Microenvironment of thymic myoid cells in myasthenia gravis

The microenvironment of myoid cells (MyCs) was studied in myasthenia gravis (MG) thymitis with lymphoid follicular hyperplasia (LFH) (nine cases) and with diffuse B cell infiltration (one case), and compared with findings in the thymuses of non-myasthenic control subjects (ten cases). Double immunostaining was used to demonstrate MyCs labelled by anti-desmin together with other thymic components such as keratin-positive epithelial cells, Ki-M 1-positive interdigitating reticulum cells (IDCs), Ki-M 4-positive follicular dendritic reticulum cells, Ki-M 6-positive macrophages, CD22-positive B-cells, CD1-positive cells, CD3-positive T-cells or HLA-DR-positive cells. Round or elongated MyCs were confined to the thymic medulla and were surrounded by CD3-positive T-cells and CD22-positive B-cells. In MG thymitis MyCs were localized in the vicinity of, but not inside germinal centres (GCs). MyCs were always HLA-DR-negative, but were invariably embedded in a cellular micromilieu with strong HLA-DR expression. A remarkable feature of MG thymitis was that the great majority of MyCs were in intimate contact with intramedullary IDCs. Morphometric studies confirmed that such contacts were significantly less frequent in thymuses from non-myasthenic subjects. This indicates that an IDC-dependent antigen-presenting process for T-cells may actively involve MyCs in MG thymitis.     

Mitogenic heparin-binding lectin-like protein from cloned thymic myoid cells

A mitogenic heparin-binding (reactive) lectin-like protein (HBP) was purified from the extract of a cloned rat thymic myoid cell R615B2 by a one-step procedure of affinity chromatography on a heparin--Sepharose CL-6B column. Four distinct peptide bands with molecular weights of 10,000, 13,000, 13,700, and 14,600 were detected on SDS-polyacrylamide gel electrophoresis. This protein is mitogenic at concentrations of as low as 1.1-70.0 ng/ml for peanut lectin-nonagglutinated thymocytes and splenocytes from euthymic mice and rats but not for splenocytes from nude mice. These results indicate that thymic myoid cell-derived HBP is an important signal for one particular step in T-cell differentiation.     

Effects of thymic myoid cell culture supernatant on cells from lymphatic tissues

Conditioned media (MCM) of cloned thymic myoid cells (IT45R92, R613Ad, and R615B2) were used to investigate their possible involvement in thymic biological events. Those myoid cells produced in a culture medium biological activities capable of stimulating the growth of thymocytes, spleen cells, and bone marrow cells of mice and rats. Surface markers detected on spleen cells proliferating in MCM were characteristic of monocyte-macrophage lineages (C3R, Fc gamma R, asialo GM1) and T-cell lineages (Thy 1) but not B cells (sIgG). Chromatographic studies also suggested that the biological activities of MCM could be separated into two different molecular entities, such as a colony-stimulating activity and an interleukin 1-like activity which supported the growth of monocyte-macrophage lineages and T-cell lineages, respectively. These results indicate that thymic myoid cells produce cytokines important for the regulation of intrathymic interleukin cascade by which clonally differentiated thymic lymphocytes may be expanded into a sizable pool.     

Changes of subcellular localization of Thy-1 antigen during thymic myoid cell differentiation

Using a quantitative enzyme immunoassay, Thy-1 antigen expressed by a rat myoid cell line R615B2 was detected mainly on the cell surface at a single cell stage, whereas at the stage of forming myotubes, Thy-1 was found predominantly in the cytoplasm. The muscle specific creatine kinase activity also increased in association with the shift of Thy-1 from the cell surface to the cytoplasm, suggesting biological significance of Thy-1 redistribution in muscle differentiation from single cells to multinucleated cells.     

