DR (Ia-like) antigens on human melanoma cells. Serological detection and immunochemical characterization.

11 cultured human melanoma cell lines were tested for the expression of DR antigens by using specific allo- and xenoantisera in an indirect rosette microassay. Four of these melanoma cell lines expressed DR antigens, but in lower amounts than expressed on cultured human B-lymphoid cells. Rabbits injected with the DR-positive melanoma cells produced antibodies that were serologically and immunochemically reactive with B-cell-derived DR antigens. Immunochemical studies indicate that melanoma cell-derived DR antigens have a two-chain structure with 34,000 and 27,000 mol wt components. The melanoma cell-derived DR beta-chain at 27,000 mol wt is slightly smaller than that of the Victor cell DR beta-chain whose mol wt is 29,000.

Interaction of histocompatibility (HL-A) antibodies and complement with synchronized human lymphoid cells in continuous culture.

The interaction of histocompatibility (HL-A) antibodies and complement with synchronized human lymphoid cells in continuous culture has been investigated. The sensitivity of cultured lymphoid cells to HL-A antibody-mediated lysis in the cytotoxic test, the extent of activation of the complement system, the degree to which labeled complement components are bound, and the ability of these cells to absorb HL-A alloantibodies do not vary significantly during the cell growth cycle. The constancy of histocompatibility antigen expression throughout the growth cycle of cultured cells suggests that these cell surface markers are an essential part of membrane cytoarchitecture and could well play a critical role in determining the normal function of the cell membrane.

Variation in susceptibility of a human lymphoid cell line to immune lysis during the cell cycle. Lack of correlation with antigen density and complement binding.

Cultured human lymphoid cells RPMI 8866 at different stages of their growth cycle vary in their susceptibility to lysis by rabbit, human, and guinea pig complement activated by HL-A antibodies or heterologous antibodies directed to membrane antigens; cells in G(1) phase are the least sensitive to lysis. To investigate the cause of differential susceptibility of cells RPMI 8866 to lysis, the expression of HL-A determinants and the ability of cells to react with complement were investigated. No change was detected in the density of HL-A antigens on RPMI 8866 cells in synchronous growth as determined by quantitative microabsorption assays, isotopic antiglobulin tests and yields of soluble HL-A antigens. Cells did not vary during the growth cycle in their ability to interact with complement components and in their capacity to activate the complement system through the classical or alternate pathway. These data suggest that variability in lytic susceptibility is due to changes in the structure of the cell membrane or in its ability to repair complement induced damage at certain intervals during the cell cycle. Therefore, this cell line constitutes a useful model to investigate the final steps of the cytolytic reaction.

Human histocompatibility determinants and virus antigens: effect of measles virus infection on HLA expression.

Histocompatibility antigens on the surface of human lymphoblastoid cells were quantified by a microadsorption technique. During the course of measles virus infection, no quantitative or qualitations in surface HLA antigens were observed. In contrast, infection with poliovirus type 1 or vesicular stomatitis virus, or treatment with puromycin (50 microgram/ml) resulted in a significant decrease in surface HLA. These experiments suggest that an inhibition of host protein synthesis rather than the insertion of virus-specificied antigens into the membrane results in a net decrease in amounts of this cell surface antigen. The HLA antigens also appear to be both functionally and structurally distinct from measles virus surface antigens. Pretreatment of cells with HLA-directed antibody did not prevent the infection of these cells by measles virus, thus HLA antigens appear unrelated to the measles virus receptor site on the plasma membrane. Electron microscopic studies revealed that measles virus maturation occurs at membrane sites devoid of demonstrable HLA. Furthermore, HLA antigens could not be detected on the surfaces of mature infectious virions.

Myosin and actin content of human skeletal muscle fibers following 35 days bed rest.

Biopsy samples were taken from the vastus lateralis muscle of seven male subjects pre- and post-35 days bed rest (BR). The myosin heavy chain (MHC) isoform distribution of the samples was determined by densitometry of MHC bands separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Individual muscle fibers were dissected from biopsy samples pre-BR (n=143) and post-BR (n=144). They were studied as regards cross-sectional area (CSA), myosin content by quantitative electrophoresis and myosin actin (M/A) ratio by densitometry of myosin and actin bands of individual muscle fibers. All fibers were typed according to their MHC isoform content determined by SDS-PAGE. A decrease in MHC-1 relative content and an increase in MHC-2X content of whole muscle samples were found, suggesting a slow to fast shift in muscle phenotype. Consistently, fiber type distribution was shifted toward type 2X and 2AX fibers. Muscle fiber atrophy occurred at variable extent among fiber types. Myosin concentration was significantly lower in type 1 and type 2A muscle fibers post-BR than pre-BR, whereas M/A ratio did not vary. The latter findings indicate a disproportionate loss of myosin compared with fiber CSA and a proportional loss of myosin and actin.

