exocrine acinar cells, like parotid cells, have difficulty in maintaining their functions in cell lines or in primary cultures. For this reason, molecular studies on exocrine cell functions are unsatisfactory. To examine the mechanisms whereby the functions of parotid acinar cells are maintained, we attempted to establish a system for primary culture and transfection of exogenous genes. Acinar cells were dispersed from rat parotid glands by digestion with enzymes and were cultured in a medium containing rat serum. Most of the cultured cells had secretory granules that contained amylase, suggesting that they were derived from acinar cells, although they spread on the dish surface and formed filopodia. The cultured cells retained both granules and the ability to release amylase in response to -adrenergic and cholinergic agonists, even 48 h after dispersion. However, the total amount of amylase in the cells decreased rapidly from 24 to 48 h after dispersion. These results suggested that amylase synthesis was more damaged than the machinery for exocytosis during culture in vitro. VAMP2 gene fused with enhanced green fluorescence protein was transfected into the dispersed acinar cells, and VAMP2 protein was expressed and localized to amylase-containing granules, as normally seen for endogenous VAMP2 protein. This indicated that new granules were generated, and that protein sorting was functional. The cells cultured by this method maintained their functions for at least 48 h. They can be used for examining the effects of exogenous genes on parotid acinar cell functions, such as regulated exocytosis and the maturation of secretory granules.This work was supported in part by Grants-in-aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (11771148, 13771104, 16390534, 16591868), by Nihon University Multidisciplinary Research Grant for 2001 and 2002, by a Suzuki Memorial Grant of Nihon University School of Dentistry at Matsudo (General Individual Research Grant for 2000 and 2002 and Joint Research Grant for 2003), and by a Grant-in-Aid for a 2001 Multidisciplinary Research Project from MEXT.     

Are all lymphoid blasts in the cell cycle?

Labelling index after one or repeated intravenous injections of 3H-thymidine was measured for various subpopulations of lymphatic cells in different canine lymphoid compartments and correlated with cell morphology. High doses of tritiated thymidine were injected and exposure times of up to 211 days were used. The labelling indices of lymphoid blasts were comparable in all tissues investigated. Labelling index varied from 100% in immunoblasts to 4% in small-sized lymphocytes. Approximately 80% of immunoblasts were labelled 1 h after 3H-thymidine application and 100% labelling was obtained after 12 h repetitive 3H-thymidine labelling. In contrast with mediumsized and large lymphocytes, immunoblasts seem to be rapidly proliferating cells in the dog with almost no G_o cells. Supported by the Deutsche Forschungsgemeinschaft DFG Grant SFB 112     

Adoptive immunotherapy with peripheral blood lymphocytes cocultured in vitro with autologous tumor cells and interleukin-2

A clinical trial of adoptive immunotherapy was carried out with peripheral blood lymphocytes (PBL), cocultured in vitro with autologous tumor cells and interieukin-2 (IL-2), in 14 patients with advanced melanoma. PBL from these patients were cocultured with irradiated autologous tumor cells for 7 days, which was followed by expansion in IL-2-containing medium. These lymphocytes were returned to the patient along with intravenous IL-2 at doses up to 2×10⁶ IU m^–2 day^–1. A dose of 300 mg/m² cyclophosphamide was administered to each patient intravenously 4 days prior to each treatment. Following coculture, the lymphocytes were primarily CD3⁺ T cells and they expressed varied degrees of cytotoxicity against autologous melanoma cells. In 9 patients the activated cells were al least 80% CD4⁺ and in 2 cases they were mostly CD8+. Some of the activated cells exhibited suppressor or helper activity in a functional regulatory coculture assay. No major therapeutic response was observed in this study. Minor responses were observed in 2 patients. Toxicities were those expected from the IL-2 dose administered.This work has been supported by an American Cancer Society Institutional Research Grant (ACS-IRG 91-230), by the University of Connecticut Clinical Research Center (grant 0021), and by the Hartford Hospital Research Fund (grant 1017-20-018). Dr. Sporn is a recipient of American Cancer Society Clinical Oncology Career Development Award 90-230     

