ABSTRACT
Recently it was found that immunoanalysis of carcinoembryonic antigen (CEA) levels in gallbladder bile may be a sensitive method to detect colorectal liver metastases in humans. Methods used in the past for the detection of CEA in various body fluids were cumbersome and time consuming, requiring acid extraction, extensive dialysis, and column purification. Single-step, solid-phase radioimmunoassays, designed specifically for serum CEA analysis, were developed commercially to replace these methods. Parameters and methodology necessary to adapt these kits for Parameters and methodology necessary to adapt these kits for use with gallbladder bile are presented here. A combination of pretreatment procedures for bile, before radioimmunoassay, permit rapid, reproducible, and accurate measurement of CEA levels in gallbladder bile.
Subject(s)
Bile/immunology , Carcinoembryonic Antigen/analysis , Colorectal Neoplasms/pathology , Liver Neoplasms/secondary , Carcinoembryonic Antigen/blood , Humans , Liver Neoplasms/diagnosis , Radioimmunoassay/methods , Reproducibility of Results , Risk Factors , Sensitivity and SpecificityABSTRACT
While computerized tomographic (CT) scanning and intraoperative exploration are both considered accurate measures of liver involvement with metastatic disease, 10% to 30% of colorectal liver metastases remain undetected. Attempting to improve current methods for detecting colorectal liver metastases, CEA levels in gallbladder bile and serum from patients with known liver metastases were determined. One hundred per cent of patients with single and multiple metastases of various dimensions were observed to have gallbladder bile CEA levels strikingly higher than serum values (4.7 to 259 times greater, p = 0.0009). Linear regression analysis of estimated tumor volume and surface area versus gallbladder bile CEA levels predicted that very small tumors (less than or equal to 1 cm3 in volume) might produce detectable levels (9 to 41 ng/mL) of biliary CEA. For this reason, patients who lack clinical and radiologic evidence of distant metastases at the time of primary colorectal resection but who do have elevated gallbladder bile CEA levels (greater than or equal to 10 ng/mL) are being followed for the appearance of occult hepatic metastases.
Subject(s)
Bile/analysis , Carcinoembryonic Antigen/analysis , Colorectal Neoplasms/surgery , Gallbladder/immunology , Liver Neoplasms/secondary , Colorectal Neoplasms/immunology , Gallbladder Diseases/immunology , Humans , Liver Neoplasms/immunology , Pilot Projects , Regression AnalysisABSTRACT
Age-matched female C57BL/6 mice given injections i.v. of the syngeneic, poorly metastatic melanomas B16-F1 or JB/RH mount a measurable anti-melanoma antibody (Ab) response. The experiments described here examine the possibility of whether these Ab responses are in fact targeted against metastatically distinct subpopulations of melanoma. Reactivity of antisera and monoclonal antibodies (MoAb) developed by syngeneic immunization were examined by radioimmunoassay, flow cytometry, and Western blot analyses. Anti-melanoma antisera collected at various times after i.v. injection of melanoma cells were tested in radioimmunoassay against C57BL/6 melanoma cell lines and clones of well-characterized experimental metastatic activity. A strong inverse correlation between metastatic activity and Ab binding was observed. Furthermore, i.v. injection of poorly metastatic melanoma lines always resulted in strong Ab response, while highly metastatic clones did not. The relative distribution of Ab-reactive cells in melanoma populations was examined by flow cytometry. Again, the proportion of reactive cells in all melanoma cell lines and clones tested was inversely proportional to the degree of experimental metastatic activity. Hybridoma technology used to capture this selective Ab response yielded monoclonal reagents with distinct anti-melanoma specificity. Monoclonal antibody directed against nonmetastatic melanoma variants defined specific Mr 45,000 and 50,000 bands in Western blot analyses. Finally, melanoma cells nonreactive with the syngeneic Ab response were isolated after immunomagnetic bead plus magnet removal of the positive population. The experimental metastatic potential of these negatively selected cells was then compared with cells similarly fractionated with normal mouse serum or control MoAb. The results of these studies showed a clear increase in metastatic potential of anti-B16-F1 melanoma antiserum or MoAb-depleted cells. These results support the contention that host Ab responses may favor tumor progression and metastasis through selective Ab responses against the poorly metastatic population.
