Immunolocalization of PTHrP in the parotid glands of three rodents species: Clethrionomys glareoulus, Microtus arvalis and white Swiss mice.

The current study was inspired by the fact that since 2004 no report had appeared on the occurrence of this peptide in healthy parotid glands of humans and animals. The objective of the current study was to investigate the immunolocalization of PTHrP in the parotid gland of three male rodents: 6 common voles (Microtus arvalis, Pallas, 1779), 6 bank voles (Clethrionomys glareoulus, Schreber, 1780) and 6 white Swiss mice, as well as to find out any species differences in the distribution of this peptide in various types of cells of the parotid gland. Immunocytochemical reactions were performed using the ABC technique with specific rabbit antibodies against human PTHrP (34-53) (CALBIOCHEM), diluted 1:70 and 1:50. We observed positive PTHrP expression in the epithelial cells of the striated duct in all the three animal species. The expression was strong in white mouse and very strong in common vole and bank vole. In all the rodent species studied, the reaction for PTHrP was granular in nature and irregularly distributed in the cytoplasm, being definitely stronger at the base and weaker at the apex of the cells. The PTHrP expression was negative in the epithelium of the intercalated duct, interlobular duct, main excretory duct, as well as in the myoepithelial cells surrounding the excretory ducts or serous acini

Immunocytochemical evaluation of reorganisation of keratinocyte cytoskeleton induced by change in Ca 2+ concentration in culture medium

Ca2+ level-induced changes in the arrangement of cytoskeleton of cultured keratinocytes were estimated immunocytochemically, by evaluating expression of specific cytokeratins, desmoplakin and tubulin. Keratinocytes were isolated from fragments of skin of dead human foetuses. Culture of epidermal cells was performed in two phases: phase I yielded cells of high proliferation abilities in serumfree Keratinocyte SFM of low Ca2+ level (0.03 mM); in phase II differentiated cells were obtained in Dulbecco medium of a high Ca2+ concentration (1.2 mM). Immunocytochemical evaluation of phase I and II cells revealed an array of differences which involved mainly expression and distribution of specific cytokeratins, distribution of tubulin, testifying to a different microtubule arrangement and distribution of desmoplakin and indicating a tendency to form desmosomes. The changes were induced by the changes in Ca2+ level in the culture medium

The expression of metallothionein (MT) and proliferation intensity in ovarian cancers treated with cisplatin and paclitaxel

Metallothioneins (MT) represent low molecular weight proteins that are supposed to fulfil several functions. They participate in the cell cycle, protect cells from oxidative stress, control levels of heavy metals and participate in multidrug resistance processes, particularly in cases of alkylating drugs. The present study aimed at evaluation of proliferation intensity (Ki67, PCNA) in ovarian cancers treated using cisplatin and paclitaxel, as related to expression of MT. The experiments were performed on samples originating from 10 patients operated on due to ovarian cancer. The material originated from the first operations or second-look operations. All the patients were treated with cisplatin and paclitaxel. Immunocytochemical reactions using antibodies to MT, Ki67 and PCNA were performed in paraffin sections originating from the cases studied. Statistical analysis was performed using Statistica software. The studies demonstrated no relation between expression of MT on the one hand and intensity of proliferation before or after chemotherapy on the other hand (gamma correlation, p > 0.05). The results indicate that expression of MT is not related to resistance to treatment using cisplatin and paclitaxel

The expression of calcitonin, calcitonin gene-related peptide and somatostatin in the thyroids of rats of different ages

Using immunocytochemistry and in situ hybridisation expression of calcitonin, calcitonin gene-related peptide (CGRP) and somatostatin was examined in rat thyroids. The immunocytochemical reactions demonstrated the presence of the proteins under investigation at all stages of rat life. Calcitonin and CGRP produced the most numerous parafollicular cells, while somatostatin was present in a few cells only. The number of cells producing the above-mentioned hormones was found to increase in rats with the progressing of age. Hybridocytochemical techniques corroborated the results obtained using immunocytochemical techniques. The most numerous cells were found to contain mRNAs for calcitonin and CGRP. In the case of somatostatin, however, multiple parafollicular cells produced mRNA for the hormone but few cells demonstrated the presence of the corresponding protein

Analysis of the specificity and selectivity of anti-EpCAM antibodies in breast cancer cell lines

The epithelial cell adhesion molecule (EpCAM) is a membrane glycoprotein that is expressed in most normal human epithelia and overexpressed in most carcinomas. Molecule is responsible for cell-to-cell adhesion and additionally participates in signaling, cell migration, proliferation and differentiation. Therefore, EpCAM has been the target of immunotherapy in clinical trials of several solid tumors. It appears to play an important role as a target for circulating tumor cells (CTCs) capturing. The aim of this study was to investigate and compare the specificity and selectivity of different anti-EpCAM antibodies in order to their usefulness for CTCs capturing. All experiments were performed in six different types of breast cancer cell lines (MCF-7, SkBr-3, T47D, CAMA-1, MDAMB-231, BT-20) and with use of three different anti-EpCAM antibodies (EBA-1, AUA-1, 9C4). The experiments revealed that investigated antibodies differ significantly regarding the specificity of EpCAM antigen binding. The most significant role in the circulating tumor cells capturing can play the EBA-1 and 9C4 anti-EpCAM antibodies as they revealed the most specific signal. The strength and specificity of reaction was dependent not only on the type of antibody but also on the type of breast cancer cell line. On the basis of the present outcomes it can be assumed that the best solution for obtaining the most specific results could be the use of mixture of different anti-EpCAM antibodies simultaneously. In conclusion, the proper selection of anti-EpCAM antibody is crucial especially when this antigen is considered as a marker for detection of circulating tumor cells

