Fetal antigens on the surface of human lymphoid cells.

Human B-lymphoblastoid cells established in long-term culture from healthy adults carry surface components that are normally found in human fetal tissues at about 10 wk of age. These antigens are strongly expressed on neoplastic B lymphocytes but not on thymocytes or a cultured T-cell line. They are carried by a small subpopulation of normal adult peripheral blood lymphocytes as well.

Fetal antigens in nonneoplastic conditions.

During studies that showed the presence of fetal antigens on the surface of human malignant melanoma tumor cells, polyvalent antisera specific for human fetal tissues of varying ages were developed. These reagents demonstrated varying patterns of expression of fetal antigens at different ages in various tissues of the human fetus. The possibility that nonneoplastic adult cells showing either maturation arrest or excessive proliferation also might express fetal antigens led to studies of human bone marrow. Although normal bone marrow cells expressed low levels of fetal antigens, large amounts were seen on bone marrow cells of patients with anemias due to iron, B12, or folic acid deficiencies, as well as on those with leukemia. Moreover, normal adult tissues adapted to long-term culture also expressed fetal antigens. After 3 weeks in organ culture adult human skin showed morphological changes similar to those seen in fetal periderm and strongly expressed fetal antigens. In addition, lymphoblasts in long-term cultured human lymphoid cell lines established from normal donors also carried surface fetal antigens. These latter antigens were shared with neoplastic B-cells (chronic lymphocytic leukemia) but not with T-cells. Their expression varied with the cell cycle. The reexpression of fetal antigens on malignant cells is thought to signal a basic derangement in the control of differentiation which is considered to be peculiar to neoplasia. However, these studies indicate that normal adult cells also may reexpress fetal antigens under circumstances unrelated to neoplasia but associated with either maturation arrest or rapid and excessive proliferation.

The Langerhans cell.

In all mammalian species so far examined, Langerhans cells or their precursors are the only epidermal cells expressing Ia antigens or their equivalents. In man, xeno-antisera raised in rabbits against purified B-lymphocyte cell membrane antigens were utilized to stain the Langerhans cells by either fluorescent or immunoferritin methods. As high proportion of the indeterminate cells in the epidermis also expressed HLA-DR antigens, and a relationship to Langerhans cells is suggested. Confirmation of these results was obtained in mouse. Alloantisera raised against I-A and I-EC subregion products again stained only Langerhans cells. Fluorescent, immunoperoxidase, and immunoferritin methods were used and confirmation of the specificity of the reaction was achieved at the electron microscope level. Langerhans cells were shown by ATPase staining to be absent from the epithelium of the central cornea, but present in the limbus. Population of the entire corneal epithelium surface was induced by application of irritants or contact sensitizing agents such as DNCB. Grafting of corneas either deficient or populated with Langerhans cells, to skin beds, may answer the question of the influence of such cells on allograft rejection.

Neurohumoral regulation of excitation-contraction coupling in ventricular myocytes from cardiomyopathic hamsters.

STUDY OBJECTIVE: The aim was to evaluate the regulation of contractility by autonomic stimulation in the necrotic stage of cardiomyopathy in male Syrian hamsters. DESIGN: The electrical and contractile activity of isolated intracellularly stimulated ventricular myocytes has been recorded and dose-response curves to [Ca2+]o, and to beta adrenergic, alpha adrenergic, and muscarinic agonists and antagonists were examined. EXPERIMENTAL MATERIAL: Ventricular cardiomyocytes were isolated by enzymatic dissociation of hearts from 90 to 120 day old cardiomyopathic hamsters CHF 147 and from age matched non-myopathic controls: CHF 148 or golden hamsters. MEASUREMENTS AND MAIN RESULTS: The membrane potential was recorded by suction (patch) electrodes. The contractile activity was recorded by a video system as the shortenings of the myocytes. The contractile response (EC50) to beta adrenergic stimulation (isoprenaline) showed a bimodal distribution: 60% of the myopathic myocytes responded like the controls, while in the remaining 40% the sensitivity was significantly decreased. The electrical activity and beta adrenergic receptor density were not different from the controls. The alpha adrenergic stimulation (by phenylephrine) was enhanced, while response to the muscarinic agonist carbachol (in the presence of isoprenaline) was attenuated in the myopathic cells. Sensitivity to [Ca2+]o was unchanged. CONCLUSIONS: Profound changes occur in the contractile response of myocytes from cardio-myopathic hamster to stimulation by mediators of the autonomic nervous system, which at this necrotic stage are not related to any significant changes in basal contractile response to [Ca2+]o, to the electrical activity, or to the number of beta adrenergic receptors.

