Human dendritic reticulum cells of lymphoid follicles: their antigenic profile and their identification as multinucleated giant cells

The B-dependent areas of human lymphoid tissue contain non-lymphoid, non-phagocytic cells known as dendritic reticulum cells (DRC). These cells can be detected only very occasionally in routinely stained histologic sections. Recently we were able to overcome this limitation by preparing a monoclonal antibody, termed R 4/23, that reacts selectively with DRC. Thus by using an optimized immunoperoxidase method applied to frozen sections, it is possible to detect DRC in situ. To determine the antigenic profile of DRC, serial frozen sections of human tonsils were immunostained with R 4/23 and a large panel of other monoclonal antibodies or conventional antisera. In addition, touch imprints of tonsils and cytocentrifuge slides of cell suspensions with increased concentrations of DRC were immunostained with these reagents. DRC proved to be positive for mu, gamma, alpha, kappa and lambda chains, complement component C3b, C3b receptors, C3d receptors, HLA-A,B,C antigens, human Ia-like antigens, common ALL antigen (cALLa), and antigens that are characteristic of the monocyte/macrophage lineages. DRC did not express delta chains, T cell antigens, or antigens that are expressed on interdigitating reticulum cells (IDC) and Langerhans cells. DRC in touch imprints and suspensions prepared from hyperplastic tonsils were found to be giant cells often with 10 or more nuclei. In certain cases of follicular hyperplasia and of centroblastic-centrocytic lymphoma, DRC with several nuclei were also detectable in situ. These results show that (1) the phenotype of DRC differs from that of all other cell types in lymphoid tissue, (2) this phenotype most nearly resembles that of cells of the monocyte/macrophage series, thus suggesting that DRC are related to these cell lineages, and (3) DRC are multinucleated giant cells.     

Fine structural localization of alkaline phosphatase in the granular pneumonocytes of hamster lung.

The fine structural localization of nonspecific alkaline phosphatase was studied in the granular pneumonocytes (type II alveolar epithelial cells) of hamster lung by incubating sections of glutaraldehyde-fixed tissues in a medium containing lead ions and sodium beta-glycerophosphate or alpha-naphthyl acid phosphate. The specificity of the reaction was tested by exposing the sections to inhibitors of alkaline phosphatase. The results showed that alkaline phosphatase activity was present in the inclusion bodies of granular pneumonocytes. The enzyme reaction was strong in the membrane lining the inclusion bodies and a weaker reaction was generally detectable in the inclusion contents. Although only a proportion of the inclusion bodies showed enzyme activity, there was no obvious correlation between the reactivity of the inclusions and their intracellular position or size. The other organelles were unreactive. The finding of alkaline phosphatase activity within the inclusion bodies of granular pneumonocytes is an enigma as these organelles are generally considered to be lyosomes.     

A simple method of staining juxtaglomerular granules in the rat kidney for high resolution light microscopy

A simple method for the demonstration of juxtaglomerular granules in Epon embedded semithin (0.5-1 micrometer) sections has been developed as follows: sections are prepared as for routine electron microscopy except that before dehydration, the tissues are immersed in 0.5% uranyl acetate in Veronal acetate buffer (pH 5.0) overnight at room temperature. After sectioning on an ultramicrotome, the semithin sections are briefly stained with toluidine blue-pyronin Y. After staining, the section is rinsed in running tap water and then air dried. Under a light microscope with a 40 X or a 100 X objective, the juxtaglomerular granules appear as deep purple particles and are thus easily separated from the bluish cytoplasm of the juxtaglomerular cells. Cellular organelles in other cells of the kidney were also clearly stained and their fine structure distinguishable.     

A Simple Method of Staining Juxtaglomerular Granules in the Rat Kidney for High Resolution Light Microscopy

A simple method for the demonstration of juxtaglomerular granules in Epon embedded semithin (0.5-1 μm) sections has been developed as follows: sections are prepared as for routine electron microscopy except that before dehydration, the tissues are immersed in 0.5% uranyl acetate in Veronal acetate buffer (pH 5.0) overnight at room temperature. After sectioning on an ultramicro-tome, the semithin sections are briefly stained with toluidine blue-pyronin Y. After staining, the section is rinsed in running tap water and then air dried. Under a light microscope with a 40 × or a 100 × objective, the juxtaglomerular granules appear as deep purple particles and are thus easily separated from the bluish cytoplasm of the juxtaglomerular cells. Cellular organelles in other cells of the kidney were also clearly stained and their fine structure distinguishable.     

