Subcellular localization of vitellogenin in crustacean adipocytes by the unlabelled antibody enzyme method

The subepidermal fat body of the Amphipod Crustacean Orchestia gammarellus shows ultrastructural modifications related to vitellogenin synthesis. In the adipocytes of vitellogenic females, the rough endoplasmic reticulum is well developed whereas in those of males and non-vitellogenic females it is almost entirely absent; lipids and glycogen are, on the contrary, less abundant. The unlabelled antibody enzyme method shows the presence of vitellogenin in the dense bodies of the adipocytes of vitellogenic females. Adipocytes of males and non-vitellogenic females are not immunoreactive.     

Immunohistochemical identification of gastrointestinal neurotensin cells in human embryos

The endocrine gastro-entero-pancreatic (GEP) system of 10 human embryos was studied with special reference to neurotensin-immunoreactive cells. These cells are first present in the ileal and jejunal mucosa of 12 to 13 week old embryos. Thereafter the neurotensin-immunoreactive cells are found regularly in these segments of the gut with an increasing number towards the terminal ileum. At about the twentieth week of gestation, the neurotensin cells are detected also in the lower duodenum, i.e. the distribution pattern is more extensive in this age than in younger embryos or in adults.     

Clastogenic effects of bleomycin, cyclophosphamide, and ethyl methanesulfonate on resting and proliferating human B- and T-lymphocytes

The effects of bleomycin (BM), cyclophosphamide (CP), and ethyl methanesulfonate (EMS) on the frequencies of chromosomal aberrations were tested in mitogen-stimulated highly purified human B- and T-lymphocytes. In unstimulated G0/G1 B- and T-lymphocytes the clastogen induction of chromosome fragments was investigated in prematurely condensed chromosomes (PCC) induced by cell fusion with xenogenic mitotic cells. BM, CP (with metabolic activation), and EMS induced a significant increase in chromosome aberrations in proliferating human B- and T-lymphocytes. There were no significant differences in the BM-induced aberration rates between the cell populations. CP and EMS induced more aberrations in T- than in B-lymphocytes. In the PCC tests, BM-exposed G0/G1 lymphocytes showed dose-dependent high yields of chromosome fragments. No significant differences between B- and T-lymphocytes were observed. CP and EMS induced no clear increase in fragments in either cell population.     

Immunohistochemical characterisation of the monoclonal antibody BLCA-38 for the detection of prostate cancer

Background: Monoclonal antibodies (MAbs) can be used to detect, image and treat cancers. This study aimed to characterise the binding of BLCA-38 MAbs to human prostate cancer cell lines, human prostate cancer biopsy samples and normal tissues to enable future targeted studies. Methods: BLCA-38 antigen expression on cancer lines was determined by flow cytometry; that on patient specimens from normal tissues and cancers was tested by immunohistochemistry using fresh frozen tissues or paraffin-embedded tissues that had undergone antigen retrieval. Results: Cell surface BLCA-38 antigen expression was seen on DU-145, PC-3, PC-3 M and PC-3 M-MM2 prostate cancer lines, but LNCaP, MDA PCa 2a or MDA PCa 2b lines were negative. Other human lines, including 8/12 bladder cancer and A431 vulval epidermoid cells, but not breast cancer lines, expressed BLCA-38 antigen. Staining occurred in glandular epithelial cells in the majority of frozen, and paraffin-embedded prostate cancer tissues and was occasionally seen in prostatic intraepithelial neoplasia (PIN). No staining was observed in normal cadaver tissues or in benign areas from various other cancer tissues. Conclusions: The BLCA-38 antibody binds to the majority of human prostate cancers but not to normal cells, and has potential for targeting novel therapies in patients with this disease.     