An immunochemical method for the detection of the expression of human gene loci in human-rodent somatic cell hybrids with special reference to the GPI locus

Species-specific antibodies, prepared against unpurified human and Chinese hamster fibroblast extracts, were used to identify the parental origins of enzymes in human-hamster somatic cell hybrids. Results of the detection of the expression of the human glucosephosphate isomerase gene locus (GPI) by electrophoretic and immunochemical techniques were concordant in 17 instances. The human GPI synthesized by fibroblasts derived from skin explants and by somatic cell hybrids retaining the human GPI locus, regardless of whether the human parental cells were lymphocytes or fibroblasts, appeared to be antigenically identical.This work was supported by the Medical Research Council of Canada (Grant MA-4061). Personnel and operating support were provided by The Children's Hospital of Winnipeg Research Foundation, Inc.     

Cell-mediated immunity in human acute myeloblastic leukemia

Summary Lymphocyte stimulation tests with autologous myeloblasts were performed in 31 patients with nonlymphatic acute leukemia. Twenty-five patients were receiving chemotherapy combined with immunotherapy; six received chemotherapy only. Thirteen nonleukemic patients with various disorders and six healthy control patients were also studied.It was found that 4/15 patients in 9/38 tests had autologous lymphocytes stimulated by autologous circulating myeloblasts. Bone marrow cells also effected stimulation, significantly more often if the marrow was taken in relapse than when it was taken in remission.However, so-called immunotherapy with allogeneic leukemic myeloblasts and BCG could not be shown to increase these frequencies. Nor did it significantly increase the degree of stimulation measured as DNA synthesis in lymphocytes.Moreover, nonleukemic bone marrow cells from patients with other disorders also stimulated autologous lymphocytes in 1/13 patients. No recognition of autologous myeloblasts was observed when the responding lymphocytes were taken in incomplete remission or during the month preceding relapse.with the technical assistance of T. Lehtinen and A. M. SjögrenSupported by the Swedish Cancer Research Foundation Grant no. 699-B76-04XA     

Cytochemical observations on the development of antibody-forming cells in popliteal lymph nodes by an improved immunoperoxidase method

Summary Precursors of cells forming antibody to horseradish peroxidase (HRP) became detectable in lymph nodes of rats and rabbits after the sensitivity and the specificity of the previously used immunocytochemical procedure were increased. The sensitivity of the method was increased by shortening the time of fixation of the tissue from 24 h to 5 h, and by exposing the antibody in the tissue section to a higher concentration of the antigen for a longer time period than previously. The specificity of the method was enhanced by inhibiting the endogenous peroxidase activity with phenylhydrazine. Five to 8 days after the injection of the antigen, many precursors of plasma cells showed the specific reaction for the antibody in the medulla of the lymph node. Most of the precursor cells were smaller than mature plasma cells and had small, eccentrically located nuclei. The smallest of the precursor cells had the appearance of lymphocytes. From 8 days to several weeks after the injection of the antigen, mature plasma cells characterized by an intense antibody reaction coexisted in the medulla with more weakly stained precursor cells. Lymphocytes, blast cells, and transitional cells containing the specific antibody appeared in germinal centers of the cortex not earlier than 2–3 weeks after the injection of the antigen. From 2–3 weeks to several months after the injection of the antigen, a considerable number of small and medium-sized lymphocytes in the diffuse cortex and in the sinuses contained the anti-HRP antibody. In some animals, a strong antibody reaction occurred in the cytoplasm of these lymphocytes. In other animals, a relatively weak reaction was observed in the perinuclear areas and on the cell surfaces. It is suggested that these lymphocytes may be memory cells.This work was supported by General Research Support Grant BR-5366 from the General Research Support Branch, Division of Research Facilities and Resources, National Institutes of Health     

Lymphocytes from children and infants mediate natural cell mediated cytotoxicity, (cytotoxicity)

Summary Lymphocytes from infants and young children were tested for natural cell mediated cytotoxicity (NCMC) against K562 and CCRF-CEM. NCMC by lymphocytes from pediatric donors of all ages was equivalent to that mediated by lymphocytes from adults. Since it has been suggested that the biological function of NCMC is to effect immunological surveillance against cancer, the appearance of NCMC effector cells early in development is consistent with early mobilization of the policing mechanism.This work was supported by Grant CA 25765 from the National Cancer InstitutePartially supported by a Grant from the Concern Foundation.     

