In vitro differentiation and antigenic changes in human melanoma cell lines.

Malignant transformation of melanocytes may be associated with changes in the expression of HLA antigens and melanoma-associated antigens (MAA). To determine whether these changes reflect the differential expression of HLA antigens and MAA by melanocytes at different stages of differentiation, we have studied the effect of the reversible induction of differentiation by fibroblast interferon (interferon beta) and/or 12-O-tetradecanoyl-phorbol 13-acetate (TPA) on the expression of HLA antigens and MAA by the melanoma cell lines DU-2, FO-1 and HO-1. The three melanoma cell lines differed in their sensitivity to the differentiating and antiproliferative activity of these two compounds and displayed an increased growth suppression and induction of differentiation, when incubated with the combination of TPA and interferon beta. Incubation of the three melanoma cell lines with interferon beta, TPA or their combination resulted in a differential modulation of the expression of membrane-bound high-molecular-mass melanoma-associated antigen, 115-kDa MAA, 100-kDa MAA, intercellular adhesion molecule 1, HLA class I antigens and gene products of the HLA-D region. Each melanoma cell line displayed a unique pattern of antigenic modulation when exposed to the two differentiating agents alone or in combination. No direct relationship was found between the effects of interferon beta and/or TPA on the growth and differentiation of the three melanoma cell lines and the expression of HLA antigens or the MAA evaluated in the present study. These findings argue against a direct role of any of the antigens tested in the reversible induction of human melanoma cell differentiation in the in vitro system.

Enhanced in vitro growth suppression of human glioblastoma cultures treated with the combination of recombinant fibroblast and immune interferons.

In the present study we have evaluated the effect of recombinant human fibroblast, IFN-beta ser, and immune, IFN-gamma, interferon, alone and in combination, on the proliferation of fifteen early passage human glioblastoma cell cultures. Explant cultures were established from glioblastoma tumor tissue obtained at the time of surgery. After sufficient outgrowth, cultures were dispersed with trypsin/versene and maintained as independent cell lines. IFN-beta ser induced a greater than or equal to 50% reduction in the 7 day growth of 6 of the 15 cultures. The majority of cultures, 9 of 15, displayed less than or equal to 50% growth suppression in comparison with control cultures after 7 days exposure to 2000 Units/ml of IFN-beta ser. When treated with 2000 Units/ml of IFN-gamma, only 1 of the 15 glioblastoma cultures exhibited a greater than or equal to 50% reduction in growth. In contrast, when treated with the combination of IFN-beta ser plus IFN-gamma, 1000 Units/ml of each interferon preparation, 12 of 15 cultures were inhibited by greater than or equal to 50% after 7 days growth. The combination of interferons was effective in suppressing glioblastoma growth both in cultures displaying relative sensitivity and those exhibiting innate resistance to either or both types of interferon when employed alone. One glioblastoma culture, G-7, was studied through 45 passages and displayed the same sensitivity at different passages to growth inhibition when exposed to IFN-beta ser, IFN-gamma or both interferons. Based on previous clinical studies indicating that IFN-beta or IFN-gamma when administered alone to patients do not generally alter the clinical progression of malignant gliomas, the present results suggest that the combination of IFN-beta plus IFN-gamma may prove more effective than either agent alone in the clinical treatment of patients with glioblastoma multiforme.

Immunohistochemical localization of procainamide in normal, ischemic, and necrotic canine myocardium during acute experimental myocardial infarction.

This report represents the first application of immunohistochemical methods for localizing an exogenously administered drug. Intravenously administered procainamide was localized in normal, ischemic, and necrotic myocardium in 23 dogs. Rabbit antiprocainamide antibodies were used in an avidin-biotin-peroxidase complex staining method. Normal myocardium demonstrated diffusely positive immunostaining for procainamide, as did the cardiac conduction system and vascular endothelial cells. Necrotic myocardium demonstrated markedly reduced to absent immunostaining. By contrast, in regions of myocardial ischemia without necrosis, immunostaining for procainamide was similar to that in the normal myocardium. Procainamide myocardial tissue levels were reduced in necrotic and ischemic zones compared to normal (p less than 0.05) only in those animals in which procainamide was administered after rather than before the onset of coronary occlusion. The demonstration of the absence of drug binding in the necrotic cells suggests that myocardial tissue levels or radiolabelled assessment of drug distribution can be misleading when nonhomogeneous tissue is sampled. The immunohistochemical technique provides additional information about the regional and cellular distribution of procainamide that is complementary to the information obtainable by radiolabelling microspheres and from biochemical assays.

Effect of recombinant human fibroblast interferon and mezerein on growth, differentiation, immune interferon binding and tumor associated antigen expression in human melanoma cells.

