Expression of phenotypic markers of mast cells, macrophages and dendritic cells in gallbladder mucosa with calculous cholecystitis.

The study aimed at quantitative analysis of expression involving markers of mast cells (tryptase), monocytes/macrophages (CD68 molecule) and dendritic cells (S100 protein) in gallbladder mucosa with acute and chronic calculous cholecystitis. Routinely prepared tissue material from the patients with acute (ACC) (n = 16) and chronic calculous cholecystitis (CCC) (n = 55) was evaluated. Three cellular markers were localized by immunocytochemistry. Their expression was quantified using spatial visualization technique. The expression of tryptase was similar in acute and chronic cholecystitis. CD68 expression in ACC was significantly higher than in the CCC group. Expression of S100 protein was significantly higher in CCC as compared to the ACC group. No significant correlations were disclosed between expression of studied markers and grading in the gallbladder wall. A weak negative correlation was noted between expression of CD68 and number of gallstones in the CCC group. The spatial visualization technique allowed for a credible quantitative evaluation of expression involving markers of mast cells (MCs), monocytes/macrophages (Mo/Ma) and dendritic cells (DCs) in gallbladder mucosa with ACC and CCC. For the first time mucosal expression of S100 protein-positive DCs was evaluated in calculous cholecystitis. The results point to distinct functions of studied cell types in the non-specific immune response in calculous cholecystitis.

Expression of mRNA for cytokines (TNF-alpha and IL-1alpha) in human cytomegalovirus (HCMV) and hepatitis B virus (HBV) infections.

The study was aimed at detecting cellular sources of transcripts for two cytokines, TNF-alpha and IL-1alpha in infection with human cytomegalovirus (HCMV) or hepatitis B virus (HBV). The studies were performed on paraffin sections of organs (liver, pancreas, spleen, lungs) obtained upon autopsy from a child deceased due to acute inborn HCMV infection, on paraffin sections of liver biopsy, obtained from a child with HCMV-induced chronic hepatitis, and of liver biopsies obtained from children with chronic type B hepatitis (n = 13). The classical in situ hybridization was applied with digoxygenin-labeled probes and amplification by the ImmunoMax technique. In HCMV infection, the most pronounced expression of mRNA for TNF-alpha and Il-1alpha was detected in pancreatic islets (mainly in beta cells) and, then, in a decreasing sequence, in liver (in macrophages and sinusoidal endothelial cells) and in lungs (in alveolar macrophages). No expression of the two cytokines was detected in the spleen. In HBV infection, weak expression of TNF-alpha and more intense expression of IL-1alpha in the liver were observed, mainly in sinusoidal endothelial cells and in macrophages as well as in hepatocytes. These results were confirmed by immunocytochemical experiments.

Cellular expression of TNF-alpha in human cytomegalovirus (HCMV) infection.

Human cytomegalovirus (HCMV) belongs to the most frequent human pathogens. Even if the respective pathomorphological patterns are known in detail, the mechanisms which lead to persistence of the virus in its latent form, its reactivation as well as mechanisms of cell death in the symptomatic infection remain to be clarified. It is postulated that HCMV controls expression of TNF-alpha gene and the associated secondary inflammatory response. On the other hand, TNF-alpha has been shown in in vitro studies to represent a potential stimulator of HCMV major IE promoter. The present studies have been aimed at evaluation of TNF-alpha expression in HCMV-infected brain, liver, kidney and pancreas, obtained upon autopsy from children deceased due to an inborn HCMV infection. In situ hybridisation using digoxigenin-labelled oligonucleotide probe demonstrated the expression of TNF-alpha transcript in the liver (in macrophages and endothelial cells) and in pancreatic islets of Langerhans (in beta cells). Immunocytochemical studies aimed at detection of TNF-alpha protein in the material yielded negative results.

Immunocytochemical localization of PTHrP in human and rat salivary glands.