Immunohistochemical localization of serine-protease inhibitors in the human placenta

Proteases and their inhibitors play a pivotal role in developmental and differentiative processes. In the present report we investigated the immunohistochemical localization of 1-antitrypsin, 1-antichymotrypsin and inter--trypsin inhibitor in first trimester as well as in term human placentas. For this purpose polyclonal antibodies against these serine-protease inhibitors were used. All inhibitors were expressed in the villous syncytiotrophoblast of first and last trimester placentas. Placental fibrinoid was positively stained for 1-antitrypsin and inter--trypsin inhibitor throughout gestation. 1-Antitrypsin and 1-antichymotrypsin showed a strong immunostaining in the Hofbauer cells (first trimester and full term placentas). Extravillous cytotrophoblast was negative for the three protease inhibitors throughout gestation. The presence of the three inhibitors in the syncytiotrophoblast suggests a role in coagulative, invasive and immunomodulatory processes. Fibrinoid, staining for 1-antitrypsin and inter--trypsin inhibitor, could also have an important immunoprotective function. The presence of protease inhibitors in the Hofbauer cells suggests an involvement of these cells in villous remodelling and differentiative processes.     

Isolation of multicellular complexes of follicular dendritic cells and lymphocytes: Immunophenotypical characterization,electron microscopy and culture studies

Summary The morphological and phenotypical features of multicellular complexes formed by follicular dendritic cells and lymphocytes (FDC-LC) isolated from human hyperplastic tonsils and adenoids are described. FDC-LC obtained with this procedure were morphologically and immuno-phenotypically heterogeneous. In one type of FDCLC, probably obtained from germinal centers, the lymphocytes exhibited ultrastructural features of centroblasts and centrocytes. In a second type, likely derived from follicular mantles, the enclosed lymphocytes were small in size and characterized by a condensed chromatin pattern. Similar heterogeneity was observed by immuno-phenotypical analysis, which revealed a prevalence of IgD+, CD3-, MT2+ small lymphocytes in a high proportion of FDC-LC. Both types of FDC-LC contained desmoplakin immunoreactivity in a typical punctate pattern corresponding to intercellular junctions when tested with a specific antibody. These findings confirm the importance of FDC in maintaining the follicular structure and also suggest that the different zones forming lymphoid follicles (mantle zone and germinal center) are formed by lymphocytes gathered in single domains by cytoplasmic processes of FDC. These domains have strong resistance to mechanical stress, such as that used in isolation procedures. FDC-LC have also been maintained as organized multicellular clusters for short periods (more than 48 h) in agarose gel cultures.Supported in part by: Ministero Pubblica Istruzione, Italy, and A.I.R.C., Milano, Italy     

Immunohistochemical studies on a human thymic epithelial cell subset defined by the anti-cytokeratin 18 monoclonal antibody

Summary Two monoclonal antibodies respectively recognizing cytokeratins (CK) 18 and 19 were applied to the human thymic epithelium (in vivo and in vitro) in normal and pathological conditions, including 12 thymomas. We observed that in both normal and hyperplastic thymuses (from patients with myasthenia gravis) virtually the entire epithelial network was CK19-positive as were the majority of cells growing in culture. In four thymomas, however, the expression of cytokeratin 19 was not detected by immunofluorescence. On the other hand, CK18 was expressed by a discrete subset of medullary thymic epithelial cells in normal and in hyperplastic thymuses. Among the thymomas a large majority was either negative or contained few isolated CK18-positive cells scattered within the tumour. Conversely, in the two undifferentiated epithelial thymomas, virtually all the tumoral network was strongly labeled with the anti-CK18 monoclonal antibody. The present investigation thus not only defines the human thymic epithelial cell subset on the basis of differential cytokeratin expression but also indicates that anti-CK antibodies with single cytokeratin specificities can be regarded as useful tools to study the heterogeneity of thymomas.     

Cellular subsets of the milky spots in the human greater omentum

Summary The cellular composition of the human milky spots was investigated on surgically removed specimens of the greater omentum of three 8-month-old infants operated on for neuroblastoma. Monoclonal antibodies and immunohistochemical methods for recognition of macrophages, B-lymphocytes and T-lymphocytes and toluidine-blue staining for mast cells were used. The mean number of cells in one milky spot amounted to 570±33. This cell population was composed of 47.5% macrophages, 29.1% B-lymphocytes, 11.7% T-lymphocytes and 6.1% mast cells. Since inflammation was absent in the material investigated, the numerical data found in the present paper could be regarded as representative cell levels of normal milky spots.     

