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C Sorg

Publications and source records attributed to C Sorg.

At least 199 records · Page 11Linked to original sources

A tumor-associated antigen expressed in melanoma cells with lower malignant potential.

The antigen K-1-2, detectable by a MAb is found in nevi and melanomas. It is associated with melanoma cells of low invasive and metastatic potential as shown by immunoperoxidase studies with cell lines, biopsies and autopsies: K-1-2 occurs in melanoma cell line SK-Mel 25, but not in cell line A-375. A-375 has a higher malignant potential than SK-Mel 25 because, in contrast to SK-Mel 25, it produces plasminogen activator and grows in nude mice. K-1-2 was frequently strongly expressed (greater than or equal to 50% cells positive) in flat (less than 1.5 mm) and less frequently in medium and thick primary tumors. In thick primary melanomas K-1-2 positive cells were confined to the junctional zone or to marginal, flat areas of the tumor. Only rarely does K-1-2 occur in metastases. Strong expression of the K-1-2 antigen was found less often in primary melanomas, which develop early metastases, than in tumors that had not metastasized during an observation period of 18 months. In 5 patients with disseminated metastatic disease, metastases strongly expressing K-1-2 and those negative for this marker or containing only a minor percentage of K-1-2 positive cells were observed simultaneously or at different times. These findings suggest that a change from high malignancy to low malignancy--as observed in animal systems--may also occur in human melanoma.

Animals↗

Selective toxicity of neocarzinostatin-monoclonal antibody conjugates to the antigen-bearing human melanoma cell line in vitro.

Monoclonal antibodies (IgG1) against high molecular weight antigen A-1-43 on human melanoma cell line A-375 were successfully linked to the anti-tumour protein neocarzinostatin (NCS) using the heterobifunctional reagent N-succinimidyl 3-(2-pyridyldithio)-propionate (SPDP). The conjugate retained both the reactivity of the antibody and the toxicity of the drug. The antigen-bearing cell line A-375, antigen-lacking cell line MeWo and normal skin fibroblasts were exposed to NCS-monoclonal antibody conjugates. As negative control, cells were also treated with free NCS and NCS coupled to normal mouse IgG1 antibodies. Inhibition of 3H-thymidine uptake after treatment was used to measure the biological activity of the cytotoxic drug complex or substance, respectively. Comparing the inhibition dose for 50% uptake (ID50) it was found that the monoclonal antibody-drug complex is about 100 times more toxic for the antigen-bearing cell line than free NCS or normal mouse IgG1-NCS. This high toxicity is due to a local increase of drug concentration on these cells. With the two cell lines lacking the appropriate antigen no significant differences in the ID50 values were observed. A selectivity factor of 40-50 was obtained by comparing the cytotoxic effect of the monoclonal antibody-NCS conjugate upon the antigen-bearing as opposed to the antigen-lacking cell type. These data demonstrate, that the toxicity of NCS can be directed by monoclonal antibodies to human tumour cells carrying the corresponding surface antigen.

Antibiotics, Antineoplastic↗

A monoclonal antibody to a differentiation antigen present on mature human macrophages and absent from monocytes.

A monoclonal antibody is described that has been generated in the mouse against cultured human blood monocytes/macrophages. The antibody, designated 25F9, belongs to the IgG1 subclass, detects antigens of m.w. 86,000, and does not react with freshly isolated blood monocytes but reacts with monocytes after 3 days of culture. The expression of the 25F9 antigen on macrophages increases with culture time. Furthermore, the antibody is negative on platelets, granulocytes, lymphocytes, and a large number of human cell lines except the two melanoma lines MeWo and Mel 57. In cryostat sections of normal human tissue (skin, lung, liver, thymus) and of inflammatory or neoplastic tissue (cutaneous lymphoma, eczema, BCG-granuloma, and melanoma), the antibody reacts with scattered macrophages in the dermis but not with epidermal Langerhans cells, with alveolar macrophages, with liver Kupffer cells, and with scattered macrophages in the cortex and medulla of thymus. In eczema, BCG-granuloma, and cutaneous lymphoma, only a few infiltrating macrophages were stained. On the other hand, a large number of macrophages and melanophages reacted positively in melanoma. In some cases melanoma cells also stained weakly positive. Thus, the antibody detects a differentiation antigen preferentially expressed on mature, tissue-fixed macrophages and absent from blood monocytes.