Single muscle fiber properties in aging and disuse.

Since the middle of the 1980s, it was understood that myosin, the motor of contraction, can be expressed in several isoforms. The isoforms of the myosin heavy-chain (MHC) portion of the molecule were found to be mostly responsible for the diversity in the contractile and energetic properties of muscle fibers. In humans, three MHC isoforms are expressed in limb muscles (MHC-1, MHC-2A and MHC-2X) and they generate three pure fiber types (types 1, 2A and 2X) and two hybrid types (types 1-2A and -2AX). Type 1, 2A and 2X fibers widely differ with respect to most of their contractile and energetic properties, and a change in their relative distribution within muscles is known to modulate their functional properties in vivo through a "qualitative" mechanism. On the basis of the MHC regulation of muscle fibers properties, it is expected that a given fiber type develops the same force and shortens at the same speed regardless of the physiologic and pathologic conditions under which the muscle works. Surprisingly, several evidences have been accumulating to show that in aging and disuse, the properties of a muscle fiber type can change with no change in its myosin isoform content. This short review considers the latter phenomenon and the possible underlying mechanisms.

Expression of C4 on human lymphoid cells and possible involvement in immune recognition phenomena.

Immunochemical studies revealed the presence of the fourth component of complement (C4) on surfaces of human lymphoid cells. Antiserums to C4 inhibited the mixed lymphocyte reaction and the mitogenic response to phytohemagglutinin, suggesting a role for membrane-associated C4 in the afferent phase of immune recognition phenomena.

Salt extraction of soluble HL-A antigens.

Extraction of cultured human lymphoid cells with hypertonic salt solutions (3 molar potassium chloride) resulted in high recoveries of membrane-associated histocompatibility (HL-A) antigens in soluble form with potent activity and marked immunologic specificity. The active principle was purified by preparative acrylamide-gel electrophoresis. Application of the hypertonic salt extraction method is now yielding sufficient HL-A antigen to begin the elucidation of the molecular basis of transplantation individuality.

Utilization of cultured human lymphoid cells for detection of humoral sensitization in prospective recipients of kidney transplants.

Prospective recipients of kidney transplants were tested for lymphocytotoxicity; from these we selected 102 sera that lacked cytotoxic antibodies against peripheral lymphocytes from at least 80 unrelated subjects. To detect humoral sensitization, we then reacted these with 17 cultured human lymphoid cell lines having different HL-A phenotypes. Cytotoxic antibodies reacting with these cultured cells were now detected in some of the sera. These antibodies were not directed against HL-A antigens, yet mediated lysis of target cells in the presence of rabbit but not of human or guinea pig complement. Furthermore, they activated the classical pathway of the rabbit complement system. Later, a significant association was found between occurrence of cytotoxic antibodies and rejection of the transplant. Thus, cultured human lymphoid cells, because of their great susceptibility to complement-mediated lysis, appear to be useful in detecting humoral sensitization in candidates for kidney grafts.

Cytotoxic antibodies to cultured melanoma cells in the sera of melanoma patients.

By means of the complement-dependent microcytotoxicity test, cytotoxic antibodies to melanoma cells in long-term culture were detected in 34 of 90 sera from melanoma patients. The incidence of cytotoxic antibodies in melanoma patients was significantly greater than in subjects free of malignant disease but not significantly greater than in patients with other types of cancer. The sera were cytolytic to melanoma cells only in conjunction with rabbit complement, and they reacted with the pabel of melanoma cells in a distinct fashion. No association was found between presence of cytotoxic antibodies and the occurrence of metastasis.

Natural antibodies to human lymphocytes in murine strains.

Murine strains vary greatly in their content of natural antibodies to human lymphocytes; sera from NZB, B10.BR/TSn, DBA/J, and B10.S mice reacted with more than 90% of the panel of human lymphocytes, yet those from other strains did not react. These levels of natural antibodies to human lymphocytes did not correlate with the H-2 type of mice, but appeared to be a dominant character with incomplete penetrance. Since sera from germfree mice also contained natural antibodies to human lymphocytes, their formation was not influenced substantially by bacteria.

Purification and immunologic evaluation of human melnoma-associated antigens.