Differentiation of rat thymic myoid progenitor cell line established by coculture with human T-lymphotropic virus type-I-producing human T cells

A thymus-derived myoid precursor cell line (ST1), which differentiates to myoid cells in the growth arrest condition, was established by the cocultivation of F344 rat thymic cells with human T-lymphotropic virus type-I (HTLV-I)-producing human lymphoid cells. No integration of HTLV-I was detected in ST1 cells by Southern blot hybridization. In a differentiation culture condition such as confluent culture or serum starvation, ST1 cells began to fuse, creating multinuclear giant cells, with the induced expression of MyoD1 and various muscle-specific antigens, including alpha-sarcomeric actin, skeletal muscle myosin, myoglobin, desmin, and acetylcholine receptor. Ultrastructural investigation revealed that differentiated ST1B cells created aggregates of thick and thin filaments with Z-band-like composition, then formed sarcomeric structures and tubular honeycomb arrays. Finally, these cells spontaneously contracted with a frequency of 0.5-2.0 Hz and synchronized with adjoining cells. Transplantation of ST1B cells into nude mice produced a small tumor nodule, showing clear differentiation to skeletal muscle cells. ST1B cells did not indicate any colony-forming activities in soft agar, demonstrating that ST1B cells retain some of the physiologically normal phenotypes. This rare cell line is promising for use in various physiological and pathological investigations including functional research of thymic myoid cells and the pathological role in autoimmune diseases, as well as animal model experiments of cell therapy related to muscular degenerative disorders or regeneration of injured muscles.     

Establishment of myoid cells from bone marrow

A peculiar adherent cell clone (R613BM) was established under muscle tissue free conditions from bone marrow of a Wistar rat. The cloned cell line was able to form myofibrils and expressed nicotinic acetylcholine receptors specific for skeletal muscles. The muscle specific characteristics have been maintained consistently for more than five years. These results suggest that bone marrow contains a precursor cell which has the potency to differentiate into muscle cells.     

Cytochemical and biochemical characterization of testicular peritubular myoid cells

Testicular peritubular myoid cells secrete a paracrine factor that is a potent modulator of Sertoli cell functions involved in the maintenance of spermatogenesis. These cells also play an integral role in maintaining the structural integrity of the seminiferous tubule. To better understand this important testicular cell type, studies were initiated to characterize cultured peritubular cells using biochemical and histochemical techniques. The electrophoretic pattern of radiolabeled secreted proteins was similar for primary and subcultured peritubular cells and was unique from that of Sertoli cells. Morphologic differences between Sertoli cells and peritubular cells were noted and extended with histochemical staining techniques. Desmin cytoskeletal filaments were demonstrated immunocytochemically in peritubular cells, both in culture and in tissue sections, but were not detected in Sertoli cells. Desmin is proposed to be a marker for peritubular cell differentiation as well as a marker for peritubular cell contamination in Sertoli cell cultures. Peritubular cells and Sertoli cells were also stained histochemically for the presence of alkaline phosphatase. Staining for the alkaline phosphatase enzyme was associated with peritubular cells but not with Sertoli cells. Alkaline phosphatase is therefore an additional histochemical marker for peritubular cells. Biochemical characterization of peritubular cells relied on cell-specific enzymatic activities. Creatine phosphokinase activity, a marker for contractile cells, was found to be associated with peritubular cells, while negligible activity was associated with Sertoli cells. Alkaline phosphatase activity assayed spectrophotometrically was found to be a useful biochemical marker for peritubular cell function and was utilized to determine the responsiveness of primary and subcultured cells to regulatory agents. Testosterone stimulated alkaline phosphatase activity associated with primary cultures of peritubular cells, thus supporting the observation that peritubular cells provide a site of androgen action in the testis. Retinol increased alkaline phosphatase activity in subcultured peritubular cells. Alkaline phosphatase activity increased in response to dibutyryl cyclic adenosine monophosphate (AMP) in both primary and subcultured peritubular cell cultures. Observations indicate that the ability of androgens and retinoids to regulate testicular function may be mediated, in part, through their effects on peritubular cells. This provides additional support for the proposal that the mesenchymal-epithelial cell interactions between peritubular cells and Sertoli cells are important for the maintenance and control of testicular function. Results imply that the endocrine regulation of tissue function may be mediated in part through alterations in mesenchymal-epithelial cell interactions.     