Adaptation of solitary intestinal lymphoid tissue in response to microbiota and chemokine receptor CCR7 signaling

Besides Peyer's patches, solitary intestinal lymphoid tissue (SILT) provides a structural platform to efficiently initiate immune responses in the murine small intestine. SILT consists of dynamic lymphoid aggregates that are heterogeneous in size and composition, ranging from small clusters of mostly lineage-negative cells known as cryptopatches to larger isolated lymphoid follicles rich in B cells. In this study, we report that in chemokine receptor CCR7-deficient mice SILT is enlarged, although unchanged in frequency and cellular composition compared with wild-type mice. This phenotype is conferred by bone marrow-derived cells and is independent of the presence of intestinal bacteria. Remarkably, particularly small-sized SILT predominates in germfree wild-type mice. Colonization of wild-type mice with commensal bacteria provokes an adjustment of the spectrum of SILT to that observed under specific pathogen-free conditions by the conversion of pre-existing lymphoid structures into larger-sized SILT. In conclusion, our findings establish that intestinal microbes influence the manifestation of gut-associated lymphoid tissues and identify CCR7 signaling as an endogeneous factor that controls this process.     

Scanning Electron Microscopy of Plasma Etched Implant Specimens

Following surface etching of previously processed plastic embedded specimens containing hard and soft tissues and implanted biomaterials with oxygen plasma, the fine structure of the tissues can be examined by scanning electron microscopy. One micrometer plastic orientation sections (with the implant removed in processing) and 110 µl;m histological sections (with the implant in situ) were examined. Direct comparison can be made between the scanning and histological observations. An examination in situ of oral tissues next to the biomaterial was also made, care being taken to minimize damage to the specimen. The fine structure of intracellular organelles was examined in detail. The method allows consecutive gathering of histological and ultrastructural data from the same plastic embedded specimen.     

Fine needle aspiration cytology and immunocytochemistry in the diagnosis of lymphoid lesions of the thyroid gland

Immunocytochemical analysis was used in conjunction with cytomorphology to evaluate fine needle aspirates from 18 patients with nodular lymphoid infiltrates of the thyroid. The smears from 13 patients had a cytomorphology that was diagnostic of chronic lymphocytic thyroiditis; immunocytochemistry showed the lymphoid population to be composed of T cells and polyclonal B cells. Three patients had high-grade malignant lymphomas by cytomorphology; immunologic evaluation showed the B-cell phenotype of the lymphoma cells and monoclonal light chain expression. Cytomorphology could not differentiate between an inflammatory and a neoplastic lymphoid infiltrate in two cases. Immunocytochemical analysis showed that the cells of B-cell phenotype were monoclonal (neoplastic) in one case and polyclonal (inflammatory) in one case.     

A gastrointestinal-specific monoclonal antibody that may be of clinical value in cytologic material

A new monoclonal antibody (MAb), 29-10, produced by immunization of mice with cells from the SW 1116 colorectal carcinoma cell line, detected an antigen present in cytologic touch imprints of surgically resected normal and neoplastic gastrointestinal (GI) tissue, including specimens from the stomach and the colon. These imprints were fixed in 95% ethanol and stained with the avidin-biotin immunoperoxidase technique. In tested cases, 22 (100%) of 22 imprints from GI adenocarcinomas and from normal GI tissue, as well as 13 (56.6%) of 23 imprints from colonic polyps, stained positively while no staining was demonstrable in imprints from other tissues. In histologic sections, only 4 (23%) of 17 colonic adenocarcinomas and 3 (11.5%) of 26 polyps stained positively. The staining ability of MAb 29-10 was compared to that of MAb 19-9, another colorectal antibody, and was found to be markedly superior for binding of the antigen in cytologic preparations. This tissue-specific antibody may be useful in identifying malignant cells of metastatic carcinoma as to their GI tract origin.     