Direct connections between mitochondria and catecholamine-storage vesicles demonstrated by high voltage electron microscopy in rat adrenal medulla

Summary Tubular channels from mitochondria to catecholamine-storage vesicles have been demonstrated in thick sections of adrenal medullary tissue from hypoglycemia-stressed rats by the use of the high voltage electron microscope. The function of these connections is not presently known although they may serve as channels for the transport of materials such as high-energy nucleotides from one organelle to the other. The present study has examined only the adrenal medulla, but it should be considered that such connections may also exist in other neural cells and possibly other cells in which there is intracellular transport of ATP.This work was supported by NIH DRR 70-4136, WVU Medical Corporation Research Grants, Biomedical Research Support Grant 5 SOI RR05433, WVU Senate Research Grant, and West Virginia Heart Association Research Grant. We wish to thank Barbara Coalgate, Billie Pack, and the staffs of the HVEM laboratories at U.S. Steel and University of Colorado.     

Neurospora glucamylase and a mutant affected in its regulation

Neurospora glucamylase is a glucose-repressible extracellular enzyme. The enzyme was purified to homogeneity and found to have a molecular weight of 82,000 and to release glucose from either maltose or amylose. The rate of glucamylase synthesis increases more than 100-fold when cells are transferred from a glucose-containing medium to a glucose-free medium. Increased production of glucamylase begins within 30 min of the transfer. Glucamylase is rapidly secreted into the medium. A mutant affecting the ability of glucose to repress the synthesis of the glucose-repressible extracellular enzymes glucamylase and invertase has been isolated and studied. The mutant constitutively synthesizes and secretes a glucamylase which is indistinguishable from the wild-type enzyme.Funds for this research were provided by Grant PCM-8011772 from the National Science Foundation and by a grant from the Research Development Fund of The Research Foundation of the State University of New York.     

Optimal strategies in immunology III. The IgM-IgG switch

Summary During a primary immune response generally two classes of antibody are produced, immunoglobulin M (IgM) and immunoglobulin G (IgG). It is currently thought that some lymphocytes which initially produce IgM switch to the production of IgG with the same specificity for antigen. During a secondary immune response IgG is the predominant antibody made throughout the response. In this paper we address the question of why such apparently complicated modes of response should have been adapted by evolution.We construct mathematical models of the immune response to growing antigens which incorporate complement dependent cell lysis. By comparing the times required to eliminate antigen we show that under certain conditions it is advantageous for an animal to switch some of its lymphocytes from IgM to IgG production during a primary response, but yet to secrete only IgG during a secondary response. The sensitivity of such a conclusion to parameter variations is studied and the biological basis and implications of our models are fully discussed.Portions of this work were performed under the auspices of the U.S. Department of Energy. A.S.P. was also supported by the National Science Foundation under Grant No. ENG-7904852 and BRSG grant S07 RR05664-11 awarded by the Biomedical Research Support Grant Program, Division of Research Resources, National Institute of Health. A.S.P. is the recepient of an NIH Research Career Development Award 1K04 AI 00357-01. S.R. was a recipient of NIH Fellowship 5 F32 AI05107-02     