Identification and characterization of Myosin from rat testicular peritubular myoid cells

In the mammalian testis, peritubular myoid cells (PMCs) surround seminiferous tubules. These cells are contractile, express the cytoskeletal markers of true smooth muscle-alpha-isoactin and F-actin-and participate in the contraction of seminiferous tubules during the transport of spermatozoa and testicular fluid to the rete testis. Myosin from PMCs (PMC-myosin) was isolated from adult rat testis and purified by cycles of assembly-disassembly and sucrose gradient centrifugation. PMC-myosin was recognized by a monoclonal anti-smooth muscle myosin antibody, and the peptide sequence shared partial homology with rat smooth muscle myosin-II, MYH11 (also known as SMM-II). Most PMC-myosin (95%) was soluble in the PMC cytosol, and purified PMC-myosin did not assemble into filaments in the in vitro salt dialysis assay at 4 degrees C, but did at 20 degrees C. PMC-myosin filaments are stable to ionic strength to the same degree as gizzard MYH11 filaments, but PMC-myosin filaments were more unstable in the presence of ATP. When PMCs were induced to contract by endothelin 1, a fraction of the PMC-myosin was found to be involved in the contraction. From these results we infer that PMCs express an isoform of smooth muscle myosin-II that is characterized by solubility at physiological ionic strength, a requirement for high temperature to assemble into filaments in vitro, and instability at low ATP concentrations. PMC-myosin is part of the PMC contraction apparatus when PMCs are stimulated with endothelin 1.     

Avian thymic accessory cells

On the basis of morphologic criteria and ingestion of latex particles, two basic types of accessory cells can be identified from quail and chick thymuses, dendritic cells, and macrophages. By using embryonic grafting techniques, we show that cells of this lineage enter the thymus during the initial colonization of the epithelial thymic rudiment by hemopoietic cells, and within a few days differentiate into cells exhibiting properties of glass adherence, Ia expression, and formation of rosettes with thymocytes. It appears that the precursors of this lineage undergo extensive, but finite, proliferation and are eventually replaced by further influx of the accessory cell lineage. In chimeric grafts, quail thymocytes were seen forming rosettes with chick accessory cells, and vice versa, indicating, as in the interaction between the epithelial cells and thymocytes, that the molecules involved in thymocyte-accessory cell association can interact across species barriers in our system.     

Immunohistology of thymic nurse cells

The demonstration of thymic nurse cells (TNC), complexes between stromal cells and thymocytes, in cell suspensions of murine thymuses, prompted us to investigate (1) the relationship of TNC to other thymic stromal cell types defined in situ, and (2) the maturation stage of the enclosed thymocytes. To this purpose we incubated frozen sections of TNC suspensions with various monoclonal antisera directed to T cells and stromal cell types, using immunohistology. This approach enabled us to study antigen expression on the "nursing" cell itself and to analyze the phenotype of the enclosed lymphocytes in cross sections of TNC. The results show that lymphocytes enveloped by TNC express high levels of Thy-1, moderate levels of T200, and variable amounts of Lyt-1. Due to enzymatic degradation Lyt-2 expression could not be studied. The enveloped cells also bear PNA receptors, but no detectable I-A/E antigens. Expression of H-2K antigens on enclosed thymocytes varied from weak to absent. The "nursing" cells react with ER-TR4, a monoclonal antibody which detects cortical epithelial-reticular cells. In addition TNC express I-A/E and H-2K antigens. In contrast, TNC do not react with ER-TR 5 and 7, monoclonal antibodies, which detect medullary epithelial cells and reticular fibroblasts, respectively. TNC do not express the macrophage antigens Mac-1 and Mac-2. We conclude that TNC in vitro represent the in vivo association of epithelial-reticular cells with cortical thymocytes. However, the enclosed thymocytes do not constitute a phenotypically distinct subset of subcapsular or outer cortical cells.     