Subject(s)
Antibody Formation , Melanoma, Experimental/pathology , Neoplasm Metastasis , Animals , Antibodies, Monoclonal , Cell Line , Flow Cytometry , Genetic Variation , Immunization , Melanoma, Experimental/genetics , Melanoma, Experimental/immunology , Mice , Mice, Inbred BALB C , Mice, Inbred C57BLABSTRACT
The phenotypic heterogeneity of tumor cell-surface galactose expression within a cell population may dictate metastatic potential. The hepatic asialoglycoprotein receptor, whose known function is to bind to terminal galactose residues of desialylated glycoproteins and effete cells, may participate in the arrest and subsequent growth of subpopulations of tumor cells with high galactose expression. To test this hypothesis, murine colon carcinoma cells (CT-26) were sorted, using the galactose-specific lectin, soybean agglutinin (SBA), and fluorescence-activated cell-sorting (FACS) technology, into two subpopulations--one low in surface galactose and one high in surface galactose. After intrasplenic injection of tumor cell subpopulations, liver metastasis was found to be proportional to the degree of tumor cell-surface galactose expression. These data suggest that tumor galactose expression and hepatic recognition may be important components of a specific mechanism of colorectal liver metastasis.
Subject(s)
Adenocarcinoma/metabolism , Colorectal Neoplasms/metabolism , Galactose/metabolism , Adenocarcinoma/pathology , Adenocarcinoma/secondary , Animals , Cell Membrane/metabolism , Cell Separation , Colorectal Neoplasms/pathology , Female , Flow Cytometry , Liver Neoplasms/secondary , Mice , Mice, Inbred BALB C , Neoplasm Transplantation , Tumor Cells, CulturedABSTRACT
Previously, all carcinoid tumors were considered to occur sporadically. We present an extremely rare series of three first-degree relatives in two generations with proximal duodenal carcinoid tumors and other family-associated neoplasms. This unusual circumstance, which has never been described previously, represents the sixth case of familial clustering of carcinoid tumors reported in the world's literature.
Subject(s)
Carcinoid Tumor/genetics , Duodenal Neoplasms/genetics , Adult , Haplotypes , Humans , Male , Middle Aged , PedigreeABSTRACT
A phase II trial was conducted with 15 patients (mean age of 65.7 years) with locally recurrent or intransit melanoma of the extremities. After total outflow occlusion with pneumatic tourniquet, the cell-cycle nonspecific anti-neoplastic agent cis-diamminedicholoroplatinum (CDDP) was infused intra-arterially in a mean dose of 26.7 mg/m2 per infusion (2.6 infusions per patient). The aim of this study was to determine the efficacy of CDDP infusion for control of intransit and recurrent melanoma of the extremities. Three to four weeks postinfusion, all visible residual disease was resected. Partial remissions were observed in ten patients (67%); five patients achieved stable disease status. No patient had complete regression of disease. At an average follow-up interval of 18.3 months (range 4-44 months), the mean local/regional disease-free survival was 14.8 months. Eighty per cent of patients (twelve of 15) had local/regional control of disease at an average follow-up of 14.8 months after CDDP infusion and surgical resection. Of five melanoma-related deaths, three patients had had no local/regional recurrence at the time of their demise. Three compartment syndromes resulted as a complication of the infusional therapy and occurred within 1-3 days of the treatment. In vitro growth of melanoma from lymph nodes draining the infused area was seen in all subjects studied. Outgrowth from tumor within the tourniquet infusion area was observed in two patients, both of whom experienced recurrences clinically at 24-months' postinfusion. Pharmacokinetic data of total CDDP concentrations from tissue and blood (n = 4) were available from pretreatment to 1 hour post-therapy. Biopsy data from patients pre- and post-treatment suggest substantial tumor uptake of CDDP as compared to local or distal normal skin, with minimal CDDP loss to the systemic circulation. Pharmacologic and clinical data of this phase II trial suggest that intraarterial infusion with tourniquet outflow-occlusion augments tumor tissue levels of CDDP within the infused extremity and enhances local control of high-risk and intransit disease.
