Differences in cytotoxic effects of activated murine peritoneal macrophages and J774 monocytic cells on metastatic variants of B16 melanoma.

The cytotoxic effects of activated peritoneal macrophages and the J774 reticulum cell sarcoma cell line on B16 melanoma cells of differing metastatic potential were investigated in vitro. The melanoma target cells were sublines of low (B16-F1) or high (B16-F10) lung colonization potential as well as a subline of high (B16-B14b) brain colonization ability. Thioglycolate-elicited peritoneal macrophages from syngeneic C57BL/6 mice and J774 cells were activated in vitro with polyinosinic-polycytidylic acid (poly I:C) and used as effector cells. Macrophage-mediated cytolysis was determined by means of 24- to 72-hour radioactivity release assays with [125I]5-iodo-2'-deoxyuridine-labeled melanoma cells; the results indicated that the more metastatic sublines B16-F10 and B16-B14b were less susceptible to cytolysis by activated macrophages and J774 cells than was the poorly metastatic B16-F1 subline. The poly I:C-activated effector cells also released soluble cytotoxin(s), which resulted in melanoma cell lysis and growth inhibition. Cytotoxin-mediated melanoma cell cytolysis was determined by counting the number of viable mitomycin-treated target cells remaining after a 40-hour incubation period in the absence or presence of various concentrations of cell-released factors, and cytotoxin-mediated cytostasis was performed with the use of the same procedures without mitomycin pretreatment of targets. The factors released from both activated macrophages and J774 cells were more effective against the poorly metastatic B16-F1 cells than against the highly metastatic B16-F10 or B16-F14b cells. In addition, the activity of the factors from both activated effector cells was inhibited by fetal bovine serum. The J774 cells and the activated peritoneal macrophages demonstrated similar activities against B16 melanoma variants, indicating that the J774 cell line may be suitable as a model for the study of macrophage cytotoxicity. The results suggested that the potential of the highly metastatic melanoma cells to implant, survive, and grow at secondary sites may be due, in part, to their ability to circumvent host antitumor mechanisms.

Differences in the sensitivities of murine metastatic lymphoma/lymphosarcoma variants to macrophage-mediated cytolysis and/or cytostasis.

A metastasizing animal tumor model for large cell lymphoma or lymphosarcoma has been established by sequential selection in vivo of the RAW117 parental cell line in BALB/c mice for enhanced colonization of liver or lung and in vitro for lack of binding to immobilized lectins. The parental RAW117 and selected sublines and clones derived from these were compared for their sensitivities in vitro to polyinosinic:polycytidylic acid-activated syngeneic macrophages in cytolysis and cytostasis assays. Activated but not unactivated macrophages had differing effects on RAW117 sublines and clones. The least metastatic (parental) cell line was the most sensitive to activated macrophage-mediated cytolysis and cytostasis, while the most metastatic subline was the least sensitive in these assays. Intermediate metastatic sublines or cones were usually less sensitive in one or both of the assays. These antitumor activities were negligible at 24 hr in the continuous presence of polyinosinic:polycytidylic acid but were clearly apparent by 48 to 72 hr. The results suggest that differential sensitivity to host macrophage surveillance mechanisms can occur in malignant cell subpopulations.

Prevalence of diabetes and its complications in the Eastern Band of Cherokee Indians.

OBJECTIVE: To determine the prevalence of diabetes and selected complications among the Eastern Band of Cherokee Indians in North Carolina. RESEARCH DESIGN AND METHODS: Multiple IHS data systems were used to determine diabetes prevalence and complication rates. The RPMS was used to identify diabetes cases as of April 1989, degree of Indian inheritance, cardiovascular diseases (including hypertension), and retinopathy. Data on laser treatments and LEA were obtained from individual registries. Information on ESRD was obtained by a research assistant. The IHS Ambulatory Patient Care reporting system was used to calculate the number of diabetic patients for the years 1982-1987. The IHS user population was used as the denominator. Rates were age-adjusted to the 1980 U.S. population and diabetic population by the direct method. RESULTS: Using clinical records, the age-adjusted prevalence of diabetes in 1988 was 105.6/1000 people, four times the U.S. rate. Rates of diabetes were highest in the groups with the highest degree of Indian inheritance. LEAs occurred among diabetic patients at three times the rate for the U.S. Between 1985 and 1989, new cases of ESRD occurred at a crude annualized rate of 578/million, approximately six times the rate for U.S. whites. CONCLUSIONS: Diabetes presents a major burden to the Eastern Band of Cherokees. The limitation inherent in this study probably result in underestimation of the prevalence of diabetes and its complications in the community. Future studies are needed to assess the impact of medical and preventive programs on diabetes prevalence and complications in this community.

Profiles of the Endocrine Clinic: the Mayo Clinic.