Parathyroid hormone-related protein (PTHrP) was isolated from tumours and is thought to represent the main factor responsible for humoral hypercalcaemia, which accompanies neoplastic diseases. At present, the protein is known to reside in multiple tissues and organs of both humans and animals. Our study was aimed at demonstrating the presence of PTHrP in normal salivary glands (parotid and submandibular) of rats and humans. Application of immunocytochemical techniques permitted to document the presence of PTHrP in the human and in the rat salivary glands. In all cases, an intense reaction was observed in intra- and interlobular ducts. In rat salivary glands, PTHrP was also present in cells of mucous acini. In our opinion, the presence of PTHrP in the ducts indicates participation of the protein in electrolyte transport across the epithelial cells. The positive reaction noted in mucous acini of rat salivary glands may indicate accessory role of PTHrP in the secretory processes in the glands.

Immunolocalization of PTHrP in the European bison and pine vole testis and epididymis.

The present study deals with immunohistochemical localization of PTHrP in European bison and pine vole testis and epididymis. PTHrP immunoreactivity was observed in spermatogenic cells of seminiferous tubules in European bison and pine vole testis, with the strongerst reaction occurring in spermatozoa of pine vole testis and epididymal duct. We also observed PTHrP expression in vascular smooth muscle of epididymis and testis in both animal species, as well as slightly weaker reaction in endothelial cells of European bison epididymis. PTHrP was also expressed in the smooth muscle of the epididymal duct in European bison and pine vole. In conclusion, PTHrP is a multifunctional peptide showing both paracrine and autocrine action. Its presence in vascular endothelium and smooth muscle of testis and epididymis is connected with the regulation of vascular muscle tone, thus affecting blood flow in the vessels. PTHrP expression depends on a number of local factors. Moreover, we suppose that PTHrP also contributes to the proliferation and differentiation of spermatogenic cells.

Correlation of morphological alterations of light and electron microscopy in chronic type B and C hepatitis.

Chronic type B and C hepatitis involves inflammatory lesions of a variable intensity and variably advanced fibrosis. Considering current, progressively growing requirements for correct evaluation of lesions in liver biopsies, an attempt was made to appraise suitability of selected techniques for a broadened histopathological diagnosis. The lesions were evaluated at the level of light and electron microscopy. Material for the study consisted of liver biopsies obtained from adults and children (n = 60) with serological markers of chronic type B or type C hepatitis. Routine techniques of staining for light and electron microscopy, as well as the techniques of Brachet and Feulgen, were applied. HBcAg expression and HBV-DNA detection in children with chronic type B hepatitis were studied employing the avidin-biotin peroxidase complex (ABC) technique and in situ hybridisation with the ImmunoMax signal amplification. Slight or moderately intense inflammatory lesions (grading of 1 to 2 points) and a low level of fibrosis advancement (staging of 1 to 2 points) prevailed in the material, independently of the etiologic agent involved and age of the patient. Both in children and in adults, extensive lesions in the nuclear chromatin represented the common trait of chronic type B and type C hepatitis examined by light microscopy. Ultrastructural patterns confirmed the lesions and demonstrated virus-resembling particles in the cell nuclei. In HCV infection, hepatocyte cytoplasm contained tubular and horseshoe-shaped structures with lesions of mitochondria, while in HBV infection Dane's particles and tubular forms of HBsAg were detected. For cognitive reasons and due to frequently equivocal literature data, our data on ultrastructural lesions in chronic type C hepatitis seem to be of particular interest. Using the ImmunoMax signal amplification, we were able to diagnose HBV infection under light microscope and to define stage of the infection. Their sensitivity, specificity and relatively short time required for performing the tests makes them advisable in the routine diagnosis of the two infections.

Detection of DNA, mRNA and early antigen of the human cytomegalovirus using the immunomax technique in autopsy material of children with intrauterine infection.