Animals↗

Collection of large quantities of mononuclear cells from individual donors by continuous flow centrifugation leukapheresis.

Continuous flow centrifugation leukapheresis allows the continuous removal of leukocytes from the peripheral blood of individual donors. With the IBM 2997 cell separator, leukapheresis of granulocytes has been well established. On the other hand, no standard procedure for the collection of lymphocytes and/or monocytes has yet been described. Here, we investigated the conditions necessary for collection of large amounts of mononuclear cells with highest possible purity. The average yield from more than 100 healthy donors was 8 X 10(9) leukocytes per donor containing 80-100% mononuclear cells (MNC). Of the MNC, 10-55% were monocytes and 45-90% lymphocytes, depending on the donor. The rotor speed for maximal MNC collection was between 800 and 1000 rpm. Highest yields of monocytes were obtained at 900 rpm. No sedimenting agents were added to the blood. With more than 150 donors no rebound leukocytosis or lasting depression of blood cell counts was observed after leukapheresis. One patient with the Sézary syndrome who was subjected to leukapheresis for 26 months at 3-6 week intervals showed no pathological changes in his blood cell count to date. The disease was stabilized and no abnormalities in resistance to infection became apparent.

Adult↗

Functional characteristics of murine macrophages responding to migration inhibitory factors.

Mouse bone marrow cells differentiate in culture in the presence of L cell-conditioned medium to macrophages (M phi). Proliferation, release of plasminogen activator, expression of transglutaminase, random motility and response to a standard preparation of purified M phi migration inhibitory factor (MIF) was recorded daily up to 14 days. After an initial phase of proliferation, precursor cells differentiated into M phi. In the course of maturation, plasminogen activator production was transiently expressed between day 4 and 12; beginning on day 5 the cells expressed intracellular transglutaminase. Random motility of cells was high at the beginning of culture but steadily declined thereafter. The response to MIF was only expressed between day 5 and 8. However, it was possible to induce MIF responsiveness in mature, unresponsive M phi by the addition of L cell-conditioned medium. To characterize the MIF-responsive M phi type further, bone marrow-derived M phi at day 6 of culture were separated on a hypotonic Percoll gradient into three distinct cell bands. While all densities of M phi displayed random migration, only cells with a density between 1.060 and 1.065 were responsive to MIF. We conclude that the response of M phi to MIF is a phenotypic trait transiently expressed in the course of maturation.

Acyltransferases↗

Migration inhibitory factors and macrophage differentiation.

It has been described before that only certain types of macrophages are capable to respond to lymphokines and that only certain macrophage phenotypes were able to migrate and to respond to migration inhibitory factors (MIF). With respect to the dissociation of MIF activities from a series of other biological activities, and with regard to the phenotype-associated response of macrophages to MIF it was asked: What are the characteristics of the MIF-responsive macrophage phenotype and what are the functional changes induced by MIF on macrophages in addition to inhibition of random migration? Bone marrow-derived macrophages on day 6 of culture were separated by hypotonic Percoll density gradient centrifugation into three distinct bands and analyzed for a variety of functions. It was found that migrating and MIF-responsive macrophages accumulate at a certain density. These macrophages were further characterized by monoclonal antibodies generated against murine macrophage phenotypes. One marker was found to be preferentially expressed by MIF-responsive macrophages. In order to study the inducibility of MIF responsiveness, bone marrow-derived macrophages on day 16 of culture which were poorly migrating and did not respond to MIF were induced to proliferate by the addition of L cell-conditioned medium. After proliferation had subsided, MIF sensitivity was restored. The effects of MIFs other than migration inhibition, on a number of functions which had been mapped within the cell cycle, were investigated. It was found that MIF acts anti-proliferative on "young", cycling macrophages. Non-cycling, mature macrophages were shifted to a state characterized by a decreased expression of transglutaminase and plasminogen activator and an increase of certain phenotypic surface markers. It is concluded that MIFs are differentiation-inducing signals, acting on the generation of macrophages from precursors but also in the recruitment of terminally differentiated macrophages to "inflammatory" type of macrophages which are functional in the induction of immune responses.

Animals↗

Selection of the delayed hypersensitivity T effector and T suppressor cell response by antigen-presenting macrophages.

