Immunohistochemical characterization of nurse cells in normal human thymus

Summary Lymphoepithelial complexes known as thymic nurse cells (TNC) have been isolated and described in the thymus of several animal species including man. Most of the investigations on TNC have been carried out in enzymatically digested thymuses in which TNC were isolated by differential sedimentation. In the present study we demonstrate TNC in immunohistochemically stained sections of human thymus as ring-shaped cells completely enclosing thymocytes and localized not only in the cortex, but also at the corticomedullary junction where they have not been previously described. TNC expressed epithelial markers [low and high molecular weight keratins identified by 35H11 and 34E12 monoclonal antibodies, a cortical antigen shared with neuroectodermal neoplasms recognized by the GE2 monoclonal antibody, and tissue polypeptide antigen (TPA:B₁)], class II histocompatibility antigens (HLA-DR), and thymosin ₁. Double staining experiments with the nuclear proliferation-associated antigen Ki-67 and the cortical epithelium marker GE2 showed that most thymocytes enclosed in these cortical TNC were not proliferating. The antigens expressed by TNC indicate that not only cortical, but also medullary epithelial cells are part of the TNC system. The possible role of TNC in the education and maturation of thymocytes is discussed.     

Immunohistochemical characterization of cells in adult human patellar tendons.

Cells in tendons are conventionally identified as elongated tenocytes and ovoid tenoblasts, but specific markers for these cells are not available. The roles and interplay of these cells in tendon growth, remodeling, and healing are not well established. Therefore, we proposed to characterize these cells with respect to cell turnover, extracellular matrix metabolism, and expression of growth factors. Here we examined 14 healthy human patellar tendon samples for the expression of various proteins in tenocytes and tenoblasts, which were identified as elongated tendon cells and ovoid tendon cells, respectively. Matrix metalloproteinase 1 (MMP1), procollagen type I (procol I), heat shock protein 47 (hsp47), bone morphogenetic protein 12 (BMP12), 13 (BMP13), and transforming growth factor beta1 (TGFbeta1) were detected by immunohistochemistry (IHC). An image analysis of the IHC staining for proliferation cell nuclear antigen (PCNA) and apoptotic cells was performed to determine the proliferation index and the apoptosis index in elongated and ovoid tendon cells. The ovoid tendon cells expressed higher levels of procol I, hsp47, MMP1, BMP12, BMP13, and TGFbeta1 than the elongated tendon cells. Both the proliferation index and the apoptosis index of ovoid tendon cells were higher than those of the elongated tendon cells. The results suggested that ovoid tendon cells, conventionally recognized as tenoblasts, were more active in matrix remodeling. The expression of BMP 12, BMP13 and TGFbeta1 might be associated with the different cellular activities of tenoblasts and tenocytes.     

Myosin and actin containing cells in the human postnatal thymus. Ultrastructural and immunohistochemical findings in normal thymus and in myasthenia gravis.

Samples of normal human thymus of different ages (4-63 years old) were studied by immunofluorescence microscopy (using antibodies to smooth muscle myosin, to actin from the chicken gizzard, and antibodies to myosin from human striated muscle) as well as by routine electron microscopy. Thymus tissue from myasthenia gravis patients was also investigated for comparative reasons. Epithelial cells reacted with anti-smooth, but not with anti-striated muscle myosin, whereas myoid cells reacted with antibodies to striated, but not to smooth muscle myosin. Both epithelial and myoid cells displayed a strong immunoreactivity with antiactin. Corresponding to this immunoreactivity, both cell types contained bundles of thin, actin-like filaments. Myoid cells occurred in the rounded and elongated variety, and they were a normal constituent of all thymuses investigated in this study. Ultrastructurally, this non-innervated, striated muscle-like cell type possessed bundles of thin and thick filaments as well as Z lines in a rather disorganized arrangement, resembling striated muscle after denervation or various other pathologic conditions. There were no overt differences in the number and structure of myoid cells between healthy and myasthenic patients.     

