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Activated lymphocytes from experimental animals and man can release materials, termed lymphotoxins, which cause growth inhibi tion and cell lysis in vitro. These molecules, from human lympho cytes, 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 (>140,000 d) are associated with nonclassical antigen-binding receptors (R), which can be of T cell origin. The smaller forms (>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 invitro. 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.

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.

Publisher Summary This chapter discusses the separation of malignant lymphoid cells by countercurrent distribution (CCD). CCD using aqueous phases with or without charge potential differences between the phases can be used to rapidly separate malignant large cell lymphoma populations to yield viable cells that can be used in biological and biochemical experiments. It was found that the murine RAW117-P cell population used in the studies was heterogeneous in its biological and biochemical characteristics and changed in these cellular properties with time. Its partitioning behavior was characteristic of its malignancy in that cells of higher but not the highest partitioning properties in a dextran-poly(ethylene glycol) two-phase system were always more metastatic. This was shown when different CCD fractions were subjected to a second CCD fractionation in the same phase system and had partition coefficients corresponding to those obtained in the first CCD fractionation. The technique of CCD has allowed rapid separation of viable large cell lymphoma cell subpopulations differing in their biological and cell-surface biochemical properties by sensitive and reproducible procedures.

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.

In response to the need for specifying the microroughness of optical surfaces, a standard test method for measuring the effective surface roughness of metallized mirrors by total integrated scattering was developed and tested under the auspices of the American Society for Testing and Materials. The "precision" of the test was found to be better than ±15% by an interlaboratory round-robin test program.

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.

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 400R 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, cyclosphophamide 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 RAW 117 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.

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.

Metastasis via lymphatics and blood does not always result in tumor colonization of tissues and organs based strictly on anatomical considerations (Salsbury, 1975; Sugarbaker, 1952). Many blood-borne experimental metastatic tumor systems show nonrandom colonization patterns that do not correlate with the initial capillary beds encountered (Dunn et al., 1954; Parks, 1974; Fidler and Nicolson, 1976, 1977; Brunson and Nicol son, 1978, 1979; Conley, 1979). After their initial arrest, malignant cells may die, invade and grow or detach and recirculate to other sites (Zeidman, 1961; Fisher and Fisher, 1967; Fidler and Nicolson, 1976, 1977; Brunson and Nicolson, 1978, 1979) suggesting that factors other than non-specific trapping in the microcirculation determine secondary site location (Nicol son, 1978). In order to determine the host and tumor cell properties important in metastatic tumor spread and survival at distant sites, we have developed animal tumor models in syngeneic hosts that show distinct metastatic behaviors. Several of these models have been based on sequential selection of murine B16 melanoma lines for enhanced blood-borne implantation, survival and growth to obtain sublines capable of preferentially colonizing lungs (Fidler, 1973), brain (Brunson et al., 1978) or ovary (Brunson and Nicol son, 1979). Similarly we have selected in vivo murine RAW117 lymphosarcoma sublines that show enhanced liver colonization after intravenous (i.v.) administration (Brunson and Nicolson, 1978) and rat 13762 mammary adenocarcinoma sublines that show colonization of regional lymph nodes and/or lungs after implantation subcutaneously (s.c.) (Neri et al., 1979). In addition, it has been possible to select tumor cell variant sublines in vitro for loss of sensitivity to cell-mediated toxicity (Fidler et al., 1976), decreased binding to immobilized-lectins (Reading et al., 1980) and increased invasion of tissues (Hart, 1980) or veins (Poste et al., 1980).

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.

Publisher Summary This chapter examines the role of lymphotoxins in natural cytotoxicity. Lymphotoxin are cytostatic or cytotoxic glycoproeins produced in vitro by activated lymphocytes. In a study described in the chapter, human peripheral blood lymphocytes (PBL) were obtained from defibrinated peripheral blood by the Ficoll–Hypaque technique. The PBL were then cultured at a cell density of 2 × 10 6 cells/ml in RPMI-1640 medium supplemented with 10% FCS (RPMI-10%) at 37°C for 5 to 7 days. At the end of the 5 to 7 days of culture, the cPBL were then used in either a direct cell killing assay or restimulated with 10 μg of Con A/ml at a cell density of 2 × 10 6 cells/ml in fresh RPMI-10% for 5 h at 37°C. The effector cells generated by this method caused significant lysis of the NK-sensitive target cells K-562 and MOLT-4F and little or no significant lysis of the continuous B cell lines Jy or RPMI-1788.

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.

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Articulation functions were established for three groups using the Synthetic Sentence Identification (SSI) test and the Modified Rhyme Test (MRT) in three message competition ratios (MCR's) of multi‐talker noise. Mean scores on the SSI and the MRT were obtained for 35 normally hearing and six flat sensorineural and six high‐frequency sloping sensorineural hearing impaired subjects. An ANOVA revealed a significant difference between tests, groups, presenation conditions, and significant interactions between variables. SSI scores were not significantly different among the tested groups while the MRT scores were significantly different at two MCR conditions. Articulation functions were considered typical for each group tested. The results suggest that the SSI and MRT are two different tests, examining two separate areas of discrimination.