Pleiotropic actions of melatonin in testicular peritubular myoid cells of immature Syrian hamsters

BackgroundPeritubular myoid cells are emerging as key regulators of testicular function in adulthood. However, little is known about the role of testicular peritubular myoid cells (TPMCs) in the development of the male gonad. We found that, compared to testes of young adult hamsters, gonads of 21 day-old animals show increased melatonin concentration, seminiferous tubular wall thickening and a heterogeneous packaging of its collagen fibers thus raising the question whether melatonin may be involved in the regulation of TPMCs.MethodsWe established primary cultures of TPMCs from immature hamsters (ihaTPMCs), which we found express melatonergic receptors.ResultsExogeneous melatonin decreased the levels of inflammatory markers (NLRP3 inflammasome, IL1β) but increased the expression of cyclooxygenase 2 (COX2, key enzyme mediating prostaglandin synthesis) and of the glial cell line-derived neurotrophic factor (GDNF) in ihaTPMCs. Melatonin also stimulated ihaTPMCs proliferation and the expression of extracellular matrix proteins such as collagen type I and IV. Furthermore, collagen gel contraction assays revealed an enhanced ability of ihaTPMCs to contract in the presence of melatonin.ConclusionMelatonin regulates immune and inflammatory functions as well as contractile phenotype of the peritubular wall in the hamster testis.General significanceIf transferable to the in vivo situation, melatonin-dependent induction of ihaTPMCs to produce factors known to exert paracrine effects in other somatic cell populations of the gonad suggests that the influence of melatonin may go beyond the peritubular wall and indicates its contribution to testicular development and the establishment of a normal and sustainable spermatogenesis.     

Collagen biosynthesis in cultured rat testicular Sertoli and peritubular myoid cells.

The incorporation of 3H-proline into protein was regarded as a measure of total protein synthesis and the incorporation into hydroxyproline as indicative of collagen synthesis. Relative collagen synthesis (expressed as percent of total protein synthesized) by Sertoli and peritubular myoid cells cultured from 20-22 day old rat testis was estimated. In both secreted and cellular pools, relative collagen synthesis by Sertoli cells was significantly greater than by peritubular myoid cells. Coculture of Sertoli and myoid cells resulted in a significant increase in relative collagen synthesis when compared to monocultures of each cell type. Addition of serum to peritubular myoid cells resulted in a stronger stimulation of relative collagen production. Sertoli cell extracellular matrix inhibited relative collagen synthesis by peritubular myoid cells in the presence or absence of serum. Radioactivity into hydroxyproline as corrected per cellular DNA also showed similar results. Immunolocalization studies confirmed that both cell types synthesize type I and type IV collagens. These results indicate that stimulation of collagen synthesis observed in Sertoli-myoid cell cocultures is due to humoral interactions, rather than extracellular matrix, and Sertoli cell extracellular matrix regulates serum-induced increase in collagen synthesis by peritubular myoid cells.     

Histochemical and ultrastructural properties of myoid cells in the thymus of the frog

Summary Histochemical and ultrastructural properties of myoid cells in the thymus of the frog were investigated and compared with properties of skeletal muscle fibres. The histochemical reactions of phospholipids, phosphorylase, succinic dehydrogenase and adenosine triphosphatase activities in myoid cells were characterized by considerable variability. Individual myoid cells apparently possess different enzyme activities which correspond to different stages of development, maturity and degeneration of these cells. The mature mononucleated myoid cells have similar enzymatic properties to the fast muscle fibres of the frog. This finding has been extended by ultrastructural observations. Features, typical of fast muscle fibres of the frog, e.g. the presence of the M-line, straight and narrow Z-line and well developed triads were found in the majority of mature myoid cells.     

Ultrastructure of mouse ectoplacental cone cells

Ectoplacental cone of the Muridae is an excrescence formed by proliferation of the polar trophectoderm of the bastocyst. Four types of trophoblast cells are seen in mouse ectoplacental cone on day 8 of the pregnancy: (1) trophoblast-1 at the base of the cone are polyhedral, compactly arranged and contain large nucleoli, (2) trophoblast-2 in the middle of the cone enclose several heterolysosomes, erythro-and leucophagosomes, (3) trophoblast-3, also in the middle, have several membrane-bound osmiophilic granules, (4) trophoblast-4 at the periphery of the cone are oblong and enclose many pleomorphic bodies. These morphological differences appear to be correlated with functional adaptation.