Immunohistochemical characterization and distribution of Langerhans cells in normal epithelium of the uterine cervix

Langerhans cells (LCs) were shown up in normal cervical tissue obtained from 32 women whose age ranged from 25 to 68 years, using immunohistological methods. LCs were detected in metaplastic and native squamous epithelium of the cervix; they were positive to Dako-LC and OKDR antibodies and those located in the basal-suprabasal epithelial layers were also OKT6-positive. The density of the LCs was higher during the proliferative phase of the menstrual cycle. The investigation into the lymphocytes present in the stroma and in the squamous epithelium showed a population of T-lymphocytes identified as predominantly T-cytotoxic/suppressor cells, sometimes in contact with LCs. The significance of these findings is discussed.     

Intermolecular complexes between three human CD1 molecules on normal thymus cells

The first cluster of differentiation (CD1) defines at least three distinct human thymic cell-surface differentiation antigens-CD1a, CD1b, and CD1c. We looked for structural homology of the three CD1 heavy chains at their peptide level by two-dimensional peptide maps. We show here that the CD1a M _r 49 000 heavy chain and the CD1b M _r 45 000 heavy chain appear to be more homologous to each other than to the CD1c M _r 43 000 heavy chain and that only one tyrosil peptide is common to the three heavy chains. Study of the CD1 heavy chains from several individuals reveals a very limited polymorphism of these molecules. We also demonstrate here that CD1a or CD1a-like molecules and other CD1 molecules can form intermolecular complexes on the surface of normal thymus cells. Molecules that are structurally very similar to CD1a molecules are associated noncovalently either with CD1c molecules or with CD1b molecules, and only CD 1a molecules can associate covalently with CD8 molecules. In contrast, we could not find these intermolecular complexes on the surface of leukemic T-cell lines in culture.Abbreviations used in this paper CD cluster of differentiation - mAb monoclonal antibody - MHC major histocompatibility complex - NP-40 Nonidet P 40 - B2m beta-2 microglobulin - PMSF phenylmethylsulfonyl fluoride - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis - TL thymus leukemia     

Neuropeptides of human thymus in normal and pathological conditions

Human thymus of healthy subjects and patients affected by thymoma-associated Myastenia Gravis were studied in order to visualize and compare the morphological distributive pattern of four neuropeptides: vasoactive intestinal peptide, substance P, neuropeptide Y, and neurotensin. Based on our observations, we formulated hypotheses on their relations in neuro-immunomodulation under physiological and pathophysiological conditions. Immuno-histochemical staining for neuropeptides was performed and morphological and morphometrical analyses were conducted on healthy and diseased thymus. In normal thymus, a specific distributive pattern was observed for the several neuropeptide-positive nerves in different thymus lobular zones. In particular substance P-positive fibers were observed in subcapsular zone, specifically located into parenchyma, where they represent the almost total amount of fibers; neurotensin-positive fibers were observed primarily located in parenchyma than perivascular site of several thymus lobular zones, and more abundant the cortico-medullary and medullary zones. Instead VIP- and NPY-positive fibers were widely distributed in perivascular and parenchymal sites of several thymus lobular zones. In thymoma, the distribution of neuropeptide-positive fibers was quantitatively reduced, while cells immunopositive to VIP and substance P were quantitatively increased and dispersed. Observation of the perivascular and parenchymal distribution of the analyzed neuropeptides suggests evidence that a regulatory function is performed by nerves and cells that secrete neuropeptide into the thymus. The alteration of neuropeptide patterns in thymoma suggests that these neurotransmitters play a role in autoimmune diseases such as Myastenia Gravis.     

DNA-synthesizing cells in human fetal thymus

Summary Fragments and suspensions of human fetal thymus were incubated in the presence of ³H-TdR to permit study of the distribution and morphology of DNA-synthesizing cells. Results of light and EM autoradiography showed that 1. although DNA-synthesizing cells were present in the medulla, the vast majority of these cells were localized in the thymic cortex, 2. cells with the typical EM appearance of small lymphocytes and lymphoid blast cells both synthesized DNA, and 3. cells in S-phase were predominantly 8 to 12 m in size.     