Determination of the surface area and viscosity of membranes in T- and B-lymphocytes by means of fluorescent probes

The surface area of lymphocyte membranes was measured by registering F?rster's energy transfer on fluorescent probes. Pyrene served as donor, 4-(n-hydroxystyryl)-N-tetradecylpiridinium (HSP) was the acceptor. The surface area B-lymphocyte membranes was shown to be 1,2 times larger than that of T-lymphocytes. The mean value of lymphocyte membranes viscosity was measured using the excimerization effect of pyrene. This value was the same in all the cells investigated Fluorescence of the probe 3-methoxybenzanthrone (MBA) was 2-2.5 times higher in B-lymphocytes and was not proportional to the surface area of T- and B-cells membranes. MBA fluorescence may imply some differences in physical structures of these cells which are not connected with the viscosity of their membrane lipid phase.     

Immunohistochemical evidence for the presence of tryptophan hydroxylase in the brains of insects as revealed by sheep anti-tryptophan hydroxylase polyclonal antibody

Immediately following the discovery of tryptophan hydroxylase in Drosophila, we demonstrated the presence of tryptophan hydroxylase in the brain of the beetle Harmonia axyridis (Coleoptera: Coccinellidae). However, whether tryptophan hydroxylase is present in the brains of other insects is still a matter of discussion. In the current study, sheep anti-tryptophan hydroxylase polyclonal antibody has been applied to test for tryptophan hydroxylase immunoreactivity in a broader taxonomic range of insect brains, including holometabolous and hemimetabolous insects: one species each of Coleoptera, Hymenoptera, Diptera, and Blattaria, and two species of Lepidoptera. All species show consistent tryptophan hydroxylase immunoreactivity with distribution patterns matching that of serotonin. The immuno-positive results of such an antibody in brains from diverse orders of insects suggest that specific tryptophan hydroxylase responsible for central serotonin synthesis is probably present in the brains of all insects. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work was supported by grants from the National Natural Science Foundation of China (grant no. 30470546) and the Natural Science Foundation of Jilin Province (grant no. 20030550–7).     

Ultrastructural identification and distribution of the adhesion molecules ICAM-1 and LFA-1 in the vascular and extravascular compartments of the human palatine tonsil

Summary Immunohistological analysis of sections prepared from human palatine tonsils revealed marked differences in the distribution of the adhesion molecule, leucocyte function antigen-1 (LFA-1) and its counter receptor, intercellular adhesion molecule-1 (ICAM-1). Light microscopy showed that LFA-1 was restricted to the leucocytes, particularly the lymphocytes. In contrast, staining of ICAM-1 was predominantly confined to the vascular endothelium with the greatest expression seen on the morphologically distinct high endothelial venules in the parafollicular areas; these are the sites that appear to support lymphocyte migration. Electron microscopy revealed that ICAM-1 was present on the luminal and lateral surfaces of the high endothelium and absent from the abluminal surface supported by basal lamina. The ICAM-1 was also absent from those surfaces of the endothelium that were in close contact with intravascular lymphocytes. Other cells stained by the anti-ICM-1 antibody included dendritic cells, plasma cells and epithelial cells in the reticulated crypt epithelium and in the upper strata of the non-keratinised stratified squamous epithelium. The high expression of LFA-1 was most prominent on lymphocytes, low on antigen-presenting cells and activated lymphoid cells, and not detectable on plasma cells, epithelial and endothelial cells. We propose that LFA-1/ICAM-1 binding participates in mediating the transendothelial migration of lymphocytes across the high endothelial venules of palatine tonsil.     