Immunophenotype of thymoma-associated lymphoid cell component of T-cell type. A new analytic procedure in keeping with structural heterogeneities

The phenotype of the lymphoid cell component of 35 thymomas was investigated by analyzing cryostat sections and lymphocyte suspensions. The morphology in each case was determined by examining multiple tissue samples from different parts of the tumor. Structural heterogeneity was shown in 14 thymomas, and a homogeneous morphology of cortical or medullary or mixed types in the others. To assess whether this heterogeneity was correlated with differences in the lymphoid phenotype, we analyzed both lymphocyte suspensions and frozen sections from the same samples. Phenotypical differences in the suspensions of each thymoma in the heterogeneous group were noted and similar differences were also observed in the cryostat sections. Phenotypical abnormalities were found in some thymomas. They consisted of the simultaneous expression of cortical and medullary markers, which was most marked in the heterogenous mixed-type thymomas invading the lung. Furthermore, the global phenotype was tested on a pool of lymphocyte suspensions in all thymomas. This procedure distinguished cortical, medullary and intermediate cortico-medullary immunophenotype models which closely correlated with the tumor histology. It was concluded that, due to the frequent structural and immunological heterogeneity of thymomas, correct assessment of their lymphoid component requires a two-step analysis. This comprises: 1) individual suspensions from samples taken from different areas of the same thymoma, and 2) a pool of these suspensions. The first step will reveal the different immunological characteristics. In the second, the lymphocyte phenotype, which may vary widely throughout the tumor, will be represented in its totality. These findings may be of great help in predicting clinical patterns, especially possible malignant evolution.     

Template matching as a tool for annotation of tomograms of stained biological structures

In recent years, electron tomography has improved our three-dimensional (3D) insight in the structural architecture of cells and organelles. For studies that involve the 3D imaging of stained sections, manual annotation of tomographic data has been an important method to help understand the overall 3D morphology of cellular compartments. Here, we postulate that template matching can provide a tool for more objective annotation and contouring of cellular structures. Also, this technique can extract information hitherto unharvested in tomographic studies. To evaluate the performance of template matching on tomograms of stained sections, we generated several templates representing a piece of microtubule or patches of membranes of different staining-thicknesses. These templates were matched to tomograms of stained electron microscopy sections. Both microtubules and ER-Golgi membranes could be detected using this method. By matching cuboids of different thicknesses, we were able to distinguish between coated and non-coated endosomal membrane-domains. Finally, heterogeneity in staining-thickness of endosomes could be observed. Template matching can be a useful addition to existing annotation-methods, and provide additional insights in cellular architecture.     

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.     

Subcellular visualization of light microscopic specimens by laser scanning microscopy and computer analysis: a new application of image analysis.

To identify subcellular organelles or to observe their pathological changes in sections prepared for light microscopy, immuno- and/or enzyme histochemical staining for the marker substances or enzymes of those subcellular organelles are frequently employed. With conventional light microscopes (CLM), however, it is hardly possible to determine whether or not the target organelles are properly stained and to confirm their fine structure. In the present study, the laser scanning microscope (LSM) was employed to obtain highly contrasted images of histochemically stained subcellular organelles at the limit of resolution in light microscopy. To refine or characterize those images, images built up as electronic signals in LSM were further processed in the Image Analysis System (IAS) with pipeline. Thus, the approximate figures of subcellular organelles such as microtubules, endoplasmic reticula, secretory granules, and mitochondria were visualized in brightfield on sections prepared for light microscopy (paraffin, frozen sections and cultured living cells). The validity of the images obtained by LSM or LSM-IAS was confirmed by immunoelectron microscopy when possible. The LSM images of histochemically stained suborganelles of various cells were definitely improved (refined and/or strengthened) by processing them with IAS.     