The present study focuses on the immunomax technique in association with the avidin-biotin-peroxidase complex (ABC) technique and a non-isotopic variation of in situ hybridisation (ISH) for optimal microscopical detection of human cytomegalovirus (HCMV). The studies were performed on an archival paraffin material originating from five children deceased due to intrauterine infection. The results of immunocytochemical and hybridocytochemical studies, with or without amplification using biotinylated tyramine, were compared with the routine histopathological results and results obtained using the polymerase chain reaction (PCR). Early antigen (EA)-HCMV was demonstrated in approximately twice as many cells as detected in the routine staining and also in cells that seemed morphologically intact. The hybridocytochemical studies confirmed the presence of HCMV DNA in cells that were positive in the immunocytochemical tests and, in addition (using the ISH-immunomax technique), in cell nuclei of intact myocardial myocytes. In general, fewer cells manifested the presence of HMCV mRNA than the presence of HCMV DNA. The immunomax technique was found to be more sensitive than the techniques of classical immunocytochemistry or of ISH. The former technique permitted the documentation of a higher number of HCMV replication sites than could be detected using the latter techniques. However, the clinical course of HCMV infection or the cause of death of the children was not directly related to the intensity of HCMV expression in tissues.

Immunocytochemical localisation of PTHrP (parathormone-related peptide) in myoepithelial cells of human sweat and parotid glands.

PTHrP is a HHM-inducing peptide. It exhibits certain structural similarity to PTH and the two hormones may act through the same receptors. PTHrP is known to be produced in various tissues as well as during development. In this study we decided to immunocytochemically demonstrate PTHrP in normal skin and squamous cell carcinomas as well as in parotid glands (normal, inflamed and neoplastic). In the skin, PTHrP expression was demonstrated in epidermis and in smooth muscle cell layer of blood vessels. In squamous cell carcinomas, the expression was noted in foci of keratinization. In parotid glands, the peptide was localised in excretory ducts and in blood vessels, while inflammation of the gland and its tumours resulted most frequently in the less intense immunoreaction. The results are consistent with those of other authors. The novel observations include demonstration of PTHrP expression in myoepithelial cells of sweat glands and in parotid glands, where it may be involved in the control of their contractile activity.

Polyhormonal aspect of the endocrine cells of the human fetal pancreas.

Histological studies were performed on 30 pancreases obtained from normal human fetuses aged between the 9th and 38th week. For immunocytochemistry, the avidin-biotin-peroxidase method was used to identify and colocalise insulin, glucagon, somatostatin, pancreatic polypeptide and proliferating cell nuclear antigen. In the 9th week, cells containing all investigated peptides were present. During the fetal period, two populations of endocrine cells have been distinguished, Langerhans islets and freely dispersed cells. The free cells were polyhormonal, containing insulin, glucagon, somatostatin and pancreatic polypeptide, and were localised in the walls of pancreatic ducts throughout the whole gland. During the development of the islets we have observed four stages: (1) the scattered polyhormonal cell stage (9th-10th week), (2) the immature polyhormonal islet stage (11th-15th week), (3) the insulin monohormonal core islet stage (16th-29th week), in which zonular and mantle islets are observed, and (4) the polymorphic islet stage (from the 30th week onwards), which is characterised by the presence of monohormonal cells expressing glucagon or somatostatin. Bigeminal and polar islets also appeared during this last stage. The islets consisted of an insulin core surrounded by a thick (in the part developing from the dorsal primordium) or thin rim (part of the pancreas concerned with the ventral primordium) of intermingled mono- or dihormonal glucagon-positive or somatostatin-positive cells. The most externally located polyhormonal cells exhibited a reaction for glucagon, somatostatin and pancreatic polypeptide. Apart from the above-mentioned types of islets, all arrangements observed in earlier stages were present. Proliferating cell nuclear antigen-positive cells (single in the large islets and more numerous in the smaller ones) were predominantly observed in the outermost layer. Taken together our data indicate that, during the human prenatal development of the islet, endocrine cells are able to synthesise several different hormones. Maturation of these cells involved or depended on a change from a polyhormonal to a monohormonal state and is concerned with decreasing proliferative capacity. This supports the concept of a common precursor stem cell for the hormone-producing cells of the fetal human pancreas.