Immunohistochemical and ultrastructural localisation of peptide-containing nerves and myocardial cells in the human atrial appendage

Summary The innervation and myocardial cells of the human atrial appendage were investigated by means of immunocytochemical and ultrastructural techniques using both tissue sections and whole mount preparations. A dense innervation of the myocardium, blood vessels and endocardium was revealed with antisera to general neuronal (protein gene product 9.5 and synaptophysin) and Schwann cell markers (S-100). The majority of nerve fibres possessed neuropeptide Y immunoreactivity and were found associated with myocardial cells, around small arteries and arterioles at the adventitial-medial border and forming a plexus in the endocardium. Subpopulations of nerve fibres displayed immunoreactivity for vasoactive intestinal polypeptide, somatostatin, substance P and calcitonin gene-related peptide. In whole-mount preparations of endocardium, substance P and calcitonin gene-related peptide immunoreactivities were found to coexist in the same varicose nerve terminals. Ultrastructural studies revealed the presence of numerous varicose terminals associated with myocardial, vascular smooth muscle and endothelial cells. Neuropeptide Y immunoreactivity was localised to large electron-dense secretory vesicles in nerve terminals which also contained numerous small vesicles. Atrial natriuretic peptide immunoreactivity occurred exclusively in myocardial cells where it was localised to large secretory vesicles. The human atrial appendage comprises a neuroendocrine complex of peptidecontaining nerves and myocardial cells producing ANP.     

Immunohistochemical evidence for the heterogeneity of maternal and fetal vascular endothelial cells in human full-term placenta

The heterogeneity of endothelial cell surface antigen expression was studied in 5 human full-term placentae by means of indirect immunohistochemistry using 9 monoclonal antibodies and by staining with fluorescent-conjugated Ulex europaeus lectin, both of which are widely used endothelial cell markers. (1) A highly specific, homogeneous staining of fetal and maternal placental vessels of all sizes and anatomical regions was observed by the monoclonal antibodies PAL-E, QBEND10 and 1F10. These antibodies were even more specific than Ulex europaeus lectin, factor VIII antibody and von Willebrand factor antibody, which cross-reacted with some non-endothelial cells and structures. The reactivity of PAL-E, QBEND10 and 1F10 with residual surface cells of the basal plate strongly suggests an endothelial origin of these cells. (2) In contrast to other organs, PAL-E, QBEND10 and HM 15/3 strongly stained endothelial cells of the macrovascular system in the human placenta. This might indicate an organ-associated heterogeneity of fetal endothelial cells. (3) Monoclonal antibodies against receptors for transferrin and IgG (FcRII) labeled the endothelial cells of fetal placental vessels with increasing intensity distal to the insertion of the umbilical cord. The vessels of the umbilical cord itself were unreactive. This might suggest a heterogeneity of macro- and microvascular endothelial cells.     

Immunohistochemical identification of calcitonin gene-related peptide and substance P in nerves of the bovine parathyroid gland

Summary Although peptide neurotransmitters have been shown to modulate hormone secretion in many glands, there are very few studies of neurotransmitters in the parathyroid gland. Bovine parathyroid glands were collected at a local abattoir, fixed with paraformaldehyde, sectioned using a cryostat, and stained by indirect immunohistochemistry for calcitonin gene-related peptide and substance P. We were able to positively identify both neuropeptides. Nerve fibres containing calcitonin gene-related peptide and substance P were identified in contact with the tunica media of arteries and arterioles and dispersed throughout the stroma of the gland. While many of the fibres encircled parenchymal lobules, no intimate contact with the peripheral chief cells was observed. All immunoreactive fibres were found to contain both neuropeptides. Since calcitonin gene-related peptide and substance P are vasodilators, they may increase blood flow within the gland. In addition, the neuropeptides may diffuse from perilobular nerve fibres into the parenchyma, thereby modulating secretion of parathyroid hormone.     

Quantitation of immunoglobulin- and peptide hormone-producing cells in gastrointestinal mucosa. Comparison of direct immunofluorescence and the unlabelled antibody peroxidase--antiperoxidase method

Direct immunofluorescence (DIF) and the unlabelled antibody peroxidase--antiperoxidase (PAP) methods were compared on a quantitative basis with regard to visualization of IgA immunocytes and gastrin cells in human gastric mucosa, and secretin cells in canine duodenal mucosa. With both DIF and PAP, two serial sections from 13 biopsy specimens were evaluated for each cell type--thus keeping tissue preparation the same with both staining methods. The three cell types were well visualized regardless of method, and there was no significant difference between cell numbers recorded with the DIF or PAP. When blind duplicate counts were obtained with an interval of three weeks, comparisons of weighted differences and the Kendall's rank correlation test indicated good precision; the reproducibility of duplicate enumerations with each method was comparable to that between the two methods. It was concluded that DIF and PAP are equally applicable for studies of these three cell types under the conditions used in this investigation.     