The T effector lymphocytes of delayed type hypersensitivity reactions (TDH) are regulated by a complex T suppressor (Ts) cell circuit. Induction of TDH cells requires Ia+ adherent cells as antigen-presenting cells. Little is known about the antigen presentation of the induction of Ts cells. We describe an experimental model in which TDH and Ts cells are induced separately by different antigen-presenting macrophages grown from bone marrow stem cells. Bone marrow derived macrophages grown in L cell-conditioned medium for various periods and labeled with 2,4-dinitrobenzene sulfonic acid differ in their ability to induce TDH and Ts cells in vitro. The functional activity of the two T subpopulations was assessed in vivo by epicutaneous challenge or sensitization with 2,4-dinitrofluorobenzene of mice receiving the in vitro educated cells. Ear swelling or suppression of swelling was recorded. It could be shown that 5-7 day bone marrow-derived DNP-labeled macrophages preferentially induced Thy 1+ Lyt 1+ antigen-specific TDH cells; 7-10 day old antigen-presenting bone marrow-derived macrophages induced preferentially Thy 1+ Lyt 2+ antigen specific Ts cells. Characterization of various phenotypic markers revealed different surface antigen expression and functional differences such as MIF responsiveness or transglutaminase activity on the two macrophage populations. These data support the concept that activation of the Ts regulatory circuit may require antigen presentation by specialized antigen presenting cells, characterized by certain surface and functional markers and different from those inducing preferentially TDH cells.

Animals↗

HLA-DR antigen expression in primary melanomas of the skin.

Ninety-three primary malignant melanomas of the skin were typed immunohistologically for the expression of HLA-DR on tumor cells. HLA-DR-positive stroma cells were HLA-DR-negative or only locally positive in most cases. In 36 (39%) of the lesions more than 10% of the tumor cells were stained by two monoclonal antibodies against the nonpolymorphic portion of HLA-DR. HLA-DR-positive tumor cells were often accumulated at the advancing front of the melanoma. The occurrence of HLA-DR-positive tumor cells was related to tumor thickness and level of invasion. Substantial numbers of HLA-DR-positive tumor cells were found in half of the tumors thicker than 1.5 mm and in only 18% of flatter lesions. The highest percentage of HLA-DR-positive tumors was found in the group of melanomas invading the reticular dermis (level IV). The majority of tumors (18/24) that had metastasized within an observation period of 0-32 months were HLA-DR-positive. Regarding the mononuclear cell infiltrate, no correlation between the degree of overall infiltrate and the expression of HLA-DR by the tumor cells was found. The infiltrate within the tumor, however, was more often marked in HLA-DR-positive than in HLA-DR-negative melanomas.

Antibodies, Monoclonal↗

[Chemical and functional characterization of lymphokines].

Macrophages are heterogeneous with respect to a number of constitutive and inducible functions. In order to study the underlying biological principle, a bone marrow liquid culture system was adopted in which bone marrow cells proliferate and differentiate into macrophages. It was found that maturing macrophages express various constitutive or inducible functions in an ordered sequence. The kinetics of their appearance and disappearance are dependent on the proliferative activity of macrophages. Macrophages in "late" G-1 of the cell cycle express constitutive functions like plasminogen activator production and are inducible by bacterial lipopolysaccharides, Poly I:C and lymphokines to release interferons. The response to lymphokines like migration inhibitory factor (MIF) and chemotactic factors is also transiently expressed during maturation. Using purified MIF, its influence on proliferation, differentiation and activation of macrophages was investigated. The changes induced were monitored following the expression of marker enzymes and of phenotype associated cell surface antigens using monoclonal antibodies. The results showed that functional changes induced by MIF on macrophages are limited and are not related to certain macrophage activating activities (MAF). As determined by flow cytofluorometry, transglutaminase expression and proliferation is consistently down-regulated by MIFs. This together with the shift and the expression of surface antigens indicates that MIFs provide a differentiation signal for a "young" macrophage to become more mature.

Animals↗

The expression of tumor-associated antigens in primary and metastatic human malignant melanoma.