Subject(s)
Cisplatin/administration & dosage , Extremities , Melanoma/prevention & control , Neoplasm Recurrence, Local/prevention & control , Skin Neoplasms/prevention & control , Adult , Aged , Catheters, Indwelling , Cisplatin/therapeutic use , Drug Evaluation , Female , Follow-Up Studies , Humans , Infusions, Intra-Arterial/adverse effects , Infusions, Intra-Arterial/methods , Lymphatic Metastasis , Male , Melanoma/mortality , Middle Aged , Neoplasm Recurrence, Local/mortality , Remission Induction , Skin Neoplasms/mortality , Tourniquets , Tumor Cells, Cultured/drug effectsABSTRACT
In the present study, we report a more detailed biochemical analysis of the B16 melanoma, metastasis-associated, Met-72 antigen. Specifically, we have examined (1) the molecular forms of Met-72 isolated during synthesis, surface expression and 'shedding' and (2) the cell-surface expression of Met-72 during the cell cycle. These experiments show that the 72 kD species originally described has an isoelectric point of between 6.3 and 6.9, but is the desialylated derivative of an 83 kD native molecule whose isoelectric point ranges between pH 4.9 and 5.6. In addition, a 90 kD glycoprotein doublet was immunoprecipitated from biosynthetically labelled B16 melanoma cells, but does not appear to be a precursor of the 83 kD or 72 kD molecule. These findings have led us to interchangeably use the terminology Met-72 and Met 72/83. The latter terminology more accurately describes the physical forms which can be identified by different labelling procedures. When culture supernatants from 3H-leucine labelled cells were subjected to anti-Met-72 immunoprecipitation, a 35 kD species was identified as a possible 'shed' product of these cells. Met-72/83 expression during the cell cycle was analyzed by flow cytometry and found not to be restricted to any particular stage. In addition, experiments were performed to determine whether low levels of Met-72 expression on poorly metastatic B16 melanomal clones was a direct result of low levels of synthesis, or if other control mechanisms regulated intracellular pools of Met-72 prior to cell-surface expression.
Subject(s)
Antigens, Neoplasm/biosynthesis , Melanoma, Experimental/immunology , Animals , Antibodies, Monoclonal/analysis , Antigens, Neoplasm/analysis , Antigens, Surface/analysis , Antigens, Surface/biosynthesis , Cell Cycle , Clone Cells/immunology , Electrophoresis, Polyacrylamide Gel , Female , Flow Cytometry , Mice , Mice, Inbred C57BL , Neoplasm Metastasis , Precipitin Tests , Solubility , Tritium , Tumor Cells, CulturedABSTRACT
Metastatic variants of the B16 melanoma displaying high experimental metastatic potential have been shown to express high levels of a 72,000-dalton glycoprotein (Met-72) on their cell surface (Kimura AK, Xiang J: J Nat Can Inst 76:1247-1253, 1986). Monoclonal antibodies (MoAb) directed against the Met-72 determinant have been used in this study as immunohistochemical reagents on preparations of fresh B16 melanoma tumors and their metastases. These immunohistochemical analyses have utilized frozen sections, impression smears, and cytospin preparations of fresh tumors harvested at various time points during tumor growth, to view the presence and location of Met-72-positive metastatic variants within tumor masses. Biotinylated anti-Met-72 MoAbs were reacted with freshly dissociated tumor cells from a B16 melanoma ovarian metastasis. These cells were then reacted with fluorescein isothiocyanate (FITC)-streptavidin and analyzed by flow cytometry. A discrete population of positively staining cells was detected and isolated by cell sorting techniques. Met-72-positive cells were then cloned and reanalyzed after several weeks of in vitro expansion and found to have high experimental metastatic potential to ovaries. Frozen sections of subcutaneous tumors and their metastases were analyzed by immunoperoxidase techniques. A consistent finding in these studies has been that the few tumor cells which showed high intensity of Met-72 staining were positioned perivascularly and at the invading front of B16 melanoma tumors.