Double-positive CD21+CD27+ B cells are highly proliferating memory cells and their distribution differs in mucosal and peripheral tissues

Background

Several B-cell defects arise in HIV infected patients, particularly in patients with chronic infection and high viral load. Loss of memory B cells (CD27⁺ B cells) in peripheral blood and lymphoid tissues is one of the major B cell dysfunctions in HIV and simian immunodeficiency virus (SIV) infection. Despite several studies, definitive identification of memory B cells based on CD27 surface expression has not been described. Similarly, the rates of cell turnover in different B cell subpopulation from lymphoid and mucosal tissues have not been well documented. In this study, we demonstrate the presence of memory B cell populations and define their distribution, frequency and immunophenotype with regards to activation, proliferation, maturation, and antibody production in normal rhesus macaques from different lymphoid tissues.

Methodology/Principal Findings

Thirteen healthy, uninfected rhesus macaques were selected for this study. CD20⁺ B cells were isolated from peripheral blood and sorted based on CD27 and CD21 surface markers to define memory B cell population. All the B cell subpopulation was further characterized phenotypically and their cell turnover rates were evaluated in vivo following bromodeoxyuridine (BrdU) inoculation. Double positive (DP) CD21⁺CD27⁺ B cells in both peripheral and lymphoid tissues are memory B cells, able to produce antibody by polyclonal activation, and without T cell help. Peripheral and lymphoid DP CD21⁺CD27⁺ B cells were also able to become activated and proliferate at higher rates than other B cell subpopulations. Increased turnover of tonsillar memory B cells were identified compared to other tissues examined.

Conclusions/Significance

We suggest that this DP memory B cells play a major role in the immune system and their function and proliferation might have an important role in HIV/SIV mediated B cell dysregulation and pathogenesis.     

In situ embedding method of cells grown in beem capsules for immunoelectron microscopic studies of oncornaviruses.

A technique of in situ embedding of cells grown in BEEM capsules has been devised for immunoelectron microscopic studies of oncornaviruses. As compared to other immunoelectron microscopic procedures, this technique is less time and reagent-consuming. The quality and specificity of this method were tested on well-characterized mouse mammary tumor virus (type B virus) and murine sarcoma virus (type C virus particles). This method gave good results in labeling of the virus particles with ferritin or peroxidase in the cells of mouse tissue cultures. In an application of this method, peroxidase labeling of type B virus particles was obtained in frozen sections of normal prostatic tissues of C3H/Dm and A/Dm strain mice treated with rabbit antiserum to mouse mammary tumor virus from A/Dm strain mouse milk. These results indicate that this method is useful and reliable for immunoelectron microscopy studies of oncornaviruses in tissue culture cells and also in frozen sections of tissues.     

Keratin-positive reticulum cells in fine needle aspirates and touch imprints of hyperplastic lymph nodes. A possible pitfall in the immunocytochemical diagnosis of metastatic carcinoma.

Fine needle aspirates and touch imprints of 36 hyperplastic (reactive) lymph nodes were tested for the presence of keratin and desmin. Keratin-positive cells with morphologic characteristics corresponding to extrafollicular (fibroblastic) reticulum cells were found in 18% of the fine needle aspirates and 42% of the touch imprints. The number of keratin-positive reticulum cells varied from 1 to greater than 30 per slide. Desmin-positive cells with similar morphology were found in 23% of fine needle aspirates and 37% of touch imprints, and the number of such cells per slide ranged from 2 to greater than 70. The relatively frequent occurrence of keratin-positive reticulum cells in these preparations from hyperplastic lymph nodes should be taken into account if keratin antibodies are used to search for carcinoma micrometastases.     

Scanning electron microscopy of plasma etched implant specimens

Following surface etching of previously processed plastic embedded specimens containing hard and soft tissues and implanted biomaterials with oxygen plasma, the fine structure of the tissues can be examined by scanning electron microscopy. One micrometer plastic orientation sections (with the implant removed in processing) and 110 microns histological sections (with the implant in situ) were examined. Direct comparison can be made between the scanning and histological observations. An examination in situ of oral tissues next to the biomaterial was also made, care being taken to minimize damage to the specimen. The fine structure of intracellular organelles was examined in detail. The method allows consecutive gathering of histological and ultrastructural data from the same plastic embedded specimen.     