It is evident that clinical endocrinology, as a discipline, is entering a particularly exciting period in its evolution. Knowledge gained from basic and clinical research is being translated at the bedside for the benefit of our patients. The emergence of new drugs and novel treatment strategies has equipped clinical endocrinologists with the tools to more successfully combat many old enemies, such as diabetes and osteoporosis. Realization of full benefit from these exciting new tools requires a practice model in which the clinical endocrinologist's role is preeminent and is coordinated and integrated with those of practitioners drawn from other disciplines. The Mayo Division of Endocrinology, Metabolism, and Nutrition provides one such model of highly integrated care. We believe that as the pace of knowledge regarding basic mechanisms of disease and their treatment quickens, such integrated divisions will prove well suited to deliver the highest quality care to people with endocrine disorders.

Brain meninges tumor formation by in vivo-selected metastatic B16 melanoma variants in mice.

The murine melanoma subline B16-F1 of low brain- and lung-colonizing potential has been used to obtain brain-colonizing variant sublines by sequential selection in vivo for their abilities to form brain meningeal tumors. After fourteen and fifteen selections in syngeneic C57BL/6 mice sublines B16-B14b and B16-B15b, respectively, were established in culture. These were then assayed in vivo by injection of single tumor cell suspensions into the tail vein (i.v.), left ventricle of the heart (i.c.) or left common carotid artery (i.a.), and the resulting tumors were examined histologically. Injection of subline B16-B14b or B16-B15b resulted in brain meningeal tumor formation in the dura mater and leptomeninges with invasion into underlying brain parenchyma and also some brain ventricular tumors at the sites of i.a. injection. Lung colonization ability remained in the brain-selected sublines, although it was remarkably reduced in i.a. tumor cell-injected animals. The brain meningeal tumors that formed were of three types: intravascular, nodular or infiltrative. Injection of tumor cells i.v. resulted mainly in the establishment of the intravascular type of brain meningeal tumors with eventual perivascular invasion, while injection i.a. or i.c. resulted mostly in nodular or infiltrative type brain meningeal tumors. The B16-B14b brain meningeal tumors formed were small (less than 1 mm in diameter) and usually nonpigmented, while B16-B15b tumors were generally large (up to 7 mm in diameter) and pigmented. Host reactions towards B16-B14b and B16-B15b tumors at meningeal sites differed. The former B16 subline was characterized by extensive fibrosis with some immunocytic cell infiltration in and around the meningeal tumors, while the latter subline did not elicit such host reactions. In contrast, tumors in brain parenchyma failed to evoke observable host reactions, and there was little evidence of immunocyte cell infiltration or glial cell alterations.

In vivo and in vitro properties of malignant variants of RAW117 metastatic murine lymphoma/lymphosarcoma.

Using the RAW117 lymphoma/lymphosarcoma system syngeneic to Balb/c strain mice, variant sublines have been selected for enhanced blood-borne liver colonization in vivo or for lack of binding to immobilized lectins in vitro. The kinetic organ distributions of intravenously injected, 3H-thymidine-labelled RAW117 parental cells and a subline sequentially selected ten times for enhanced liver colonization were similar, suggesting that the differences in malignancy between these two cell lines were not due to dramatic differences in organ localization properties. Examination of the malignant properties of the selected sublines and cell clones derived from these in immune-impaired animals indicated that host immune status was important in determining the quantity of experimental metastases in this system. Although impairment of T-cell or NK-mediated anti-tumor responses by using 400 R 60Co-irradiated or Balb/c nude (nu/nu) mice suggested that certain immunologic responses were not effective in preventing experimental metastasis, impairment of macrophage function with chlorine, silica, trypan blue, carrageenan, cyclophosphamide or pristane were effective and resulted in enhanced malignancy of the parental RAW117 line. In contrast, impairment of macrophage function had little or no effect on the experimental metastatic properties of highly malignant RAW117 sublines or clones. In vitro humoral responses or cell-mediated immunologic assays using lymphoid cells from normal or tumor-bearing hosts failed to demonstrate antibody-mediated or antibody-dependent cell-mediated cytotoxicity (ADCC), T-cell or NK-cell responses against RAW117 cells. However, poly I: C activated macrophages were more effective against parental RAW117 cells than against a highly metastatic subline in cytolysis and cytostasis assays suggesting that the highly metastatic RAW117 cells can more readily escape macrophage-mediated host defenses.

Lymphotoxins - a multicomponent system of growth inhibitory and cell-lytic glycoproteins.