Subject(s)
Biomarkers, Tumor/analysis , Melanoma/pathology , Membrane Glycoproteins/analysis , Tumor Cells, Cultured/pathology , Animals , Antibodies, Monoclonal , Female , Melanoma/diagnosis , Mice , Mice, Inbred C57BL , Neoplasm Metastasis , RadioimmunoassayABSTRACT
Two widely used B16 melanoma cell lines of low and high lung colonizing potential (B16-F1 and B16-F10) were compared in their ability to induce platelet aggregation. The results of these experiments showed a reproducible difference in platelet aggregating activity of these two cell lines which directly correlated with their lung colonizing potentials. However, when clones were derived from these heterogeneous cell lines and tested for experimental metastatic potential, platelet aggregating ability and Met-72 expression, no correlation could be attached to the platelet aggregating activity of the clones. Results of these experiments provide direct evidence that platelet aggregation is not an accurate index of experimental metastatic potential of tumor cell clones, nor is it an essential trait of all metastatic cells. The ability of tumor cells to induce platelet aggregation is examined and discussed in the context of cellular heterogeneity.
Subject(s)
Melanoma/secondary , Platelet Aggregation , Animals , Clone Cells/pathology , Female , Melanoma/blood , Mice , Mice, Inbred C57BL , Neoplasm Metastasis/bloodABSTRACT
Analysis of a number of B16 melanoma clones has revealed a high correlation between metastatic activity and the quantitative expression of a 72,000 dalton glycoprotein, Met 72. In the present study, metastatic tumor cell variants have been directly isolated from a heterogeneous, poorly metastatic melanoma (B16-F1) by anti-Met 72 monclonal antibodies and cell sorting procedures. These studies provide direct proof that Met-72 antigens are in fact surface markers of B16 melanoma metastatic variants and may provide the means of monitoring their presence, influence and autonomy during tumor progression.
Subject(s)
Antibodies, Monoclonal , Melanoma, Experimental/pathology , Animals , Antibodies, Neoplasm/immunology , Antigens, Neoplasm/analysis , Cell Separation , Flow Cytometry , Lung Neoplasms/secondary , Melanoma, Experimental/analysis , Melanoma, Experimental/immunology , Membrane Proteins/analysis , Mice , Neoplasm Proteins/analysisABSTRACT
In vitro cultures of a highly metastatic B16 melanoma clone (BL6-10) were found to undergo dramatic changes in morphology and differentiation upon transfer to another culture medium. Specifically, BL6-10 melanoma cells which had been originally selected and adapted for growth in Eagles' Hanks' amino acid supplemented media with 10 per cent newborn calf serum were amelanotic and epitheliod in shape. When these cells were shifted into Dulbecco's modified Eagles medium with 10 per cent fetal calf serum, they became highly melanotic and of spindle/dendritic morphology within 4 days of culture. These morphological changes as well as other parameters were all characteristic of established criteria of melanoma differentiation. Alterations in the differentiation state of our highly metastatic variant, BL6-10, did not result in any change in tumorigenicity but did have profound effects on metastatic potential. All of the morphological and functional characteristics of the differentiated melanoma were found to be reversible by re-plating the cells in their original growth medium and 4 days of in vitro growth. These studies have allowed us to follow and more firmly establish Met-72 antigen expression as a surface marker for metastatic cells of the B16 melanoma, and have provided direct experimental evidence that the less differentiated, Met-72 positive melanoma form is the dominant cell type capable of metastatic potential.