Immuno-ultrastructural localization of B-cell-specific monoclonal antibodies B1 and B2

Monoclonal antibodies B1 and B2 are thought to recognize B-lineage restricted antigens, and have been used to define stages of B-cell maturation and characterize B-cell lymphomas. Immunostaining on cryostat sections has revealed a puzzling dendritic or extracellular pattern of staining for B2 within germinal centers and neoplastic follicles. In this study B1 and B2 are localized precisely on hyperplastic and neoplastic lymphoid tissues using immuno-ultrastructural techniques on cryostat sections, cell suspensions, and cell monolayers. B1 and B2 were localized to cell surfaces, including microvillous surface projections, on small and large transformed normal and neoplastic B lymphocytes. B2, in addition to staining in lymphoid cells, was localized to anastomosing cytoplasmic processes of dendritic histiocytes. These findings explain the apparently extracellular localization of B2 in cryostat sections and indicate that patterns of staining for B2 may represent a combination of staining on lymphoid cells and dendritic histiocytes.     

Electron microscopic study on the initiatory cell-activation of procambium at commencement of germination in radish cotyledons

Most fine structural studies of seed germination have been begun at 24 hr after imbibition of water and lasted for several days. However, studies on the initial period beginning with water uptake are few. In the present study, fine structural changes in radish seed cotyledonal tissues which occur within the first 24 hr of germination were investigated. Remarkable morphological and developmental changes of the cell organelles were profound in procambial cells in a matter of hours, while no cytological changes were noticed yet in mesophyll (storage tissue) cells. The endoplasmic reticulum and Golgi bodies were somewhat proliferated, a rapid development even, of proplastids occurred, and the increment of mitochondria in numbers and profiles were particularly noteworthy. From these observations, it was interpreted that, although the procambium accounts for only a small portion of the cotyledons, the initiatory activation of procambial cells, being sensitive to the starting of water uptake, might provide certain facility for leading the activation of the more lethargic storage tissue in the beginning of germination process.     

Double immunocytochemical labeling of cell and tissue samples with monoclonal anti-bromodeoxyuridine

We describe a new monoclonal antibody (designated Bu20a) against bromodeoxyuridine (BrdU). This antibody was selected by screening against human tissues using the APAAP technique, and shows no crossreactivity with normal nuclei. It stains BrdU incorporated into the nuclei of a wide range of cell types, including human tonsil lymphoid cells, normal mouse tissues, and human tumors growing in nude mice. A double-labeling technique is described using this antibody in which cell smears or tissue sections are first labeled by an immunoperoxidase procedure for a cellular antigen (e.g., mouse or human histocompatibility class II antigen, T-lymphocyte antigen, keratin) and BrdU is then detected by indirect immunofluorescence. This procedure, which was applied to a variety of human and animal cells and tissues, is of wide potential value in analyzing the phenotype of S-phase cells and in co-localizing antigen expression and BrdU incorporation in tissue sections.     

Effects of caffeine treatment on the fine structure,silica secretion,and shell morphology of the testate amoeba Assulina muscorum

Assulina muscorum secretes morphologically altered shells when cultured in a medium with 5 mM caffeine. The siliceous scales, normally distributed in a regular overlapping pattern, are disorganized, thicker and wider than normal, and occasionally have incompletely silicified surfaces that appear irregular in profile in transmission electron microscopic ultrathin sections. The shape of the silica deposition vesicles (SDVs) in the cytoplasm is altered and they are less regularly arranged. The swollen appearances of the SDVs, and of nearby Golgi tubules, give additional evidence that caffeine affects the fine structural morphology of membranous secretory organelles and can disrupt their normal depositional activity. In addition to the greater thickness and width of the siliceous scales in caffeine-treated cells, the length and width of the shell are larger compared to controls, but the aspect ratio (length / width) is smaller. The latter is attributed to a larger increase in width relative to the increase in length of the caffeine-reated cells. Since some of the scales are deposited with the long axis laterally on the shell surface, in addition to being greater in width, this raises the interesting question of whether the morphology of the SDVs and the siliceous products influences the size and morphogenesis of the shell. Further research is needed to clarify the interaction of the SDVs with the cytoplasmic cytoskeletal system during shell morphogenesis. © 1995 Wiley-Liss, Inc.