Immunohistochemical characterization of pituitary stellate cells in rats

Pituitary stellate cells from the normal adult male rats were immunohistochemically investigated at the light microscopical level by the use of rat TSH-beta, porcine ACTH1-39, porcine ACTH17-39, rat FSH and ovine FSH antisera. They were characterized by the stellate shape and a mimic engulfment of acidophils. In the present study, they were identified to be the ACTH cells but some were TSH cells. Although most of the corticotrophs showed a peripheral fringe immunostained with the porcine ACTH17-39 antiserum, some others were stained diffusively throughout the cytoplasm. The latter cells coincided, in shape and in homogenous stainability of the cytoplasm, with the stellate TSH cells. Both cells did not correspond but were independent in distribution at the same site of the gland on the adjacent two sections. The stellate type of FSH cells could react with the ovine FSH antiserum, but not with the rat FSH antiserum. Absorption tests of the ovine FSH, procine ACTH1-39 and procine ACTH17-39 antisera were carried out by an application of procine ACTH. In consequence, the porcine ACTH)-39 and porcine ACTH17-39 antisera were absorbed efficaciously by the ACTH antigen at the dose of 10 micrograms/ml, but the ovine FSH antiserum was not enough absorbed by ACTH in the doses of less than 1 mg/ml. It was not finally concluded whether or not the single stellate cells produced ACTH and FSH.     

Heterogeneity of the first cluster of differentiation: characterization and epitopic mapping of three CD1 molecules on normal human thymus cells

In humans, the presence of two non-HLA class 1-like molecules, whose expression, similar to murine Tla, is restricted to cortical thymocytes, has been shown with monoclonal antibodies defining the first cluster of differentiation (CD1). We report here with the use of 12 anti-CD1 antibodies and a combination of technical approaches, the characterization of a third CD1 molecule. We show that we can presently define seven different epitopes on the three CD1 molecules: four epitopes are restricted to the 49,000 dalton molecule, two epitopes to the 45,000 dalton molecule, and one epitope to the 43,000 dalton molecule. We show that the association of the newly identified 45,000 dalton heavy chain with human beta2-microglobulin is weak. In addition we show the presence of a fourth non-HLA class I molecular species on the surface of normal human thymus cells.     

Immunohistochemical detection of human gastrointestinal glutathione peroxidase in normal tissues and cultured cells with novel mouse monoclonal antibodies.

This is the first report to describe the successful detection of human gastrointestinal glutathione peroxidase in normal tissues by Western blotting and immunohistochemical staining techniques. Four hybridoma clones producing monoclonal antibodies (MAbs) against the human gastrointestinal glutathione peroxidase were established from mice immunized with a gastrointestinal glutathione peroxidase-derived peptide. The MAbs did not crossreact with other members of the glutathione peroxidase family, be it cellular glutathione peroxidase, phospholipid hydroperoxide glutathione peroxidase, or extracellular glutathione peroxidase. Although the MAbs were found to react with a 24-kD protein in a Western blotting assay using gastric carcinoma cell extracts as antigen, they did not react with a B-lymphoblastoid cell extract. Immunohistochemical staining showed gastrointestinal glutathione peroxidase localized in the cytoplasm and in the nucleus of gastric carcinoma cells. Moreover, gastrointestinal glutathione peroxidase was detected in tissue extracts of human stomach, small intestine, large intestine, liver, and gallbladder by Western blotting, and its localization was immunohistochemically confirmed in the mucosal epithelia of the basal area of gastric pits and intestinal crypts.     