Development of a quantitative diagnostic method of estrogen receptor expression levels by immunohistochemistry using organic fluorescent material-assembled nanoparticles

The detection of estrogen receptors (ERs) by immunohistochemistry (IHC) using 3,3′-diaminobenzidine (DAB) is slightly weak as a prognostic marker, but it is essential to the application of endocrine therapy, such as antiestrogen tamoxifen-based therapy. IHC using DAB is a poor quantitative method because horseradish peroxidase (HRP) activity depends on reaction time, temperature and substrate concentration. However, IHC using fluorescent material provides an effective method to quantitatively use IHC because the signal intensity is proportional to the intensity of the photon excitation energy. However, the high level of autofluorescence has impeded the development of quantitative IHC using fluorescence. We developed organic fluorescent material (tetramethylrhodamine)-assembled nanoparticles for IHC. Tissue autofluorescence is comparable to the fluorescence intensity of quantum dots, which are the most representative fluorescent nanoparticles. The fluorescent intensity of our novel nanoparticles was 10.2-fold greater than quantum dots, and they did not bind non-specifically to breast cancer tissues due to the polyethylene glycol chain that coated their surfaces. Therefore, the fluorescent intensity of our nanoparticles significantly exceeded autofluorescence, which produced a significantly higher signal-to-noise ratio on IHC-imaged cancer tissues than previous methods. Moreover, immunostaining data from our nanoparticle fluorescent IHC and IHC with DAB were compared in the same region of adjacent tissues sections to quantitatively examine the two methods. The results demonstrated that our nanoparticle staining analyzed a wide range of ER expression levels with higher accuracy and quantitative sensitivity than DAB staining. This enhancement in the diagnostic accuracy and sensitivity for ERs using our immunostaining method will improve the prediction of responses to therapies that target ERs and progesterone receptors that are induced by a downstream ER signal.     

Differential role of reactive oxygen species in the activation of mitogen-activated protein kinases and Akt by key receptors on B-lymphocytes: CD40, the B cell antigen receptor, and CXCR4

Background Antibodies produced by B-lymphocytes play a key role in the host defense against infection. The development, survival, and activation of B cell is regulated by multiple receptors including the B cell antigen receptor (BCR), which detects the presence of pathogens, CD40, which binds co-stimulatory molecules on activated T cells, and chemokines such as SDF-1 (CXCL12) that play key roles in B cell development and trafficking. Signaling by many receptors results in the generation of reactive oxygen species (ROS) that function as second messengers by regulating the activity of redox-sensitive kinases and phosphatases. We investigated the role of ROS in signaling by the BCR, CD40, and CXCR4, the receptor for SDF-1. We focused on activation of ERK, JNK, p38, and Akt, kinases that regulate multiple processes including cell survival, proliferation, and migration. Results Using the anti-oxidants N-acetyl L-cysteine (NAC) and ebselen to deplete intracellular ROS, we identified a differential requirement for ROS in the activation of ERK, JNK, p38, and Akt by these receptors. We found that CD40 activated JNK, p38, and Akt via redox-dependent pathways that were sensitive to ROS depletion by NAC and ebselen. In contrast, BCR-induced activation of ERK, JNK, p38, and Akt was not affected by ROS depletion. We also found that CXCR4-induced Akt activation was ROS-dependent even though activation of the ERK, JNK, and p38 MAP kinases by CXCR4 occurred via ROS-independent pathways. Conclusion The differential requirement for ROS in the activation of ERK, JNK, p38, and Akt by the BCR, CD40, and CXCR4 likely reflects the multiplicity of upstream activators for each of these kinases, only some of which may be regulated in a redox-dependent manner. These findings support the idea that ROS are important second messengers in B cells and suggest that oxidants or anti-oxidants could be used to modulate B cell activation.