Anti-spermine monoclonal antibodies (ASPM-1 and ASPM-2 MAbs) against N-(γ-maleimidobutyryloxy) succinimide (GMBS)-conjugated spermine have been demonstrated to be useful for polyamine (PA) immunocytochemistry (ICC) (Fujiwara et al. 1994). Based on the evaluated antibody specificity of the MAbs we studied the immunostaining mechanism in the PA ICC using glutaraldehyde-conjugated Spm (Spm-GA-BSA conjugates) as simulation examples of tissue sections fixed with glutaraldehyde (GA) by enzyme-linked immunosorbent assays (ELISA). It was demonstrated that ASPM-1 bound to the GA conjugates to almost the same degree as to Spm-GMBS-BSA conjugates used for screening the MAbs, but that ASPM-2 did so about ten-times less than to the GMBS conjugates. However, no immunoreaction occurred in either case with paraformaldehyde (PFA)-conjugated Spm. This completely agrees with the results of the present PA ICC, showing an immunoreaction for PAs in a Hela cell line and a variety of cells in the rat adenohypophysis when fixed with GA or a mixture of GA and PFA, but none with PFA alone. The present study also suggests that the Spm-GA-BSA conjugates consist of at least two types of Spm derivatives, one conjugating Spm through only one of the two primary amino groups in the molecule, and another in which Spm has reacted with GA at both the primary amino groups. The former may react primarily with ASPM-1 MAb and the latter with ASPM-2 MAb. This may confirm our previously proposed idea that PAs are converted to a variety of PA derivatives during the fixation process of PA ICC. The model ELISA procedures should prove useful in assessing immunostaining mechanisms in ICC.

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Histatin is a group of histidine-rich polypeptides specific to parotid and submandibular gland secretions with several different biological functions such as stabilization of mineral-solute interaction in oral fluid, and antibacterial and antifungal actions. The authors generated polyclonal antibody to histatin by using purified histatin 5 as an immunogen and assayed the immunoreactivity by enzyme linked immunosorbent assay (ELISA) and immunoblotting. The antibody was further used to localize histatin in normal and tumors of salivary glans, pleomorphic adenoma, Warthin's tumor, adenoid cystic, acinic cell, mucoepidermoid, papillary cystadenocarcinoma and undifferentiated carcinoma by immunohistochemical methods. The normal major and minor human salivary glands showed an intense immunoreactivity in ductal cells, trace immunoreactivity in serous acini and no immunoreactivity in mucous acinar cells, suggesting histatin in mainly produced by ductal cells and to a lesser extent by serous cells. A consistent immunoreactivity of histatin in ductal segments of normal glands and a variable expression in the tumor cells of all the neoplastic lesions examined may implicate a role of this polypeptide in normal salivary gland function and salivary tumors. In addition, the findings may implicate common precursor cells, however, differing in the state of differentiation in normal and neoplastic conditions.

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The dynamics of gap junction protein connexins (CXN) in the developing rat exorbital lacrimal gland cells were examined by immunofluorescence and electron microscopy. Indirect double-staining immunofluorescence histochemistry of unfixed frozen sections of the exorbital lacrimal gland showed that the gap junctions in the rat exorbital lacrimal gland cells were composed of proteins which were immunologically equivalent to CXN 26 and 32. Six days after birth, a slight immunoreaction of CXN 26 was detected, but the immunoreaction of CXN 32 was negligible. Diminutive gap junction plaques were identified on protoplasmic fracture faces (P face) of freeze fracture replicas. The immunoreaction of CXN 32 was first detected on postnatal day 11. The immunoreactions of CXN 26 and 32 were detected at almost the same site of the acinar cell lateral aspect. The immunoreaction was not detected in the duct epithelium of the lacrimal gland. As the age advanced, the immunoreactivity of CXN 26 and 32 increased in both quantity and intensity. Seventeen days after birth when the rat eyelids opened, the intensity and distribution of CXN 26 and 32 immunoreactivity were approximately equal to those of the rats of postnatal day 25. Electron microscopy of ultrathin sections and freeze fracture replicas also revealed gap junctions. Twenty-five days after birth, nearly all cells showed distinctly positive immunoreactions of CXN 26 and 32, the feature similar to that of adult rats. These findings showed that gap junctions gradually increased in number postnatally and reached almost the adult level between postnatal days 17-25. The number of secretory granules in acinar cells increased with age. Gap junctions increased in size with age as revealed by freeze fracture replicas. These findings indicate that gap junctions develop as the secretory activity of acinar cells is up regulated in the exorbital lacrimal gland.