Expression of Ia antigens on Langerhans cells in mice, guinea pigs, and man.

In all mammalian species so far examined, Langerhans cells or their precursors are the only epidermal cells expressing Ia antigens or their equivalents. In man, xenoantisera raised in rabbits against purified B lymphocyte cell membrane antigens were utilized to stain the Langerhans cells, by either fluorescence or immunoferritin methods. A high proportion of the indeterminate cells in the epidermis also expressed HLA-DR antigens, and a relationship to Langerhans cells is suggested. Confirmation of these results was obtained in mouse. Alloantisera raised against I-A and I-EC subregion products again stained only Langerhans cells. Fluorescence, immunoperoxidase, and immunoferritin methods were used, and confirmation of the specificity of the reaction was achieved at the electron microscope level. Langerhans cells were shown, by ATPase staining, to be absent from the epithelium of the central cornea, but were present in the limbus. Population of the entire corneal epithelium surface was induced by application or irritants or contact sensitizing agents such as dinitrochlorobenzene. Grafting of corneas either deficient or populated with Langerhans cells, to skin beds, may answer the question of the influence of such cells on allograft rejection.

Ultrastructural studies of keratinized epithelia of the mouse. IV. Quantitative studies of lysosomes.

Electron microscope cytochemical and simple morphometric studies have permitted an estimate to be made of the absolute numbers of lysosomes present in various cell layers of murine epidermis. Most lysosomes appear to be present in basal layer keratinocytes with few being detected in Langerhans cells or in granular layer keratinocytes. The observation that lysosomes are not numerous in any of the strata is discussed with respect to an alternative explanation for the presence of diffuse acid hydrolase activity in the granular layers.

Ultrastructural studies of keratinized epithelia of the mouse. III. Determination of the volumes of nuclei and cytoplasm of cells in murine epidermis.

Simple morphometric analyses were applied to mouse epidermal specimens prepared for electrom microscopy. Mean values were obtained for the dimensions of cells and nuclei in basal, suprabasal, and granular layers. These measurements were applied to simplified models representing the shapes of cells in the three strata. A fourfold increase in cytoplasmic volume was observed as cells passed from the basal to granular layers. During this transition, the nuclear volume did not decrease significantly.

Antigens specified by the Tla locus are expressed on the surface of murine Langerhans cells.

A monoclonal antibody against the murine thymus leukemia antigen TL, was employed to demonstrate the presence of the antigen on the surface of dendritic cells in murine epidermis of Tla-positive strains, B.10A and A.TH. Immunofluorescence and immunoperoxidase staining of EDTA-separated epidermal sheets demonstrated dendritic cells with a distribution pattern and density comparable to that noted for anti-IAk staining. Tla-negative mouse strains such as A.TL, C3H/HeJ, and C57BL/6 did not show any staining of dendritic epidermal cells. Epidermal cell suspensions similarly contained 2-4% cells with discrete surface staining with anti-TL antibody. Capping was noted in these cells. Once again positive results were noted only in appropriate Tla-positive strains. Control staining was carried out in all cases on frozen sections of thymii from mice. Thymocytes in the cortical zones and some dendritic cells at the corticomedullary junction were stained. TL antigen in mouse appears to be analogous to T-6 antigen previously detected on human Langerhans cells.