The combination of recombinant human fibroblast interferon (INF-delta) and the antileukemic compound mezerein (MEZ) results in a synergistic suppression in the growth of human melanoma cells and a concomitant increase in melanin synthesis. In the present study we have further analyzed this synergistic interaction and have also evaluated the effect of IFN-delta and MEZ, alone and in combination, on recombinant human gamma interferon (IFN-gamma) binding and Class I HLA and melanoma associated antigen (MAA) expression in the HO-1 human melanoma cell line. Single cell clones isolated from the HO-1 cell line varied in their sensitivity to the antiproliferative effects of IFN-delta and MEZ. With all twelve clones, however, the combination of IFN-delta plus MEZ was more growth inhibitory than either agent used alone, even in HO-1 subclones displaying relative resistance to IFN-delta. By continuous growth in gradually increasing concentrations of IFN-delta, a variant population of HO-1 cells, HO-1 delta R-D, was generated which was more resistant to the antigrowth effects of IFN-delta than the original HO-1 parental cell line. In the IFN delta R-D cell line the combination of IFN-delta plus MEZ synergistically suppressed growth. Exposure of HO-1 cells to 2500 units/ml IFN-delta or 50 ng/ml MEZ for 96 hr resulted in no change or an increase in the binding of labelled IFN-gamma to surface receptors, whereas the combination of IFN-delta plus MEZ increased IFN-gamma binding 2-to-4-fold in HO-1 cells. This increase was the result of an increase in the number of receptors on treated cells coupled with a protection against a decrease in receptors observed for confluent untreated cells. Changes in IFN-gamma binding resulting from treatment with IFN-delta plus MEZ were not associated with alterations in the binding affinity of INF-gamma to its receptor. Changes were also observed in the expression of HLA Class I antigens and MAAs following treatment of HO-1 cells with IFN-delta, MEZ or IFN-delta plus MEZ. IFN-delta and MEZ increased the expression of HLA Class I antigens a 96 kd MAA defined by MoAb CL203, a 100 kd MAA defined by MoAb 376.96 and a 115 kd MAA defined by MoAb 345.134 but decreased the expression of a high molecular weight-melanoma associated antigen (HMW-MAA) defined by MoAb 325.28S.(ABSTRACT TRUNCATED AT 400 WORDS)

Distribution of fibrinogen and albumin in normal, ischaemic, and necrotic myocardium during the evolution of myocardial infarction: an immunohistochemical study.

The role of mediators of inflammation in the pathogenesis and evolution of myocardial infarction has attracted increased interest as interventions which inhibit the inflammatory response after coronary artery occlusion have been shown to decrease infarct size. The distribution of fibrinogen and albumin in ischaemia myocardium after closed chest balloon occlusion of the left anterior descending coronary artery was studied by immunohistochemical techniques in 34 dogs, and compared to morphological evaluation of cellular injury. In myocardium which was ischaemic but not necrotic (that is, glycogen loss and the absence of light and electron microscopic and tetrazolium staining evidence of necrosis, n = 8 dogs) no accumulation of these proteins was detected within the ischaemic zone. In myocardium which was necrotic by morphological criteria (n = 26 dogs), fibrinogen and albumin were detected in the necrotic fibres as early as 3 h after coronary occlusion using both the peroxidase-antiperoxidase and avidin-biotin immunostaining methods. Non-ischaemic myocardium never showed positive staining. The presence of fibrinogen and albumin in myocardial fibres appears to be specific for indicating irreversible injury.

Limitations of postmortem assessment of human coronary artery size and luminal narrowing: differential effects of tissue fixation and processing on vessels with different degrees of atherosclerosis.

Numerous studies have utilized histologic sections of coronary arteries as the standard for testing the validity of the angiographic determination of coronary artery dimensions. However, little attention has been given to artifactual dimensional changes that occur during fixation and histologic processing of tissues (dehydration, clearing, embedding, sectioning and staining). Using planimetric techniques, the dimensional changes that occurred with fixation and processing were quantitated in 61 coronary artery segments with minimal or moderate to severe atherosclerosis obtained from 12 patients studied at autopsy. In vessels with minimal atherosclerotic narrowing, fixation and processing resulted in a decrease in total vessel cross-sectional area and luminal cross-sectional area (p less than or equal to 0.05), whereas absolute wall area (total vessel cross-sectional area minus luminal cross-sectional area) did not change (p = NS). These disproportionate changes resulted in an alteration in the relation between lumen and wall areas so that luminal cross-sectional area decreased from 47.6 +/- 8.5% of the total vessel cross-sectional area observed before fixation to 36.2 +/- 7% after processing (p less than or equal to 0.05). In vessels with moderate to severe atherosclerosis, both the total cross-sectional area and wall area decreased after fixation and processing (p less than or equal to 0.05), but luminal area did not change (p = NS). As a result, the percent luminal cross-sectional area in these vessels increased from 21.1 +/- 10.1% before fixation to 28.7 +/- 9.7% after processing (p less than 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical localization of 6-keto-PGF1-alpha in canine coronary vasculature.

The localization of the prostacyclin metabolite, 6-keto-PGF1-alpha, in canine coronary vasculature was accomplished using immunohistochemical techniques (avidin-biotin method of immunoperoxidase staining). Six-keto-PGF1-alpha was localized to the intimal endothelial cell layer of epicardial and intramyocardial arteries and veins. No specific staining was seen in the the media or adventitia of canine coronary vasculature, or in capillaries, or myocardial fibers. To our knowledge these studies represent the first immunohistochemical demonstration of the endothelial cell localization of the prostacyclin metabolite, 6-keto-PGF1-alpha. The described technique allows the cellular localization of prostaglandin metabolites in histologic sections.