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The present review describes our own experience in the application of immunoperoxidase staining to routine histo- and cytodiagnosis. A) Use of commercially available antibodies: 1) Mycobacterial infection (tuberculosis, atypical mycobacterial infection and leprosy) was demonstrated by using a BCG antiserum, with much higher sensitivity than Ziehl-Neelsen's acid-fast method. 2) Chlamydial and bacterial epididymitis was distinguished by immunostaining for C. trachomatis and E. coli. The chlamydial antigens were identified in pap-stained cytologic preparations after bleaching the dyes in acid alcohol, while prostatic malacoplakia was clearly positive for the E. coli antigens. B) Use of patients' sera: Diluted patients' sera became convenient probes for indirect immunoperoxidase localization of pathogens in paraffin sections, particularly when cellular tissue reaction was evident histologically. Examples included Staphylococcal pyoderma, cat scratch lymphadenitis, cryptococcosis, sporotrichosis, amebic dysentery, cutaneous leishmaniasis, and liver ascariasis. Endogenous human IgG in sections was scarcely detected by the peroxidase labeled secondary antibody. Similarly, sera of animals experimentally infected with Treponema pallidum and Toxoplasma gondii were applicable to human material. C) Immunostaining and non-isotopic in situ hybridization: Comparison was made in human specimens infected by cytomegalovirus (CMV), human papillomavirus (HPV) and Epstein-Barr virus. In the latter two oncogenic viruses, the viral antigens were less frequently detectable than the viral genomes in cervical severe dysplasia and Hodgkin's disease. D) Ultrastructural visualization of pathogens in routine material: The antigens of C. trachomatis, E. coil, CMV and HPV were seen directly in paraffin sections by applying pre-embedding immunoelectron microscopy. This approach was useful to confirm the presence of pathogens within the lesions and the specificity of the antibodies. The viral genomes were also identifiable at the ultrastructural level.

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Mucins are high molecular weight glycoproteins having oligosaccharides attached to serine or threonine residues of the core protein backbone. Synthesis and secretion of mucin are common features of glandular epithelial tissues. Alterations in the glycosylation of mucins have been described in cancer, however little is known about the expression of different types of mucin core protein. To evaluate whether mucin antigens expression is correlated with the biological behavior of neoplasms or not, we examined the expression of mucin carbohydrate antigens associated with the earliest steps in mucin glycosylation (Tn and sialosyl-Tn) and core protein antigens associated with MUC1 and MUC2 gene products (DF3 and MRP) in tumors of several organs using immunohistochemical methods. Simultaneous expression of Tn and sialosyl-Tn was observed in adenocarcinomas of the pancreas, intrahepatic bile-duct and ovary, in 100%, 92% and 100% of the cases respectively. It was not frequently observed in the breast cancers (10%), although a close association of Tn expression with lymph node metastasis was observed in the scirrhous type of breast cancer. In the pancreatic and intrahepatic bile-duct carcinomas, a pattern of DF3(+) and MRP(-) was found in 100% of the invasive carcinomas with a poor prognosis, whereas DF3(-) and MRP(+) pattern was seen in 79% of the non-invasive carcinomas with a favorable prognosis. In the breast cancers, DF3 was expressed in 95% but MRP was not expressed. In the ovary, MRP was expressed only in the mucinous tumors, but not in the serous tumors. Ovarian serous adenocarcinomas showed DF3(+) and MRP(-) pattern in 100%. On the other hand, ovarian mucinous adenocarcinomas showed DF3(+) and MRP(-) pattern in 45% of the cases, and DF3(+) and MRP(+) pattern in 55%, while benign mucinous adenomas did not show DF3(+) and MRP(+) pattern. These results suggest that the patterns of expression of several mucin antigens are associated with the biological properties of neoplasms, and are useful to predict prognosis of the patients.

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mRNA in situ hybridization has provided important information in the various fields of biology and medicine but it is not easy to incorporate the technique to diagnostic pathology laboratories, compared to immunohistochemistry. The technique is relatively cumbersome and time-consuming and it is important for pathologists to determine in what situations mRNA in situ hybridization can provide important information in surgical pathology materials. Biological significance of mRNA in situ hybridization employing surgical pathology materials are summarized as follows; i) determine whether immunoreactivity observed by conventional immunohistochemistry represent the gene products produced, stored or bound to receptor, ii) study the localization of the gene products with rapid intracellular half-life, iii) examine the localization of expression when DNA sequences are known but no reliable antibodies are available for immunohistochemistry. and iv) analyze the expression at mRNA and protein levels if there are discrepancies between these two levels of expression. Surgical pathologists should consider these aspects above prior to performing and/or ordering mRNA in situ hybridization. Prompt fixation is also important for specimen preparation regardless of fixatives employed. Choice of the probes and detection methods may influence the results but it is important for pathologists to determine the most appropriate methods in his or her own laboratories based on the facility and budget. In general, the preservation of mRNA in tissue specimen and sensitivity of detection systems largely influence the results of mRNA in situ hybridization on surgical pathology materials.