Comparative epidermal Langerhans cell migration studies in epidermal and epidermal/dermal equivalent grafts.

Immigration of Langerhans cell precursors from the peripheral blood to the skin was studied in human grafts placed on severe combined immunodeficient (SCID) mice. Monocyte fractions of human blood were injected intraperitoneally to SCID bearing either reconstituted (Langerhans cell free) epidermal sheets (E) or living skin equivalents (E/D) consisting of both epidermis and dermis. A range of immunocytochemical and ultrastructural markers was employed to monitor the colonization of the grafts, i.e., CD1a/c, Birbeck granules. In situ hybridization with probes against Alu sequences of human DNA were employed together with immunostaining for MHC class I mouse and human antigens to document graft survival. Although unequivocal LC were detected within E grafts, including both human (CD1a positive) and murine (NLDC-145 positive), no migration was achieved in the E/D situations.

Comparison of S-100 and OKT6 antisera in human skin.

The monoclonal antibody OKT6 and antisera against S-100 protein have both been advocated as immunologic markers of Langerhans cells in the skin. S-100 antiserum has an advantage in its ability to stain Langerhans cells in paraffin tissues. In order to evaluate whether these antibodies stain equivalent numbers of Langerhans cells in skin, we compared the staining patterns of S-100 antiserum and OKT6 antibody on biopsy specimens from 40 patients with leprosy using immunoperoxidase techniques. Utilizing OKT6 antibody, greater numbers of positive Langerhans cells were found in the epidermis in tuberculoid leprosy, reversal reaction, and erythema nodosum leprosum than in lepromatous leprosy. However, these differences were not observed with the S-100 antiserum and, overall, fewer cells were found as compared with the OKT6 antibody. In the dermis both antibodies stained "dendritic cells" that were found encircling granulomas in tuberculoid leprosy and reversal reaction. Staining in lepromatous leprosy granulomas, in contrast to the epidermal staining pattern, revealed rare OKT6-positive cells, while S-100 cells were numerous and were more diffusely distributed throughout the granuloma. Our results indicate that antiserum to S-100 protein and OKT6 antibody stain morphologically similar cells (dendritic cells), but do not provide comparable results concerning distribution and frequency of these cells.

Macrophages, but not Langerhans cell-like cells of dendritic lineage, express the CD36 molecule in normal human dermis: relevance to downregulatory cutaneous immune responses?

The CD36 molecule has been shown to be associated with subsets of peripheral blood monocyte/macrophages and, in cells isolated from either ultraviolet (UV)-irradiated or diseased skin, to induce downregulatory immune responses. Although macrophages are certainly present within normal human dermis, whether they normally express CD36 is still a matter of debate. In this study, we investigated dermal CD36-expressing macrophages in situ using the gold immunoelectron microscopic technique on tissue ultracryosections. This is a very sensitive and specific method, and its results clearly reflect the in vivo immunophenotypic constitutive situation. Macrophages in normal human dermis were variously shaped from round to dendritic and were localized either immediately beneath the epidermis, in perivascular areas, or in intervascular zones. Macrophages showed consistent gold-positive staining on their cell surface. In contrast, other dermal cells, including fibroblasts, lymphocytes, and mast cells, as well as dermal fibers, were not decorated with gold; dermal Langerhans cell-like dendritic cells (LC/DC), though they did show gold labeling in some intracytoplasmic organelles, did not show any gold particles along their plasma membranes. Therefore, although macrophages in normal human dermis exhibit variability with regard to their localization and shape, they regularly and constitutively expressed CD36. CD36 molecules may be considered a useful marker for macrophages in normal human dermis and may furthermore confer on macrophages, or a subpopulation thereof, intriguing functional properties (e.g., downregulatory capacity versus upregulatory capacity subserved by LC/DC) within the cutaneous immune system.

Evidence for atrophy and apoptosis in the ventral prostate of rats given the 5 alpha-reductase inhibitor finasteride.