Heterogeneity of epithelial cells in the human thymus

Summary To evaluate interrelationships among epithelial cells, and between morphology and function in the microenvironment, we studied the ultrastructural morphology of epithelial cells in sections of human thymus from donors aged 2 months to 31 years. Six types of epithelial cells were observed: subcapsular-perivascular (type 1); pale (type 2); intermediate (type 3); dark (type 4); undifferentiated (type 5); and large-medullary (type 6). Cells of types 2, 3 and 4 were found throughout the organ. The type-2 to -4 epithelial cells may represent various stages in a differentiation process. In this, type-2 cells are very active and type-4 cells are possibly degenerating elements. Type-4 cells can also contribute to Hassall's corpuscles. Type-5 cells were located mainly in the cortico-medullary region and showed the morphological characteristics of undifferentiated elements. Type-6 cells were located exclusively in the medulla and displayed characteristics of cellular activity. Small Hassall's corpuscles consisted of type-6 epithelial cells; in larger corpuscles many nuclei of type-6 cells were found. Cells of types 2 and 6 contained tubular structures (diameter approximately 20 nm).Concerning the function of thymus epithelial cells, the features associated with protein synthesis observed in cellular types 2 and 6 make them likely candidates for humoral factor-producing and/or secreting elements. In addition, type-2 and -3 cells in the cortex appear to contribute to a special pattern of epithelium-lymphocyte interaction (thymic nurse cells), as demonstrated by the intracytoplasmic location of lymphocytes in the epithelial cells. The various steps in intrathymic T-cell maturation occur at locations in a microenvironment composed of morphologically distinct epithelial cells.     

Immunohistochemical characterization of brush cells in the rat larynx

The immunohistochemical characteristics of brush cells in the laryngeal mucosa were examined using immunohistochemistry for various immunohistochemical cell markers including villin at the light and electron microscopic levels. Cells that were immunoreactive to villin were barrel-shaped with thick cytoplasmic processes extending toward the lumen of the laryngeal cavity. Immunoelectron microscopic observations revealed thick and short microvilli with long rootlets of microfilaments. Numerous small clear vesicles and small finger-like cytoplasmic processes were observed in the apical process and lateral membrane, respectively. Double immunofluorescence showed villin-immunoreactive cells were not immunoreactive for the markers of solitary chemosensory cells, GNAT3 and phospholipase C, β2-subunit (PLCβ2), or for that of neuroendocrine cells, synaptosome-associated protein 25kD. Furthermore, immunoreactivities for cytokeratin 18 (CK18) and doublecortin like-kinase 1 in the perinuclear cytoplasm of villin-immunoreactive cells. However, some CK18-immunoreactive cells were immunoreactive to GNAT3 but not to villin. Regarding sensory innervation, only a few intraepithelial nerve endings with P2X3, SP, or CGRP immunoreactivity attached to villin-immunoreactive cells. In the present study, brush cells in the rat laryngeal mucosa were classified by immunoreactivity for villin, and were independent of other non-ciliated epithelial cells such as solitary chemosensory cells and neuroendocrine cells.     

Immunohistochemical studies of neurochemical markers in normal human buccal mucosa

The content of various substances, such as regulatory peptides, hormones and structural proteins, was investigated in normal buccal mucosa using indirect immunofluorescence. Thin nerve fibres, which from a morphological point of view were most probably sensory, showed immunoreactivity for substance P (SP), calcitonin gene-related peptide (CGRP), neuropeptide K (NPK) and neurokinin A (NKA). Also galanin (GAL), -melanocyte stimulating hormone (-MSH) and somatostatin (SOM) stained thin fibres were found in the propria, which were, however, few in number and the -MSH staining was weak. CGRP, vasoactive intestinal polypeptide (VIP), peptide histidine isoleucine amide (PHI) and neuropeptide Y (NPY) immunoreactive nerve fibres were observed in close connection to blood vessels. SOM positive cells with processes were found, mostly scattered, in the connective tissue. A population of cells within the epithelium also showed somatostatin immunoreactivity. Protein S-100 (S-100) stained distinct populations of cells at two separate locations. In the propria, cells with one or two slender processes were seen, being mostly single but sometimes forming groups. In the epithelium, dendritic cells with many processes with or without spines were observed, mainly located to the basal layer of the lamina epithelialis. Single nerve fibres and nerve bundles were also stained. Neurofilament (NF) positive fibres, singly and in bundles, as well as endorgan-like structures were seen. Neuron-specific enolase (NSE) and protein gene product 9.5 (PGP 9.5) both stained the same structures, namely single fibres, nerve bundles, nerves surrounding vessels and innervating muscles and glands (if present in the section), as well as Merkel cells. Also with these two markers endorgan-like structures were seen. No clear innervation of the epithelium could be observed with the markers used. No methionine-enkephalin (ENK) or synaptophysin (SYN) immunoreactive material was found.     