Monoclonal antibodies were raised against cultured melanoma cells and selected for their reactivity with melanoma-associated antigens expressed on various melanoma lines. Of 34 monoclonal antibodies, which were all reacting with melanoma lines only 19 antibodies bound to fresh melanoma tissue. Of the 19 antibodies 10 displayed a broad cross-reactivity with normal cells and structures, whereas 9 had a restricted reactivity. Of these antibodies five types were defined, which detected antigens (nevocellular I, nevocellular II, neural, endothelial, basal cell), which were found on certain normal tissues and structures and on certain tumor phenotypes. On the basis of an extensive study on melanoma biopsies of different stages it became evident, that the endothelial, the basal cell and also HLA-DR antigens were significantly more expressed in high risk melanomas. The basal cell antigen was preferentially expressed in locoregional metastases. By immunization with fresh melanoma biopsies different specificities of monoclonal antibodies were obtained as compared to immunization with established cell lines.

Animals↗

Regulation of plasminogen activator secretion, interferon induction and proliferation in murine macrophages.

The purpose of this work was to study the interrelationship of proliferation and secretion of plasminogen activator (PA) and interferon (IFN) by murine macrophages. For induction of macrophage proliferation and secretion of PA, concanavalin A (Con A) was used. Secretion of IFN was induced by polyinosinic polycytidylic acid complex. The glucocorticoid dexamethasone acetate (DA) (10(-6)-10(9) M) inhibited Con A-stimulated secretion of PA and synthesis of DNA as evaluated by incorporation of [3H]thymidine. DA did not inhibit IFN induction. Preincubating macrophages with DA for 45 h reduced basal proliferation and secretion of PA but did not reduce responsiveness to Con A. Also retinoic acid, a modulator of carcinogenesis was used in inhibition studies because of its known antagonistic effects on lymphocyte mitogenesis. In macrophages a biphasic effect of retinoic acid (1 X 10(-5) - 5 X 10(-5)M) was found: (a) inhibition of DNA synthesis and secretion of PA during the first 45 h of incubation, and (b) enhancement of DNA synthesis (but not PA secretion) after 72 h. Secretion of IFN was not affected. It is suggested that secretion of PA but not IFN is linked to cell cycle traverse of macrophages.

Animals↗

Detection of phenotypic differences on human malignant melanoma lines and their variant sublines with monoclonal antibodies.

The hybridoma technique was used to generate monoclonal antibodies against a wide spectrum of melanoma-associated surface antigens. Mice were immunized against the human melanoma lines Mel A-375, SK Mel-25, and Mel S-5 (subclone of SK Mel-25), which differ with respect to a number of biological and biochemical properties. Spleen cells were fused with P3 X 63-AG8.653 myeloma cells. Twenty hybridomas producing antibodies that were negative on platelets, leukocytes, and monocytes but positive on melanoma cells were isolated and recloned. The specificity of antibodies was investigated on 30 human melanoma and nonmelanoma lines. Five groups of antibodies could be distinguished by their reactivity (1) with few melanoma lines and embryonic fibroblasts; (2) with melanoma, neuroblastoma, and teratoma; (3) with melanoma, neuroblastoma, glioblastoma, teratoma, and carcinoma; (4) with melanoma, teratoma, and carcinoma; and (5) with melanoma, neuroblastoma, teratoma, glioblastoma, carcinoma, embryonic fibroblasts, and B-lymphoblastoid cells. The antigen expression was qualitatively and quantitatively different from cell line to cell line. No evidence for melanoma-specific antigens was found. Eight antibodies were isolated detecting phenotypic differences on sublines of SK Mel-25.

Antibodies, Monoclonal↗

Heterogeneity of primary and metastatic human malignant melanoma as detected with monoclonal antibodies in cryostat sections of biopsies.

Monoclonal antibodies were generated against established melanoma cell lines and characterized by their reactivity with various sublines. The antibodies selected for their reaction with melanoma-associated antigens were tested on cryostat sections of melanoma tissue from various stages and on other tumors. The reactivity with normal tissues was also determined. Of 30 antibodies reacting with melanoma cell lines 11 did not react with melanoma biopsies. Of the remaining 19 antibodies nine displayed broad cross-reactivity with normal cells and structures and other benign or malignant tumor cells. Among the remaining antibodies five types were defined that detected antigens (nevocellular I, nevocellular II, neural, endothelial, basal cell) found on certain normal tissues and structures and on certain tumor phenotypes. Even though there seems to be a tendency for some antigens to be preferentially associated with certain stages of melanoma, it has not yet been possible to establish any clear-cut correlation between the expression of one of the differentiation antigens and a particular stage or malignancy potential of melanoma.