Castration causes cell loss in the rat ventral prostate through a process called apoptosis. Although 5 alpha-reductase inhibition also causes prostate cell loss, the mechanisms involved have been debated. To investigate this question further, we have evaluated the histological responses of the rat ventral prostate to both castration and 5 alpha-reductase inhibition. Rats were left intact, castrated, or given the selective 5 alpha-reductase inhibitor finasteride. After 4, 9, 14, and 21 days the prostates were excised, the androgen and DNA content determined, and the tissue was subjected to histological and histomorphometric analysis. Finasteride and castration decreased prostate weight at day 21 by 65% and 93%, respectively. Castration decreased DNA content (micrograms per prostate) by a maximum of 88% at 14 days. Finasteride had no significant effect on DNA content after 4 days and decreased DNA content by a maximum of 52% at 14 days. When castrate prostate sections were stained for tissue transglutaminase, a marker of apoptotic cell death, a maximum of 23% of epithelial cells were stained by day 14 with a return to control levels by day 21. Finasteride caused a less intense increase in staining in which 16% of epithelial cells stained for tissue transglutaminase on day 9 with a return to baseline by day 14. When prostate sections were stained for DNA breaks, another marker of cell death, castration, caused a peak of staining on day 4 with 6% of epithelial cells staining and a return to near control levels by day 21. Finasteride-induced staining was less intense with peak staining at day 4 (0.7% of epithelial cells) and a return to control values by day 9. Morphometrics were used to assess the effect of castration and finasteride on prostate duct size and epithelial cell mass. After 4 days of finasteride treatment, the mean ductal mass decreased by 47%, with no significant change thereafter. The mean epithelial cell mass decreased by 15% on day 4 and 60% on day 9, with no further decrease thereafter. Castration caused a more rapid and greater decrease in both morphometric parameters with a 95% reduction in the mass of prostate ducts and a 93% decrease in epithelial cell mass by day 9. We conclude that castration induces a more profound involution of the rat ventral prostate than does 5 alpha-reductase inhibition. Cell loss occurs in both groups, but the degree of cell loss is less with finasteride.(ABSTRACT TRUNCATED AT 400 WORDS)

Evidence for atrophy and apoptosis in the prostates of men given finasteride.

Finasteride, a 5 alpha-reductase inhibitor, decreases prostate size and improves symptoms in men with benign prostatic hyperplasia. However, little is known about prostate histopathology in men taking finasteride. To determine the mechanism by which finasteride reduces prostate size, tissue was collected at the time of prostatectomy from men taking either no medication (n = 10) or 5 mg finasteride daily for 6-18 days (n = 6; group 1), 23-73 days (n = 5; group 2), or 3 months to 4 yr (n = 5; group 3). To assess whether finasteride causes epithelial atrophy, morphometric measurement of epithelial cell and duct width was used. The mean epithelial cell width in control prostates (mean +/- SEM, 21 +/- 0.7 microns) decreased with duration of treatment to 19 +/- 1 microns in group 1, 15 +/- 2 microns in group 2, and 8 +/- 0.3 microns in group 3. Mean duct width decreased from 135 +/- 6 microns in the control prostates to 128 +/- 10 microns in group 1, 103 +/- 3 microns in group 2, and 63 +/- 6 microns in group 3. To assess whether prostate cell death was occurring, sections were in situ end labeled for DNA breaks and immunostained for tissue transglutaminase (tTG), a marker of apoptosis (programmed cell death). The percentage of epithelial cells staining for DNA breaks was 0.4 +/- 0.2 in control prostates, 2.8 +/- 0.9 in group 1, 1.7 +/- 0.5 in group 2, and 0.7 +/- 0.3 microns in group 3. Anti-tTG staining of epithelial cells was graded on a scale of 0-4. In control prostates, 3 +/- 1% of the ducts were grade 3 or 4 (> 50% of epithelial cells staining). In finasteride-treated prostates, 2 +/- 2% of the prostates in group 1, 13 +/- 4% of the prostates in group 2, and 0.5 +/- 0.5% of the prostates in group 3 were grade 3-4. These results indicate that a progressive decrease in epithelial cell size and function occurs during the first several months in the prostates of men treated with finasteride. The staining for DNA breaks and the tTG staining also indicate that an increased rate of apoptosis is occurring transiently in these prostates. We conclude that finasteride causes prostate involution through a combination of atrophy and cell death.