Subject(s)
Lung Neoplasms/secondary , Melanoma/secondary , Animals , Antibodies, Monoclonal , Cell Differentiation , Cell Division , Cell Line , Cell Membrane/immunology , Culture Media , Epitopes/immunology , Female , Flow Cytometry , Lung Neoplasms/immunology , Melanoma/immunology , Mice , Mice, Inbred C57BL , Microscopy, Electron , Phenotype , Photomicrography , RadioimmunoassayABSTRACT
In the present study, both poorly and highly metastatic clones derived from the C57BL/6 mouse B16 melanoma were used with cyclophosphamide in an attempt to elicit host antibody responses against cell surface markers expressed on highly metastatic tumor variants. The immunizations, performed in both syngeneic and xenogeneic combinations in Lewis rats, resulted in the production of 3 mouse and 2 rat monoclonal antibodies (MoAb) that preferentially react with highly metastatic clones derived from the B16 melanoma. These MoAb all immunoprecipitated a 72,000-dalton, cell surface-expressed glycoprotein, referred to as Met-72. In this study, 2 of the mouse anti-Met-72 MoAb were examined in detail for a) tumor specificity, b) reactivity against normal mouse tissue by in vivo absorption, and c) their ability to discriminate highly metastatic clones derived from the B16 melanoma.
Subject(s)
Antigens, Neoplasm/analysis , Antigens, Surface/analysis , Melanoma/immunology , Animals , Antibodies, Monoclonal/immunology , Antibody Specificity , Cell Line , Cyclophosphamide/pharmacology , Immunization , Mice , Mice, Inbred C57BL , Molecular Weight , Neoplasm Metastasis , Phenotype , Rats , Rats, Inbred LewABSTRACT
We recently described the production and characterization of an Ab-specific monoclonal antibody (mAb), designated K14.83-11, which specifically enhanced the alloreactivity of normal T lymphocytes, as well as highly selected Ab-specific T-cell lines and clones against the appropriate Ab molecule. In the present report, we have studied both the binding specificity and immunoenhancing activity of K14.83-11 in a number of genetic combinations to better characterize the possible modes through which this mAb achieves its T cell-enhancing activity. Results reported here (1) confirm and extend previous findings revealing the possible importance of the immunoglobulin G3 isotype of K14.83-11 in its immunoenhancing activity, and (2) show a distinction in the determinant binding and subsequent ability to elicit an enhancing effect. Surprisingly, K14.83-11 proved ineffective in enhancing autologous or Ab-restricted reactions. This system, then, may provide the means of comparing the functional importance of Ab expression in self-restricted and allogeneic reactions.
Subject(s)
Antibodies, Monoclonal/immunology , H-2 Antigens/immunology , T-Lymphocytes/immunology , Animals , Antibody Specificity , Epitopes , Growth Substances/biosynthesis , H-2 Antigens/genetics , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fc Fragments/immunology , Isoantigens/immunology , Lymphocyte Activation , Lymphokines/biosynthesis , Mice , Mice, Inbred StrainsABSTRACT
An anti-I-Ab monoclonal antibody, designated K14.83-11, was produced in a fusion between SP 2/0 Ag-14 myeloma cells and spleen cells from a B10.D2/n mouse primed in vivo against C57BL/10. Unlike other anti-I-Ab monoclonal antibodies thus far described, K14.83-11 was found to have a combination of features involving specificity, isotype, and function, unique among existing anti-I-A reagents. K14.83-11 exhibited a strong binding to the I-Ab gene product, with only slight cross-reactivity to the I-Ap/q family of allelic products and no reactivity towards I-Ak,d. When analyzed for isotype, K14.83-11 was found to be of a rare IgG3 isotype. With respect to biologic activity, K14.83-11 not only failed to produce the expected inhibition of specific anti-I-Ab T cell reactivity in vitro, but instead produced a striking enhancement of T cell responses against the I-Ab gene product. The possible relationship of IgG isotype and function was suggested when the immunoenhancing effect of K14.83-11 on reactive T lymphocytes was reversed to that of suppression with highly purified F(ab)2 fragments obtained by pepsin digestion.