Activated lymphocytes from experimental animals and man can release materials, termed lymphotoxins , which cause growth inhibition and cell lysis in vitro. These molecules, from human lymphocytes, are glycoproteins which can be divided into five molecular weight classes. These forms are heterogeneous, for each MW class can be further subdivided into multiple charge subclasses. It is now clear certain MW classes are interrelated and form a system of cell toxins. The larger classes (greater than 140,000 d) are associated with nonclassical antigen-binding receptors (R), which can be of T cell origin. The smaller forms (less than 90,000 d) do not express R function and are derived from the larger forms, possibly by enzymatic action. Two MW classes, one receptor-associated and one non-receptor associated, have been purified to homogeneity and their peptide composition is being studied. Functional studies reveal the larger MW forms derived from alloimmune cell populations; can induce selective and nonselective destruction of cells in vitro. Antibodies which inhibit the in vitro cell lytic ability of various human LT forms can block different classes of human lymphocyte cell killing reactions in vitro.

Sodium-stimulated glutamate transport in osmotically shocked cells and membrane vesicles of Escherichia coli.

Three phenotypically distinct strains of Escherichia coli B were studied: one in which the transport of glutamate was strongly stimulated by sodium, one in which the transport was relatively independent of sodium, and one which did not transport glutamate. Membrane vesicle preparations from the three strains followed the behavior of whole cells with respect to sodium-stimulated transport. Although glutamate-binding material could be released from cells by osmotic shock, its affinity for glutamate was not significantly influenced by sodium. Furthermore, the shocked cells retained sodium-stimulated transport. The accumulated results suggest that the sodium-activated glutamate transport system resides in the cytoplasmic membrane and that releasable binding protein(s) is not intimately involved in its function.

In vivo and in vitro production and detection of monoclonal antibodies to surface components on metastatic variants of murine tumor cells.

Mouse-mouse and rat-mouse hybridoma cell lines secreting complement-dependent cytotoxic or cell-binding monoclonal antibodies have been produced against cell surface components of two murine metastatic tumor systems: B16 melanoma and RAW117 lymphosarcoma. We have used both in vivo and in vitro spleen cell culture methods for immunization and have made a number of methodologic alterations to increase the yield and survival of hybridomas including media osmolality, pH, serum type, macrophage feeder layers and supplemented amino acids, vitamins and metabolites. Using complement-dependent cytotoxicity or lectin immobilization of tumor cells to polystyrene via a water-soluble carbodiimide for an enzyme-linked immunosorbant assay we were able to rapidly screen hybridoma cultures for specific antibody secretion.

Characterization of murine monoclonal antibodies to Escherichia coli J5.

Twenty-eight independently derived monoclonal antibodies (MAb) directed against Escherichia coli J5 endotoxin were produced and characterized. Each MAb exhibited a specific titer by both radioimmunoassay and passive hemagglutination assay. Most of the MAb were of the immunoglobulin G isotype; however, several immunoglobulin M antibodies and one immunoglobulin A antibody were produced. When characterized for their capacity to cross-react with purified endotoxin preparations from several gram-negative bacteria, 22 MAb exhibited no cross-reactivity; 6 demonstrated a limited capacity to cross-react with other endotoxin preparations. When characterized for their capacity to react with the intact organism instead of the purified endotoxin the pattern of cross-reactivity was quite different. Most of the MAb were able to react with Salmonella minnesota Re595. Eighteen were able to react with E. coli O111:B4 (the parent strain of E. coli J5), 13 MAb reacted weakly with Pseudomonas aeruginosa, and 3 reacted weakly with Klebsiella pneumonia. The data imply that MAb generated against E. coli J5 endotoxin demonstrate greater cross-reactivity when assayed against the whole bacterium than when assayed against the corresponding purified endotoxin. We were unable to demonstrate that any of the 28 MAb could passively protect mice against lethal endotoxin challenge.

Human monoclonal antibodies that protect mice against challenge with Pseudomonas aeruginosa.

Lymphocytes from healthy volunteers and from cystic fibrosis patients were transformed with Epstein-Barr virus and cultured at a limiting dilution to generate lymphoblastoid cell lines that secreted human monoclonal antibodies specific for lipopolysaccharide (LPS) from Pseudomonas aeruginosa. Three cell lines (RM5, FDD7, and 11F9) produced immunoglobulin M (IgM) antibody species that reacted specifically with P. aeruginosa Fisher immunotypes 2, 4, and 5, respectively, and with LPS extracted from these immunotypes. A fourth cell line (9H10) produced a single IgM antibody species that recognized P. aeruginosa immunotypes 3, 6, and 7 and LPS extracted from them. Monoclonal antibodies secreted by cell lines RM5, FDD7, and 11F9 protected neutropenic mice prophylactically against challenge with P. aeruginosa immunotypes 2, 4, and 5, and those secreted by 9H10 protected against P. aeruginosa immunotypes 3 and 6 but did not protect against immunotype 7. In vivo experiments indicated that antibodies protected mice against infection by increasing the rate of bacterial clearance.