Immunohistochemical characterization of neoplastic cells of breast origin

ABSTRACT: BACKGROUND: After skin cancer, breast cancer is the most common malignancy in women. Tumors of unknown origin account for 5-15% of malignant neoplasms, with 1.5% corresponding to breast cancer. We selected an immunohistochemical panel with conventional and newer markers, such as mammaglobin, for the detection of neoplastic cells of breast origin. The specific objectives are: 1) to determine the sensitivity and specificity of the panel, with an especial emphasis on the inclusion of the mammaglobin marker, and 2) to compare immunohistochemistry performed on whole tissue sections and on Tissue Micro-Array. Methods. Twenty-nine metastatic breast tumors were included and assumed as tumors of unknown origin. Other 48 biopsies of other tissues were selected and assumed as negative controls. Tissue Micro-Array was performed. Immunohistochemistry for mammaglobin, gross cystic disease fluid protein-15, estrogen receptor, progesterone receptor and cytokeratin 7 was done. Results. Mammaglobin positive staining was observed in 10/29 cases, in 13/29 cases for gross cystic disease fluid protein-15, in 20/29 cases for estrogen receptor, 9/29 cases for progesterone receptor, and in 25/29 cases for cytokeratin 7. Among the negative controls, mammaglobin was positive in 2/48, and gross cystic disease fluid protein-15 in 4/48. Conclusions. The inclusion of MAG antibody in the immunohistochemical panel for the detection of tumors of unknown origin increased the probability of detecting metastasis of breast cancer. For the diagnostic strategy with the highest positive predictive value (87.5%), hormone receptors and mammaglobin were considered in serial, non-parallel manner.     

Immunohistochemical localization of smooth muscle myosin in normal human tissues

Immunohistochemical localization of smooth muscle myosin, an immunologically distinct contractile protein, was achieved using rabbit anti-human uterine smooth muscle myosin antibodies. In immunodiffusion studies and in cryostat sections, these antibodies were highly specific and reacted with smooth muscle myosin but not with platelet, skeletal muscle, or cardiac muscle myosin. To evaluate comprehensively the structural profile of smooth muscle elements in normal human tissues, an indirect immunoperoxidase technique (peroxidase-antiperoxidase) was applied to a wide variety of specimens. Parallel studies comparing cryostat sections with fixed (10% formalin, B5, Bouin's, or Zenker's solution) paraffin-embedded tissues revealed optimal immunoreactivity, sensitivity, and specificity of staining for smooth muscle myosin using frozen tissues. Strong immunoreactivity was present in muscular tissues such as blood vessels and the muscularis of gastrointestinal and genitourinary tracts. Distinct delineation of smooth muscle elements, including individual smooth muscle cells, and their specific patterns of alignment and organization, were observed, e.g., cells comprising the muscularis mucosae and extending into the lamina propria of the gastrointestinal tract, and myoepithelial cells of skin, exocrine glands, and breast. This method provides excellent morphologic preservation and readily permits unambiguous identification of individual cells containing smooth muscle myosin.     