Melanoma-associated antigens (MAA) were isolated and their functional immunologic properties were evaluated. Spent fetal calf serum-free culture media and 3-m KCI extracts of cultured human melanoma cells grown in this medium were used as antigen sources. Ultracentrifugal flotation on KBr was used to separate MAA and HLA antigens present in the extracts or spent culture media; thus interference by histocompatibility antigens was prevented in subsequent tests of tumor antigenic activity. MAA purified in this manner retained their immunologic functions as evidenced by their ability to produce delayed cutaneous hypersensitivity reactions in patients with melanoma, specifically combine with antimelanoma xenoantibody, and elicit production of functionally specific xenoantibody. Possible structural differences between HLA antigens and MAA were considered in evaluation of the data.

Comparison of histocompatibility antigens on cultured human tumor cells and fibroblasts by quantitative antibody absorption and sensitivity to cell-mediated cytotoxicity.

Human tumor and febroblast tissue culture cells were compared to determine the suitability of fibroblasts as control cells in experiments on human tumor serology and cellular immunology. Fibroblasts expressed the same HL-A antigen profile as did melanoma cells. Furthermore, the quantitative expression of the determinants was similar on both cell types. In four of five pairs tested, the fibroblasts displayed similar sensitivity to effector cells generated by mixed lymphocyte culture as did the tumor cells from the same donor, but there were some differences in the effects of specific alloimmune effector cells at high and low effector-to-target ratios on the two types of target cells. Results indicated that fibroblasts are legitimate control target cells for studies in human tumor immunology, if screening assays are done to verify their antigenicity and sensitivity to cell-mediated cytolysis.

Structural properties and tissue distribution of the antigen recognized by the monoclonal antibody 653.40S to human melanoma cells.

The hybridoma 653.40S, constructed with splenocytes from an inbred BALB/c mouse immunized with cultured human melanoma cells, secreted an antibody that had been shown to recognize an antigenic determinant restricted to human melanoma cell lines. The monoclonal antibody (MoAb) 653.40S showed immunoprecipitation of two glycopolypeptides synthesized by melanoma cells, one with the apparent molecular weight of 280,000 and the other one with a molecular weight larger than 500.000. These two glycopolypeptides were not bridged by disulfide bonds and were peripheral rather than integral to the plasma cell membrane. Comparison of the reactivity of cells of the melanocyte lineage with the MoAb 653.40S and with the MoAb Q5/13 to human Ia-like antigens showed that the former reacted with proliferating melanocytes and melanoma cells, whereas the latter reacted only with melanoma cells. The MoAb 653.40S did not react with a large variety of surgically removed normal and tumor tissues except for some instances of basal cell and squamous cell carcinomas. These results suggested that double staining of pigmented skin lesions with the MoAb 653.40S and with an MoAb to Ia-like antigens may help to solve controversial diagnosis of melanoma. Furthermore, the MoAb 653.40S may be useful for radioimaging and immunotherapy of melanoma.

Human lymphoblastoid cell lines as effector cells in mitogen-dependent and antibody-dependent cell-mediated lysis.

Based on reports that both human and murine lymphoblastoid cell lines can effect lysis in antibody-dependent (ADCC) and mitogen-induced (MICC) cell-mediated cytotoxicity, 10 human lymphoblastoid cell lines were investigated for their ability to act as effector cells in these lytic reactions. Four cell lines promoted MICC and ADCC of chicken erythrocytes. MICC was inhibited by subjecting the effector cells to gamma-irradiation, and ADCC was partially inhibited by treating antibody-coated target cells with Protein A from Staphylococcus aureus. The latter finding suggests that antibody plays a role in the cytotoxic reaction. The lytic activity of cultured human lymphoid cells in both MICC and ADCC reactions was, however, quite variable for unknown reasons. Because of this instability human lymphoblastoid cell lines are not a suitable source of effector cells for studying the biological and molecular properties of structures responsible for the lytic reaction.

Lysis of cultured human melanoma M10 cells by polyclonal xenoantibodies to melanoma-associated antigens.

Cultured human melanoma cells M10 harvested from cultures in different phases of their growth show significant changes in the expression of melanoma-associated antigens (MAA), but they do not vary in susceptibility to lysis mediated by anti-MAA xenoantisera and effected by complement or lymphoid cells. Furthermore, melanoma cells M10 showed a significant increase in susceptibility to immune lysis following treatment with puromycin at doses that do not effect the expression of MAA. The lack of correlation between MAA density and susceptibility to immune lysis supports the contention that, under the experimental conditions used, cellular properties play a major role in the outcome of immune attack.

Cell surface markers on epithelial-Burkitt hybrid cells superinfected with Epstein-Barr virus.