Subject(s)
Antibodies, Monoclonal/physiology , Antibody Specificity , Histocompatibility Antigens Class II/immunology , Lymphocyte Activation , T-Lymphocytes/immunology , Adjuvants, Immunologic/physiology , Animals , Antibodies, Monoclonal/immunology , Binding Sites, Antibody , Cell Line , Female , Immunoglobulin G/physiology , Male , Mice , Mice, Inbred C57BLABSTRACT
B16-F1 melanoma cells were plated onto plastic tissue-culture dishes rendered non-adhesive for cells by coating with 0.12 per cent poly(2-hydroxyethyl methylacrylate), poly(HEMA). These growth conditions caused the normally flat, adherent B16-F1 cells to grow as single cells in suspension. Within 24 hours, the rounded cells formed aggregates and grew at a slower rate than control cells grown at the same density on untreated plastic dishes. Microscopic observations provided evidence that polykaryocytosis was occurring among the aggregates. Following replating onto standard adhesive tissue-culture plastic, 20-30 per cent of the aggregates were observed to contain varying numbers of multinucleated giant cells (polykaryocytes). The study has revealed a previously undescribed propensity of certain B16-F1 cells cultivated as aggregates in suspension to develop into polykaryocytes, most probably as a result of spontaneous tumor cell-tumor cell fusion. The possible relevance of this behavior in vitro to events in tumor progression is discussed. This study, however, does not support the findings of others that the metastatic capability of B16-F1 cells is increased by such non-adherent culture conditions. No increase in metastatic potential was observed for B16-F1 cells, or for a low metastatic clone (F1-7) derived from it, grown for 72 or 96 hours in a spherical configuration compared to control cells grown in a flat, adherent monolayer.
Subject(s)
Melanoma/pathology , Animals , Cell Adhesion , Cell Division , Cell Nucleus/ultrastructure , Female , Lung Neoplasms/secondary , Mice , Mitosis , Neoplasm Metastasis , Polyhydroxyethyl Methacrylate/metabolismABSTRACT
In the experiments reported here, we examine the need for cell division as a critical component in the clonal expansion of alloreactive CTL precursors. Unlike previous attempts to inhibit DNA synthesis and cell division non-specifically, we have chosen to follow the normal unimpeded development of CTL in two of the most commonly used in vivo and in vitro allograft systems. The development and relative contribution of CTL lymphoblast-associated cytotoxicity has been followed by density gradient separation and functional analysis of the various fractions of lymphocytes obtained throughout the entire course of sensitization. In addition to the physical parameters (size and density), even more convincing data have been obtained from in vivo administration of 3H-TdR during the entire allograft reaction. The results presented here clearly confirm CTL precursor proliferation in vitro but provide strong evidence that in vivo CTL normally arise via a mechanism independent of blast formation and cellular proliferation. Interpretations of these findings in relationship to the concept of "clonal expansion" for the generation of mature CTL are discussed.
Subject(s)
Hematopoietic Stem Cells/immunology , Lymphocyte Activation , T-Lymphocytes, Cytotoxic/immunology , Animals , Cell Count , Cell Division , Cell Separation , Centrifugation, Density Gradient , Female , H-2 Antigens/administration & dosage , Hematopoietic Stem Cells/cytology , Killer Cells, Natural/immunology , Kinetics , Lymphocyte Culture Test, Mixed , Male , Mice , Mice, Inbred C57BL , Mice, Inbred CBA , Mice, Inbred DBA , T-Lymphocytes, Cytotoxic/classification , T-Lymphocytes, Cytotoxic/cytology , Transplantation, HomologousABSTRACT