Studies on tissue expression of HBV in children with chronic hepatitis type B using Immunomax technique.

The study aimed at employing the Immunomax technique to detect the markers of HBV replication (HBcAg and HBV-DNA) in liver biopsy material, obtained from children with chronic hepatitis type B. In line with the currently modified classification of chronic hepatitis and with the increasing potential of antiviral therapy it seemed purposeful to supplement routine staining techniques with studies at the molecular level. Our studies demonstrated the effective detection of both the core antigen and HBV-DNA in liver tissue in children using immunocytochemical techniques and in situ hybridization, amplified with the Immunomax technique. HBcAg was detected in 26 out of 27 liver biopsies from patients with chronic hepatitis type B and with replication of the virus. HBV-DNA was detected in all study children with HBV infection and in 2 out of 5 cases of chronic hepatitis of a distinct etiology. No significant relationships could be found between the detection of tissue HBV markers on the one hand and the intensity of inflammatory lesions or severity of fibrosis on the other.

Proliferating-cell nuclear antigen (PCNA) in the human adrenal glands at the end of the embryonic period.

Studies were performed on adrenal glands of 7 human embryos between 6 and 8 weeks of intra-uterine life using clone PC-10 mouse serum (Dako, No. M879). During the 6th week, immunoreactivity for the proliferating-cell nuclear antigen (PCNA) was observed only in the central and medial parts of the developing fetal zone of the adrenal gland (75.2 +/- 3.1% of adrenocortical cells showed nuclei stained with anti-PCNA), while the peripheral part was immunonegative. One week later the second outer layer, i.e. the permanent cortex appeared. During the 8th week, the cortex formed two distinct zones: a relatively large and centrally located fetal zone and a thin rim of definitive (permanent) cortex, the later adult adrenal cortex. PCNA-positive cells were present throughout the surface of the gland being most numerous in the center (76.4 +/- 1.4%) and less numerous in the peripheral part (16.6 +/- 2.6%). A very thin layer of permanent cortex surrounding the fetal zone showed less numerous cells stained for PCNA (36.4 +/- 3.4%) as compared with the inner fetal zone. During the 8th week the proliferative cells activity was similar in both zones. The middle proliferative center of the fetal zone disappeared, and all cells of this zone had similar PCNA reactivity.

Prenatal development of the human pancreatic islets. Immunocytochemical identification of insulin-, glucagon-, somatostatin- and pancreatic polypeptide-containing cells.

Histological studies were performed on 24 pancreases of normal human embryos and fetuses aged 7 to 38 weeks. For immunocytochemistry, the avidin-biotin-peroxidase method was used to identify and localize insulin, glucagon, somatostatin, and pancreatic polypeptide (PP) cells. In 7 wk old embryos, cells containing somatostatin and PP are observed. One week later appear single glucagon-positive cells. In the 9th wk, insulin producing cells are visible. During the fetal period two populations of the investigated cells are found: Langerhans islets and dispersed cells. The latter cells containing insulin, glucagon or somatostatin are localized in the walls of pancreatic ducts throughout the whole gland, while PP-positive cells are seen mainly in the part of the pancreas, which develops from the ventral anlage (anteroinferior part of the head and adjacent part of the main pancreatic duct). During the development of islets we have observed four stages: (1) scattered cells (7 to 10 weeks); (2) grouping cells (11 to 15 weeks); (3) mantle and zonular islets (10 to 29 weeks), in which B cells located inside are surrounded by a thick zone of A, PP and somatostatin-producing cells; (4) mixed islets (from 30 weeks on) - all cells are scattered over the whole transverse section of the islet. In the developing pancreas, the glucagon- and somatostatin-containing cells are the most numerous, while the insulin and PP-containing cells occur in lesser quantities.

Immunocytochemical characterization of two thyroid medullary carcinoma cell lines in vitro.