Attempts were made to superinfect two epithelial-Burkitt hybrid cell lines, designated D98/HR-1 and D98/Raji, with Epstein-Barr virus (EBV) and to investigate the expression of some cell surfacr markers including histocompatibility antigens, and the presence of B-cell markers, such as receptors for the third complement component and for monkey red blood cells. Successful superinfection of D98/HR-1 cells with EBV was made evident by the expression of early antigen and, to a lesser extent, virus capsid antigen. Only a rare D98/Raji cell was found to be positive for early antigen. The histocompatibility antigens of the parental cell lines D98, HR-1, and Raji were expressed on the surfaces of the hybrid cells. Receptors for third complement components b and d were not detected on the hybrid cells or on the D98P OR HR-1 cell lines; they were found, however, on the Raji cells, indicating that EBV receptors and complement receptors can be separated. The significance of the infection of the hybrid cells with EBV and the expression of cell surface markers is described.

Purification of soluble human melanoma-associated antigens.

Soluble extracts of melanoma specimens were prepared by 3 M KCl extraction. Delayed cutaneous hypersensitivity reactions to these antigens were noted in 25 of 39 melanoma patients and 7 of 30 patients with other neoplasms. Only 4 of 34 melanoma patients reacted to an extract of autologous muscle. Maximum reactivity to these antigens occurred at 24 hr and was demonstrated histologically by skin biopsy. Chromatography on Sephadex G-150 resulted in two fractions that elicited delayed cutaneous hypersensitivity reactions in melanoma patients. These fractions were subjected to polyacrylamide gel electrophoresis. The first region of the Sephadex 1 gel reacted in 8 of 13 melanoma patients and only 1 of 7 patients with other neoplasms. Some activity was found in other regions of the gel. Skin test activity was confined to the second polyacrylamide gel electrophoresis region of the Sephadex II gel, to which 7 of 9 melanoma patients reacted compared with 1 of 7 patients with other tumors. Recovery of antigenic activity in excess of the total present in the original extract after partial purification indicated that inhibitors of delayed cutaneous hypersensitivity reactions present in the crude KCl extract were removed. A 20-fold increase in antigenic activity per unit protein was achieved.

Approaches for the isolation of biologically functional tumor-associated antigens.

Melanoma-associated antigens were isolated from human melanoma cells in long-term tissue culture and from the spent culture fluid of these cells propagated in chemically defined, serum-free media. The 3 M KCl extracts from such cells and their concentrated spent culture media elicited specific delayed cutaneous hypersensitivity reactions in patients with malignant melanoma but not in patients with other neoplasms. HLA antigens present in these extracts could be specifically removed by ultracentrifugation in KBr at a density of 1.23 g/ml. Purification of melanoma-associated antigens was achieved by this step, followed by ion-exchange chromatography and preparative isoelectric focusing on Pevikon C870. Another approach is described for the isolation of carcionembryonic antigens from metastatic lesions with an approximately 70% yield utilizing the least denaturing procedures, which avoid lyophilization and involve essentially 0.9% NaCl solution extraction, specific adsorption, elution from concanavalin A Sepharose, and subsequent gel-exclusion chromatography on Ultrogel AcA 22. For effective isolation of carcinoembryonic antigens freely shed from cultured cells derived from a primary colon tumor, a system was devised based on the use of Amicon hollow fiber culture units, in which cultured tumor cells were introduced in the extracapiliary spaces of such a unit. The extracapillary fluid, containing carcinoembryonic antigens but no fetal calf serum components, is removed and further purified by affinity chromatography.

Effects of resistance training on myosin function studied by the in vitro motility assay in young and older men.

It is generally believed that the maximum shortening velocity (V(o)) of a skeletal muscle fiber type does not vary unless a change in myosin heavy chain (MHC) isoform composition occurs. However, recent findings have shown that V(o) of a given fiber type can change after training, suggesting the hypothesis that the function of myosin can vary without a change in isoform. The present study addressed the latter hypothesis by studying the function of isolated myosin isoforms by the use of the in vitro motility assay (IVMA) technique. Four young (age 23-29 yr, YO) and four elderly men (age 68-82 yr, EL) underwent a 12-wk progressive resistance training program of the knee extensor muscles and to one pre- and one posttraining biopsy of the vastus lateralis muscle. The significant increase in one-repetition maximum posttraining in both YO and EL indicated that training was effective. After training, MHC isoform composition showed a shift from MHC(2X) toward MHC(2A) in YO and no shift in EL. The velocity of sliding (V(f)) of actin filaments on pure myosin isoforms extracted from single fibers was studied in IVMA. One hundred sixty IVMA samples were prepared from 480 single fibers, and at least 50 filaments were analyzed in each experiment. Whereas no training-induced change was observed in V(f) of myosin isoform 1 either in YO or in EL, a significant increase in V(f) of myosin isoform 2A after training was observed in both YO (18%) and EL (19%). The results indicate that resistance training can change the velocity of the myosin molecule.