Developing a microfluidic-based system to quantify cell capture efficiency

Micro-fabrication technology has substantial potential for identifying molecular markers expressed on the surfaces of tissue cells and viruses. It has been found in several conceptual prototypes that cells with such markers are able to be captured by their antibodies immobilized on microchannel substrates and unbound cells are flushed out by a driven flow. The feasibility and reliability of such a microfluidic-based assay, however, remains to be further tested. In the current work, we developed a microfluidic-based system consisting of a microfluidic chip, an image grabbing unit, data acquisition and analysis software, as well as a supporting base. Specific binding of CD59-expressed or BSA-coupled human red blood cells (RBCs) to anti-CD59 or anti-BSA antibody-immobilized chip surfaces was quantified by capture efficiency and by the fraction of bound cells. Impacts of respective flow rate, cell concentration, antibody concentration and site density were tested systematically. The measured data indicated that the assay was robust. The robustness was further confirmed by capture efficiencies measured from an independent ELISA-based cell binding assay. These results demonstrated that the system developed provided a new platform to effectively quantify cellular surface markers effectively, which promoted the potential applications in both biological studies and clinical diagnoses. Supported by the National Key Basic Research Program of China (Grant No. 2006CB910303), National Natural Science Foundation of China (Grant Nos. 30730032 and 10332060), National High-Tech Research and Development Program of China (Grant No. 2007AA02Z306) and Chinese Academy of Sciences Grant (Grant No.2005-1-16)     

Optimal strategies in immunology

Summary The optimal strategy available to the immune system for responding to a non-replicating thymus-independent antigen is examined. By applying Pontryagin's maximum principle to a set of mathematical models of lymphocyte populations and their antibody production, it is found that the optimal strategy of bang-bang control appears robust. In a variety of structurely related biological models, similar behaviour is observed.The models that we consider assume that antigen triggers a population of B-lymphocytes. These triggered lymphocytes can either proliferate and secrete modest amounts of antibody or differentiate into nondividing plasma cells which secrete large amounts of antibody. For biologically reasonable parameter values it is found that for low doses of antigen, immediate differentiation into plasma cells is optimal, while for high antigen doses a proliferative state followed by differentiation is the best strategy.Work performed under the auspices of the U. S. Energy Research Development Administration.Work supported by NSF Grant No. BMS 75-18897.     

Keratinization and the effect of vitamin A in aggregates of a squamous carcinoma cell line NBT II

Summary The terminal differentiation, keratinization, of a rat bladder tumor cell line, NBT II, occurred in multicellular aggregates. After aggregation, these cells did not undergo a round of mitosis before keratinization. 5-Bromodeoxyuridine added to the monolayer cell culture 2 days before aggregation completely prevented this differentiation; it was ineffective when added at the time of cell aggregation. Vitamin A prevented the keratinization of NBT II cells in aggregates but did not inhibit aggregate formation; it enhanced the number of cells engaged in DNA synthesis. This model appears to be very useful for analyzing the mechanisms of terminal differentiation and its modulation by vitamin A in tumor cells. This research was supported by Institutional Research Grant 731-01-E from the American Cancer Society and in part by Research Grant CA 14137 from the National Cancer Institute to Dr. J. Leighton.     

Initiation and characterization of a diploid cell line from larval tissues ofAedes dorsalis (Meigen)

Summary Mosquito cell cultures were initiated from the minced tissues of newly hatchedAedes dorsalis (Meigen) larvae. Continuous cell division occurred only after an adaptive period of approximately 6 months. Optimal growth of the cells required a relatively low pH of 6.5. Karyological studies showed that the cells have remained diploid (2n=6) for 60 serial passages and that the cultures are free of contaminating cells. The cultures also were shown to be free of bacteria (includingMycoplasma), fungi and virions. Subpopulations (strains) of the original parental cultures have been selected and characterized on the basis of morphology, karyology, growth rate and monolayer formation. These studies were supported in part by funds from the Office of Naval Research, by Research Grant AI03028 from the National Institute of Allergy and Infectious Diseases, and by General Research Support Grant I-SO1-FR-05441 from the National Institutes of Health, U.S. Department of Health, Education and Welfare.     