Cloned lines of the methylcholanthrene-induced DBA/2 T lymphoma Eb and its highly metastatic variant line ESb were analyzed for differences in the expression of serologically detectable cell surface differentiation markers. Flow cytofluorographic analysis of cells stained with fluorescein isothiocyanate-conjugated monoclonal rate anti-mouse Thy-1, Lyt-1, Lyt-2 and complement-dependent cytotoxicity with mouse alloantisera against Lyt-3.2 and Ly-6.2 revealed, for the parental low metastasizing line, Eb, a phenotype of Thy-1+, Lyt-1-, Lyt-2+, Lyt-3+, Ly-6+, whereas the highly metastasizing variant line typed as Thy-1-, Lyt-1+, Lyt-2-, Lyt-3-, Ly-6-. Analysis of galactose oxidase/NaB3H4-labeled glycoproteins from Eb and ESb clones by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate showed further phenotypic differences. Selective binding of radiolabeled glycoproteins to Helix pomatia or Vicia villosa-Sepharose, respectively, allowed the identification of T130 to be expressed on Eb cells and T145 to be expressed on some ESb clones. The latter antigen is expressed on murine cytotoxic T lymphocytes. Immune precipitation analysis revealed that Eb and ESb bear different molecular forms of the T200 antigen. Comparisons of iodinated surface proteins derived from tumor cells either treated or untreated with tunicamycin indicated that many of the differences in membrane proteins between Eb and ESb cells could be attributed to differences in glycosylation. Our results, derived from a defined tumor system of lymphoid origin, show that the progression from a low to a high malignant tumor line can be associated with changes in the expression of various defined cell surface differentiation antigens. The question of a possible relationship between tumor progression and cell differentiation or dedifferentiation is discussed.
Subject(s)
Antigens, Ly/genetics , Glycoproteins/genetics , Membrane Proteins/genetics , T-Lymphocytes/immunology , Animals , Antigens, Surface/genetics , Borohydrides/metabolism , Female , Galactose Oxidase/metabolism , Glycoproteins/immunology , Glycoproteins/metabolism , Lymphoma/genetics , Lymphoma/immunology , Lymphoma/metabolism , Membrane Proteins/immunology , Membrane Proteins/metabolism , Mice , Mice, Inbred DBA , Molecular Weight , Neoplasm Metastasis , Receptors, Mitogen , Thy-1 Antigens , Tunicamycin/pharmacologyABSTRACT
Subclasses of lymphocytes can be separated on gradients of non-toxic polyvinylpyrrolidone-coated colloidal silica (Percoll) by virtue of differential densities. Such gradients can yield functionally active lymphocyte populations after brief centrifugation. Gradients can be generated in a discontinuous step fashion and centrifuged in standard table-top laboratory centrifuges or as self-generating gradients during ultracentrifugation. The density medium has low viscosity and can be made isotonic for virtually any use. Gradients have proved useful in both human and experimental animal studies, and high percentage yields allow for separations from small cell numbers. Methods are described for separation of whole blood and lymphoid subpopuctions. The cytoxic capability of various density fractions was evaluated for mixed lymphocyte culture-induced allogeneic killing and spontaneous, so-called "natural" killer cell activity. The lower density associated with blast transformation allows for significant enrichments of stimulated cells from in vitro cultures. Higher thymidine incorporation, restimualtion in mixed lymphocyte reactions, and greater cytotoxic capacity are associated with these "blast" fractions.
Subject(s)
Cell Separation/methods , Centrifugation, Density Gradient , Lymphocytes/immunology , Povidone , Animals , Cytotoxicity Tests, Immunologic , Humans , Immunologic Techniques , Killer Cells, Natural/immunology , Lymphocyte Activation , Lymphocyte Culture Test, Mixed , Mice , Phytohemagglutinins/pharmacology , Rosette Formation , Silicon Dioxide , Spleen/immunologyABSTRACT
We have analyzed the surface glycoproteins of resting and in vitro activated human T lymphocytes by the galactose oxidase/NaB3H4 and the periodate/NaB3H4 labeling techniques. The labeled glycoproteins were separated by polyacrylamide slab gel electrophoresis and visualized by fluorography. A "new" glycoprotein with an apparent molecular weight of 130,000 (GP130) was strongly labeled on alloantigen-activated T blasts but only weakly or not at all on mitogen-stimulated T blasts and resting T lymphocytes. These results demonstrate that human T cells, as earlier found in the mouse system, express different surface molecules in relation to the particular mode of activation and stage of differentiation.