Immunohistochemical localization of Zn-alpha 2-glycoprotein in normal human tissues

The Zn-alpha 2-glycoprotein (Zn-alpha 2-GP) is present at a high concentration in the seminal plasma and at significant levels in other human body fluids. Its precise localization, however, has remained unclear, as well as its physiological and pathological significance. The present study reports the immunohistochemical localization of this protein in normal adult human tissues. Localization of the reactive product to anti-human plasma Zn-alpha 2-GP antibody was demonstrated in the following cells: luminal and basal cells of the prostate gland, luminal epithelial cells of the acini and of some ducts of the mammary glands, luminal cells of the secretory portion of the eccrine and apocrine sweat glands, serous cells of the salivary, tracheal, and bronchial glands, acinar cells of the esophageal glands, exocrine acinar cells of the pancreas, hepatocytes of the liver, and epithelial cells of the proximal and distal tubules in the kidney. The present results suggest that Zn-alpha 2-GP exerts some unknown but fairly widespread exocrine function and may be produced in the various epithelial cells tested. Hepatocytes are also suggested to be a source of the protein in the blood plasma.     

Immunohistochemical distribution of inter-alpha-trypsin inhibitor chains in normal and malignant human lung tissue.

The inter-alpha-trypsin inhibitor (ITI) family is a group of plasma proteins built up from heavy (HC1, HC2, HC3) and light (bikunin) chains synthesized in the liver. In this study we determined the distribution of ITI constitutive chains in normal and cancerous lung tissues using polyclonal antibodies. In normal lung tissue, H2, H3, and bikunin chains were found in polymorphonuclear cells, whereas H1 and bikunin proteins were found in mast cells. Bikunin was further observed in bronchoepithelial mucous cells. In lung carcinoma, similar findings were obtained on infiltrating polymorphonuclear and mast cells surrounding the tumor islets. Highly differentiated cancerous cells displayed strong intracytoplasmic staining with H1 and bikunin antiserum in both adenocarcinoma and squamous cell carcinoma. Moreover, weak but frequent H2 expression was observed in adenocarcinoma cells, whereas no H3-related protein could be detected in cancer cells. Local lung ITI expression was confirmed by RT-PCR. Although the respective role of inflammatory and tumor cells in ITI chain synthesis cannot be presently clarified, these results show that heavy chains as well as bikunin are involved in malignant transformation of lung tissue.(J Histochem Cytochem 47:1625-1632, 1999)     

Age-related decrease of somatostatin receptor number in the normal human thymus

The thymus exhibits a pattern of aging oriented toward a physiological involution. The structural changes start with a steady decrease of thymocytes, whereas no major variations occur in the number of thymic epithelial cells (TEC). The data concerning the role of hormones and neuropeptides in thymic involution are equivocal. We recently demonstrated the presence of somatostatin (SS) and three different SS receptor (SSR) subtypes in the human thymus. TEC selectively expressed SSR subtype 1 (sst(1)) and sst(2A). In the present study we investigated whether SSR number is age related in the thymus. Binding of the sst(2)-preferring ligand (125)I-Tyr(3)-octreotide was evaluated in a large series of normal human thymuses of different age by SSR autoradiography and ligand binding on tissue homogenates. The score at autoradiography and the number of SSR at membrane homogenate binding (B(max)) were inversely correlated with the thymus age (r = -0.84, P < 0.001; r = -0.82, P < 0.001, respectively). The autoradiographic score was positively correlated with the B(max) values (r = 0.74, P < 0.001). Because the TEC number in the age range considered remains unchanged, the decrease of octreotide binding sites might be due to a reduction of sst(2A) receptor number on TEC. The age-related expression of a receptor involved mainly in controlling secretive processes is in line with the evidence that the major changes occurring in TEC with aging are related to their capabilities in producing thymic hormones. In conclusion, SS and SSR might play a role in the involution of the human thymus. These findings underline the links between the neuroendocrine and immune systems and support the concept that neuropeptides participate in development of cellular immunity in humans.