Distribution of cytosolic and mitochondrial aspartate aminotransferase in normal, ischemic, and necrotic myocardium. An immunohistochemical study.

We utilized immunoperoxidase methods to study the distribution of both cytosolic or soluble(s) and mitochondrial (m) aspartate aminotransferase (AspAT) in normal, ischemic, and necrotic myocardium. Human myocardium was obtained from autopsy (n = 9) and surgery (n = 6). Cardiac tissue from 26 dogs with experimental myocardial infarction induced by closed-chest balloon occlusion and four dogs with myocardial ischemia without necrosis induced by a 50% reduction in left main coronary artery flow for 3 hours were studied. Duration of occlusion was 45 minutes (n = 1), 3 hours (n = 11), 5 to 6 hours (n = 10), or 15 to 24 hours (n = 4). Highly purified m- and s-AspAT and specific antibodies were prepared in our laboratory. In all cases, control experiments were performed. Microscopically normal human and dog myocardium uniformly stained for m- and s-AspAT. Necrotic myocardium from patients with infarcts showed markedly reduced immunostaining. In those dogs with myocardial necrosis, all dogs with coronary occlusion of 5 to 24 hours, and eight of 11 dogs with 3-hour occlusions, immunostaining was significantly reduced for both s- and m-AspAT in regions confirmed to be necrotic by triphenyl tetrazolium chloride and hematoxylin and eosin staining. Myocardial necrosis was confirmed in the 3-hour infarcts by electron microscopy. In the four dogs with a 50% reduction in left main flow for 3 hours, and one dog with a 45-minute coronary occlusion, ischemia was demonstrated by glycogen loss in period acid-Schiff-stained sections but there was no evidence of necrosis by electron microscopy or triphenyl tetrazolium chloride staining and there was no loss of immunostaining evident for s- or m-AspAT. Thus, s- and m-AspAT were visualized in normal and ischemic myocardium with decreased staining in necrotic tissue using immunoperoxidase techniques. Loss of both s- and m-AspAT can be demonstrated in human myocardium and in experimental canine myocardium as early as 3 hours after coronary occlusion and appears to be specific for irreversible myocardial injury. No depletion of isoenzyme can be detected by immunohistochemical techniques in tissue that is ischemic but not necrotic. Furthermore, by these immunoperoxidase techniques, loss of s- and m-AspAT from necrotic myocardium appears to be simultaneous.

Myocytolysis (vacuolar degeneration) of myocardium: immunohistochemical evidence of viability.

Human myocardium with focal myocytolysis (vacuolar degeneration, colliquative myocytolysis) was examined by routine light microscopy and by immunoperoxidase staining techniques for creatine kinase (CK) M and B, myoglobin, lactate dehydrogenase (H4)(LDH-1), and aspartate aminotransferase (AST, GOT). Sections of myocardium were selected from autopsy and surgical specimens from patients with and without clinical morphologic evidence of ischemic heart disease. Areas of coagulation necrosis showed loss of enzyme staining, while both normal and myocytolytic cells stained darkly. These results indicate that fibers with myocytolysis retain enzymes and other proteins, indicating sarcolemmal integrity, which is not present in fibers with coagulation necrosis. The implication of these findings is that fibers with myocytolysis are viable; thus, myocytolysis may be a reversible form of myocardial alteration that does not necessarily lead to cell death and eventual myocardial fibrosis.

Distribution of LDH-1 in normal, ischemic, and necrotic myocardium. An immunoperoxidase study.

To study the distribution of lactate dehydrogenase (LDH-1) (H4) in normal, ischemic, and necrotic myocardium using the peroxidase-antiperoxidase technic, the authors studied formalin-fixed paraffin-embedded sections of human (n = 11) and canine (n = 28) myocardium. All normal control myocardium showed positive immunostaining for LDH-1 (H4). In infarcts 10 hours or more old, the histologically necrotic myocardium (by triphenyl tetrazolium chloride staining) (TTC) showed markedly diminished immunostaining. In 24-dogs ischemia was induced in a closed-chest model using a balloon-tipped catheter inflated in the left anterior descending coronary artery. In dogs with 3 hours or more of occlusion, myocardium that was necrotic by TTC staining, light and/or electron microscopy, showed diminished staining for LDH-1, while normal, control myocardium stained intensely. In four dogs, ischemia was induced by a controlled perfusion apparatus by which left main coronary flow was reduced by 50%. Ischemia without necrosis was documented by demonstration of glycogen loss with no light or electron microscopic evidence of necrosis. These ischemic fibers stained intensely for LDH-1, as did controls. Thus, by immunoperoxidase staining, LDH-1 can be demonstrated in normal human and canine myocardium. In experimental models of ischemia in dogs, tissue that was ischemic but not necrotic showed no diminished staining. LDH-1 loss can be detected in necrotic myocardium as early as 3 hours after coronary artery occlusion.