The immunocytochemical characterization of cell lines originating from thyroid medullary carcinoma, i.e. human TT cells and rat rMTC 6-23 cells, was undertaken. The immunocytochemical studies were supplemented by ultrastructural studies, including ultrastructural immunocytochemistry, and by radioimmunological estimation of calcitonin secretion to the medium. In rMTC 6-23 cells (subcultures 24 to 30), no hormone presence was demonstrated immunocytochemically, which corresponded to the absence of secretory granules at the ultrastructural level. Of various proteins sought, only neuron-specific enolase could be demonstrated. Nevertheless, the cells secreted calcitonin into the medium. TT cells (passages 145 to 160) produced secretory granules. The granules contained calcitonin, calcitonin gene-related peptide, somatostatin, neurotensin, met-enkephalin, leu-enkephalin, gastrin releasing peptide, parathyroid hormone-related protein, functional proteins of the chromogranin group and synaptophysin. Other functional proteins found in the cytosol of TT cells included non-specific enolase, calbindin and tyrosine hydroxylase. Receptor for calcitriol was localized in the cell nucleus. Marker proteins were localized in the cytosol (carcinoembryonic antigen) and in the cell skeleton (alpha-tubulin, cytokeratin). Following changes in ionized calcium levels in the medium, changes in calcitonin secretion and in immunocytochemical detectability of some hormones and functional proteins were observed. TT cells demonstrated the expression of numerous hormones and functional proteins associated with calcitonin secretion. Further, the cells in their ultrastructure, immunocytochemical and secretory characteristics, resemble more closely normal parafollicular cells of the thyroid and, in our opinion, represent a more appropriate model for functional studies.

Immunocytochemical studies on endocrine cells of alimentary tract of the pig in the embryonic and fetal period of life.

The study aimed at immunocytochemical analysis of alimentary tract endocrine cells between 20th day of embryonal life and 105th day of fetal life of domestic pig. In the pancreas, presence of endocrine cells was detected already in 20th day and, at the time, the cells comprised around 3/4 all cells in primordia of the organ. Starting at that time, numerous endocrine cells produced insulin and glucagon and individual cells synthesized somatostatin and pancreatic polypeptide. In the 20th day, stomach and duodenum contained single endocrine cells but hormone production was not detected until days 27 and 30. Beginning from this days, both organs manifested rapid increase in the number of gastrin-producing cells. In each of the three organs, the number of somatostatin-producing cells exhibited most extensive changes.

Hybridocytochemical and immuno-ultrastructural study of calcitonin gene expression in cultured medullary carcinoma cells.

The study was aimed at a morphological demonstration of calcitonin (CT) gene expression in cultured TT cells, or, more specifically, hybridocytochemical detection of CT mRNA and calcitonin gene-related peptide (CGRP) mRNA and ultrastructural localization of the two hormones. The TT cells originated from medullary carcinoma of human thyroid gland. Ultrastructural studies of TT cells demonstrated a well-developed rough endoplasmic reticulum, large Golgi apparatus and low number of secretory granules. Hybridocytochemical studies showed the presence of mRNAs for CT and CGRP in all TT cells. At the ultrastructural level, double immunolabelling demonstrated that the two hormones were always expressed together in the same secretory granules. Our results provide a significant addition to the biochemical studies performed up to now and indicate that all TT cells produce both mRNAs and both hormones in parallel.

Immunocytochemical studies on pancreatic endocrine cells at early stages of development of the pig.

The studies were performed on pig embryos between 23rd and 31st day of intrauterine life. Immunocytochemical markers of neuroendocrine cells, i.e. neuron-specific enolase, chromogranin and synaptophysin as well as basal hormones, i.e. insulin, glucagon, somatostatin, pancreatic polypeptide and, additionally, serotonin and gastrin were detected in serial sections. Our studies indicate that differentiation of pancreatic endocrine cells does not take place unitemporally. At the first stage, the cells acquire the traits of neuroendocrine cells and secrete more than one hormone while final specialization toward cells secreting individual hormones take places at a later stage.