Acid phosphatase in the Golgi apparatus of cells forming extracellular matrix of hard tissues

Summary Histochemical studies using cryostat sections of fixed rodent fetal and newborn tissues indicated that acid phosphatase (APase) staining of the Golgi apparatus (GA) of cells secreting matrix for hard tissue formation was a general phenomenon. The enzyme was chiefly observed in the GA of tall secretory ameloblasts involved in enamel formation and in the GA of odontoblasts forming dentine; lysosome-like granules reactive for this enzyme were also observed in these cells. Activity was also intense in the GA and lysosomes of osteoblasts involved in intramembranous and endochondral bone formation.High levels of APase in the GA of extracellular matrix-forming cells appeared to correlate with secretory activity. The GA of most other cells, even chondroblasts forming cartilage matrix, had much less marked APase activity. Contrary to previous suggestions, it appears that APase may have a more direct role in osteogenesis than the osteolytic or resorptive action usually cited.This investigation was supported by PHS Research Grant No. DE 02668 from the National Institute of Dental Research and in part by General Research Support Grant No. RR 5333 from the General Research Support Branch of the National Institutes of Health.The authors gratefully acknowledge the excellent technical assistance of Dorothy H. Clapp and Peggy E. Yates.     

Australian Natural History Series: Albatrosses. Terence Lindsey.

Australian Natural History Series: Great Whales. John Bannister. 2008. CSIRO Publishing, Collingwood, Victoria, Australia. 142 pp. $27.45. ISBN: 978-0-64309-373-7 (softcover). Australian Natural History Series: Platypus. Fourth Edition. Tom Grant, illustrated by Dominic Fanning. 2008. CSIRO Publishing, Collingwood, Victoria, Australia. 159 pp. $27.45. ISBN: 978-0-64309-370-6 (softcover).     

Characterization of cells cultured from early lactation milks

Summary Two major types of cells can be cultured from early lactation human milks: a colony-forming epithelial cell and an adherent nondividing cell referred to as a foam cell The epithelial cells show a positive reaction with a specific antiserum reactive against membrane components of the milk fat globule, whereas the foam cells do not. The nondividing foam cells are phagocytic and can be killed by silica particles; they produce lysozyme, are resistant to trypsinization, and have Fc receptors. These properties, together with the lack of reaction with antiserum to the milk fat globule membrane, suggest that the foam cells are not terminally differential epithelial cells, but tissue macrophages. R. L. C. was supported by Grant No. Ca 19455 from the National Cancer Institute, a Yamagiawa-Yoshida Memorial International Cancer Study Grant, and the Imperial Cancer Research Fund. J. A. P. was supported by Grant No. CA 19455 from the National Cancer Institute.     

Endogenous HPRT activity in mycoplasmas isolated from cell cultures

Summary Five mycoplasma species most frequently isolated from cell cultures were tested for the presence of endogenous hypoxanthine phosphoribosyl-transferase (HPRT), activity. All of the five, cultured in cell-free medium, contained variable but significant levels of HPRT. Two strains ofM. hyorhinis exhibited a 13-fold difference in their specific HPRT activity. When infected with any of these mycoplasma species, HPRT-deficient mouse cell mutants rapidly acquired a cell-associated HPRT activity; however, the cells remained sensitive to HAT medium and resistant to 6-thioguanine. On the other hand, normal HPRT-positive cells deliberately infected with the mycoplasmas uniformly became sensitive to HAT medium. The apparent transfer of mycoplasma-specific HPRT activity to HPRT-deficient cells may be used as a sensitive measure of cell infection by these mycoplasma strains. The HPRT activities of mycoplasmas share several common properties so that they can be distinguished easily from the mammalian HPRT isozymes. Compared to the animal cell enzymes, the mycoplasmal HPRT activities are less heat stable, more strongly inhibited by 6-thioguanine, and in general migrate more slowly in electrophoresis at a neutral pH. This work was supported in part by PHS Research Grants 5 R01 GM21014 and 1 P03 GM19100 (Genetics Center Grant to Albert Einstein College of Medicine), and PHS Research Contracts N01 GM 6-2119 and N01-AG-4-2865 (to the Institute for Medical Research), from the National Institute of General Medical Sciences and National Institute on Aging. S. S. is a recipient of a Faculty Research Award from the American Cancer Society.