Antibodies, Monoclonal↗

Characterization of human lymphocyte derived chemotactic factors for mononuclear phagocytes--I. Production and detection.

Human peripheral blood mononuclear cells were isolated on a large scale by leukapheresis of either individual donors or pooled cell concentrates supplied by a local blood bank. Optimal conditions with respect to cell density, lectin (soluble and insoluble Concanavalin A; phytohemagglutinin) concentration and culture time were established for monocyte chemotactic factor (LMCF) production. LMCF was assayed on highly purified human monocytes/macrophages which had been kept in culture up to 4 days for optimal expression of response to LMCF. Chemotaxis assays were performed in a novel type multichamber assembly and migrated cells were enumerated by enzyme-linked immunosorbent assay. Based on the described methodology it is possible to produce litre quantities of LMCF and assay large numbers of samples both of which are prerequisites for chemical and functional characterizations of LMCF.

Cells, Cultured↗

Characterization of human lymphocyte derived chemotactic factors for mononuclear phagocytes--II. Chemical characterization.

Supernatants of PHA-stimulated human mononuclear cells were fractionated by Sephadex G-100 chromatography and isoelectric focusing. Chemotactic activities for human mononuclear phagocytes were identified at various molecular weight ranges. Major activities were found at mol. wt 45, 30, 25, 18, 12.5, 8 and 6 K. After isoelectric focusing chemotactic activities were recovered predominantly at pH 3.7-5.5 and 7.8-8.5. Fractionation by high performance liquid chromatography (HPLC) on the molecular sieve column I-125 and basic ion exchange column SAX 300 confirmed these results. Furthermore, after absorption of crude supernatants on the reversed-phase column RP 300, chemotactic activity was recovered quantitatively and thus could be separated from the bulk of other materials. In kinetic experiments supernatants were harvested after 5, 27, 42 and 63 hr and fractionated on a molecular sieve column. It was found that only low molecular weight chemotactic activity was released after 5 hr. After 27 hr most activity was found between 10-20,000 after 42 hr activity was found over the whole fractionation range. It is concluded that multiple molecular species of chemotactic factors acting on mononuclear phagocytes are released by activated lymphocyte cultures, whose chemical nature and function remains to be studied.

Cells, Cultured↗

Tumor angiogenic activity (TAA) production in vitro and growth in the nude mouse by human malignant melanoma.

The production of angiogenic activity by eleven human melanoma lines in vitro was compared with the extent of tumor growth in the nude mouse. Angiogenic activity was assayed by measuring the vascular response of the chick chorioallantoic membrane to serum-free supernatants. Growth in the nude mouse was determined after subcutaneous injection of cells 60 days later. Angiogenic activities ranged from negative to highly positive. In five lines angiogenic activity in vitro correlates with the extent of tumor growth in the nude mouse. In contrast, two lines did not show such a correlation, e.g. they produced large tumours without any detectable angiogenic activity. Histological examination of these two tumors revealed moderate degrees of vascularization and only low degrees of necrosis. It is concluded that the extent of tumor growth in the nude mouse is partly independent of the production of angiogenic activity by the tumor cells themselves.

Angiogenesis Inducing Agents↗

In situ analysis of the mononuclear cell infiltrate in primary malignant melanoma of the skin.

Monoclonal antibodies, directed against functionally different lymphocyte subsets, were applied on frozen sections of primary malignant melanomas and benign nevi. Positive reaction was identified by means of an immunoperoxidase method. It was found that lymphocytic infiltrate underneath and in between malignant melanoma is composed of approximately equal numbers of OKT4 positive helper and OKT8 positive suppressor/cytotoxic T cells. The majority of these lymphocytes also expressed HLA-Dr antigen, indicating an activated state. In addition HLA-Dr, OKT6 positive dendritic cells were present in the infiltrate and between the melanoma cells. Finally, melanoma cells expressed demonstrable amounts of HLA A, B, C antigens, whereas benign nevi did not. It is concluded that all ingredients for a successful immune reaction against primary malignant melanoma are on hand. This finding is in agreement with the relatively frequent occurrence of partial or even complete regression of primary malignant melanoma of the skin.

Antibodies, Monoclonal↗