Laryngeal paraganglioma. Case report with ultrastructural analysis and literature review.

Laryngeal paraganglioma is an infrequently reported tumor; only 16 examples have been recorded in the English literature. All but one laryngeal paraganglioma originated superiorly in the larynx; involvement of the ipsilateral aryepiglottic fold is common. Male patients predominate (11:5). The average age of patients at the time of diagnosis was 47 years, and symptoms had been present for an average duration of 5.8 years (range 6 months to 27 years). Attempted biopsy has resulted in significant hemorrhage in three cases. As illustrated by the present case, the Grimelius argyrophil stain is a useful diagnostic procedure. Electron microscopy confirmed the presence of neurosecretory granules with core diameters ranging from 110 to 140 nm. Surgical resection is the preferred treatment and has been possible in 14 cases; nine patients are alive and free of tumor for an average of 3 years. Compared to other head and neck paragangliomas, these have a more malignant course with a 25% mortality; tender subcutaneous metastases are commonly observed in these patients.

The relationship of mast cells and their secreted products to the volume of fibrosis in posttransplant hearts.

A series of 96 posttransplant endomyocardial biopsies taken from 11 patients was subjected to quantitative analysis of mast cells and fibrosis. Ultrastructural analysis showed that mast cell numbers were increased and there was obvious degranulation in some posttransplant hearts. Activated mast cells and their secreted products, which contain heparin and histamine, are toxic to the hearts and may contribute to interstitial and perimyocytic fibrosis. The numbers of mast cells and granules were correlated with volume of fibrosis (r = 0.63, P less than 0.025; r = 0.73, P less than 0.01). There were differences between the release of mast cell granule contents seen in the posttransplant hearts and the rapid and massive reaction of anaphylactic degranulation of mast cells. Some mast cells progressively lost their dense granule contents, displaying a variety of piecemeal degranulation that indicates a slow degranulation process. These events occurred from the first week; they lasted from weeks to months. The fibrosis developed quickly in the cases with more mast cells and degranulation. The cases with fewer mast cells and granules showed only mild increases in the volume of fibrosis. Mast cells appeared as early as the first posttransplantation week. Patients with greater numbers of mast cells underwent more severe rejection episodes. This study demonstrated that mast cells play an early and important role in the perimyocytic and interstitial fibrosis of posttransplant hearts. Mast cells may also be important in the inflammatory process of rejection reaction. The severity of fibrosis appears related to the density of mast cells and their granules.

Langerhans cells in the normal conjunctiva and peripheral cornea of selected species.

The distribution of Langerhans cells (LCs) in human corneal and conjunctival epithelial sheets was investigated by histochemical, immunofluorescence, and immuno-electron microscopic methods. The LCs stained positive with ATPase and with antibodies to HLA-DR antigen and were negative to DOPA-oxidase. Human conjunctiva showed 250 to 300 LCs/mm2 compared to 15 to 20/mm2 in the peripheral third of the corneal epithelium, approximately similar of LCs were present in Lewis rat, fewer cells in guinea pigs and mice, and no detectable cells in the chick.

Drug-metabolizing enzymes in rat liver myofibroblasts.