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Shedding of plasma membrane or ectocytosis is a ubiquitous process in eukaryotic cells. In this study, we examined the role of vesicles exfoliated from human peripheral blood leukocytes (PBL) in proliferation, expression of IL-2 receptor (CD25) and in the process of apoptosis (programmed cell death) in these cells. Vesicles derived from peripheral blood leukocytes stimulated with conconavalin (Con)-A induced agglutination, IL-2 receptor expression and proliferation in leukocytes freshly isolated from peripheral blood in a fashion similar to that induced by Con-A. These vesicles expressed Con-A on their surface. Furthermore, IL-2 receptor expression induced by concanavalin-A and by vesicles derived from Con-A activated PBL was inhibited by an antiserum to Con-A suggesting that the activation induced by vesicles is mediated by Con-A bound to their membranes. In contradistinction to these vesicles, those derived from IL-2 activated PBL, induced apoptosis in PBL freshly isolated from peripheral blood. The apoptotic leukocytes were identified by loss of cell viability, their distinct morphologic characteristics, and by reactivity with the monoclonal antibody, BM1 that reacts with apoptotic cells. These studies implicate vesicles shed from leukocytes in the proliferation, expression of IL-2 receptor and apoptosis of leukocytes.

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It has been found that 3, 4-dihydro-6-[4-(3, 4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone (Vesnarinone), a newly synthesized positive inotropic agent, has differentiation-inducing activity against various human solid tumor cells.
In the present study, we examined the effect of vesnarinone on the differentiation, apoptosis and Le^y antigen expression of human adenoid squamous carcinoma-forming cell line TYS derived from a minor salivary gland, which was grown in culture. Cultivation of TYS cells in the presence of vesnarinone (10-50μg/ml) resulted in the formation of secretory granules with salivary amylase in the cytoplasm as well as in the induction of apoptosis in the cells detached from the plastic surface and floated in the medium, in which intracellular calcium levels were significantly increased. On the other hand, appearance of large cells with numerous vacuoles having salivary amylase was observed in the attached cells, in which apoptotic change was not detected. Le^y expression was detected in the cytoplasmic membrane of cultured TYS cells, while translocation of Le^y antigen from cytoplasmic membrane to perinuclear area was observed in the attached TYS cells treated with vesnarinone. Moreover, it has been found by immunoblot analysis of TYS cell membrane proteins and cytosolic proteins that the Le^y antigen in TYS cells forms a complex with 40kd protein and the Le^y complex moves from cytoplasmic membrane to perinuclear area in the attached TYS cells undergoing acinar cell differentiation, but not apoptosis. Thus, it can be considered that the translocation of Le^y antigen in vesnarinone-treated TYS cells may be associated with the cellular differentiation, rather than with the induction of apoptosis.

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Ribosomal RNA (rRNA) sequences are present in all prokaryotic and eukaryotic cells. These sequences are highly conserved among organisms and are know to be species specific. Sequence analysis is widely utilized in diagnostic microbiology in order to identify infectious agents. A rapid in situ hybridization assay for detection of rRNA sequences in formalin or Bouin's fixed paraffin embedded tissues was developed using biotin-labeled oligonucleotide DNA probes specific for a variety of rRNA sequences. This method was used effectively to identify human, Aspergillus species, Pneumocystis carinii, and Toxoplasma gondii rRNA sequences in a protocol requiring less than 30min to complete. Utilization of site specific oligonucleotide probes specific for a variety of rRNA sequences may aid in the diagnosis of several medically important bacterial, fungal, and protozoal pathogens.

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Sites of glycosylation were investigated using postembedding and preembedding lectin staining. Positive labeling in the luminal side of perinuclear spaces and endoplasmic reticulum was observed by N-acetylgalactosamine and mannose binding lectins. Cis lamellae of the Golgi apparatus were stained by N-acetylgalactosamine and mannose binding lectins. Medial to trans lamellae were mainly labeled by galactose binding lectin and transmost lamellae were labeled by sialic acid and fucose binding lectins. The present results elucidated the route of glycoconjugate synthesis in relation to cell organelle.

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A couple of triple and dual staining methods [high iron diamine (HID)-alcian blue (AB) pH 2.5-periodic acid-Schiff (PAS) and HID-PAS] were successfully utilized to analyze mucosubstances elaborated and released by epidermal mucous cells of four Indian freshwater fish species in light microscopy. In addition, other single and dual staining methods such as PAS, HID, AB pH 1.0, AB pH 2.5, AB pH 1.0-PAS, AB pH 2.5-PAS and HID-AB pH 2.5 were employed to further analyze the same substances and to substantiate the results obtained by the HID-AB pH 2.5-PAS and HID-PAS techniques. The comprehensive uses of pre-lectin histochemical procedures including the HID-AB pH 2.5-PAS and HID-PAS methods were unusually useful to study the cytochemical properties of mucosubstances of different mucous cell types involved in the epidermis of four Indian freshwater fish species.

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