The myofibroblast is considered to be a key component in the pathogenesis of hepatic fibrosis. There is a need for therapeutic intervention in hepatic fibrosis, and, to date, the number of efficacious anti-fibrotic drugs is negligible. At best, the current therapeutic modalities reduce liver enzymes, an indicator of liver damage, but cannot reduce or prevent fibrosis. We have described the anti-fibrotic effect of pentoxifylline in an experimental model of hepatic fibrosis. Evidence suggests that, in addition to pentoxifylline itself, at least two of the metabolites of pentoxifylline are of therapeutic interest. We have reported that one of these metabolites (M-1) has a biological activity similar to that of its parent drug. The second metabolite (M-1R) has been reported to be more potent than the parent drug. Recent evidence suggests that inhibition of cytochrome P450 1A2 (CYP1A2) results in higher levels of pentoxifylline and M-1 and may be responsible for the production of the novel, potent metabolite (M-1R). We therefore investigated whether the myofibroblast, the cell with a crucial role in fibrosis, contains drug-metabolizing enzymes and thus may play a critical role in the anti-fibrotic actions of pentoxifylline. Our results showed that myofibroblasts contain aryl hydrocarbon hydroxylase activity, ethoxyresorufin O-deethylase activity, and methoxyresorufin O-demethylase activity. The results presented here also indicate that aryl hydrocarbon hydroxylase and methoxyresorufin O-demethylase activities can be increased by treatment of cells with dibenzanthracene, an inducer of CYP1A activities.

Deficiency of myeloperoxidase and abnormal chromosome 1 occurs in variant (HL60) promyelocytes.

Maturation of normal polymorphonuclear neutrophils is characterized by successive periods of granule synthesis, a process which frequently is abnormal in leukemia. Recently, the human leukemic cell line HL60, displaying a promyelocytic phenotype, has been used to study granulocyte maturation. We describe a variant line of HL60, called HL60-A7, resulting from growth in actinomycin D, which contains atypical large azurophilic granules deficient in myeloperoxidase. The products of in-vitro translation of A7 RNA contained less than 5% of the immunoreactive MPO found in the parent line. Electrophoresis of plasma membrane polypeptides radioiodinated by the lactoperoxidase technique revealed several differences. Karyotypic analysis identified a consistent chromosome 1q+ abnormality which was not found in any of the parental cells examined. This constellation of differences between HL60 and HL60-A7, i.e. MPO deficiency, abnormal granule morphology, cell surface changes, and further cytogenetic abnormalities, may point to a common site sensitive to altered regulation in some leukemic promyelocytes.

An immunoperoxidase investigation of S-100 protein in granular cell myoblastomas: evidence for Schwann cell derivation.

Five cases of granular cell myoblastoma have been studied for detection of the neuroectodermal protein S-100. Immunoperoxidase staining on paraffin sections, using an antibody raised against calf brain S-100 protein, was utilized to demonstrate positive cytoplasmic and nuclear reactivity in all cases. Negative staining in adjacent muscle and connective tissue elements was contrasted to in situ control staining of Schwann cells in peripheral nerves and staining of Langerhans cells and melanocytes in overlying stratified epithelia. These observations are interpreted as support for possible Schwann cell origin of granular cell myoblastomas.

Immunohistochemical demonstration of S-100 protein antigen-containing cells in beryllium-induced, zirconium-induced and sarcoidosis granulomas.

S-100 protein has been detected in epidermal Langerhans cells, interdigitating reticulum cells, and large mononuclear cells of cutaneous T-cell lymphomas. Experimental data imply these cells are involved in the presentation of antigens and/or maturation of T-lymphocytes. The authors studied the morphology and distribution of S-100 protein antigen-containing cells in cutaneous sarcoidosis and metal-induced granulomas, both immunogenic and foreign-body types, with light and electron microscopic immunoperoxidase technics. Immunological reaction was seen in Langerhans cells, peripheral nerves, and granulomatous lesion dendritic cells. The latter showed a large, irregular nucleus and branching cytoplasm. They intermingled with other mononuclear cells in the granulomas but not with organized epithelioid cells. Morphometric quantification of dendritic cells in the three types of granulomas revealed statistically significant differences (P less than 0.005) and electron microscopy demonstrated their typical cytoplasmic appearance, without Birbeck granules. The increased number of dendritic cells in immunogenic granulomas, and their shared antigenic components with Langerhans cells suggest they act as accessory cells in eliciting the granulomatous response.