The effect of calcitriol and its analogues on proliferation and hormone expression in cultured cells of thyroid medullary carcinomas

The study aimed at evaluating the effects of calcitriol and of its analogues on the proliferation of TT and rMTC cells (human and rat line tumour cells originating from thyroid medullary carcinoma) and at examining the effects of the substances on the secretion of the principal hormones of the cells, calcitonin (CT) and calcitonin gene-related peptide (CGRP). Cells of thyroid medullary carcinoma (human TT cells and rat rMTC cells) were cultured for 5 days in the absence or in the presence of calcitriol and of its two analogues (PRI-1906 and PRI-2191) in concentrations of 10-9 to 10-6 M. Calcitriol and the applied analogues weakly inhibited proliferation of thyroid medullary carcinoma in in vitro conditions. The evident effect of analogues on hormone secretion points to their effect on the process of CT gene expression

The expression of selected neuroendocrine markers and of anti-neoplastic cytokines (IL-2 and IL-12) in lung cancers

We have continued our studies by detecting three markers of neuroendocrine tumours of the lungs, including chromogranin A, NSE and synaptophysin, to confirm the neuroendocrine origin of lung tumours and by examining the content of two anti-neoplastic cytokines, IL-2 and IL-12 in the tumours. The studies were performed on paraffin sections of lung carcinoids (n = 13) and small cell lung carcinomas (SCLC) (n = 15). Pronounced expression of all 3 markers of neuroendocrine tumours was detected in most of the pulmonary carcinoids and in 5/15 of SCLC. Co-expression of the two cytokines (IL-2 and IL-12) in tumour cells was detected in 12/13 patients with lung carcinoid and expression of at least one cytokine in 12/15 patients with SCLC. Significantly lower numbers of cells immunoreactive to both cytokines were detected in SCLC as compared to lung carcinoids. The studies have confirmed the literature data on the lowered secretion of IL-2 in SCLC and extend the data by supplying information on the expression of IL-12. The lowered expression of the two cytokines at the time of diagnosis may represent a prognostic factor for survival in SCLC

Evaluation of apoptosis, proliferation intensity and metallothionein (MT) expression in comparison with selected clinicopathological variables in primary adenocarcinomas of the large intestine

Tumour growth and expansion are the result of proliferative activity and the capacity to eliminate cells by apoptosis and/or necrosis. The present study was aimed at comparing the apoptosis and proliferation intensity in cells of adenocarcinomas of the large intestine with the expression of metallothionein (MT), the grade of the tumour and the depth to which the tumour infiltrated the intestinal wall. The TUNEL technique and immunocytochemical reactions (expression of caspase-3, Ki-67, MT) were used to detect apoptosis. The results demonstrated augmented levels of all the variables examined, positively correlated with grade of malignancy, G, and with the depth of intestinal wall infiltration by the tumour cells. The testing of apoptosis, proliferation and MT expression may prove useful in the appraisal of the growth and progression of primary adenocarcinomas in the large intestine

Epstein-Barr virus (EBV) infection in B-cell non-Hodgkin's lymphomas in children: virus latency and its correlation with CD21 and CD23 molecules.

Epstein Barr virus (EBV) infection of human B lymphocytes in vitro results in immortalisation of the cells and augmented membranous expression of numerous B-cell activation molecules, including CD23. Other studies demonstrated that only those B lymphocytes which carry the surface CD21 (EBV receptor) become transformation-competent. Inspired by the relatively unclear relations between expression of EBV and those of CD21 and CD23 in in vivo conditions we have decided to define correlations between tissue markers of EBV and of CD21 and CD23 molecules in B-cell non-Hodgkin's lymphomas (NHLs) in children. The studies were performed on an archival tissue material originating from children with B-cell NHLs (n=26) using immunocytochemical techniques, in situ hybridisation, and PCR. Our studies confirmed the latent phase of EBV infection in all of the EBV-positive patients. Viral proteins as well as viral RNAs (EBERs) was found both in the cytoplasm, in cell nuclei and in cell membranes of mainly the transformed lymphocytes B. Expression of the latent proteins (EBNA2 and LMP1) and that of EBERs in B-cell NHLs was significantly higher as compared to children with nonneoplastic lesions. The studies demonstrated reciprocally positive correlations between expressions of CD21 and CD23 in our children, but no correlation could be demonstrated between expression of EBV tissue markers and that of CD21 and/or CD23. Positive correlation was confirmed between expression of EBNA2 and LMP1 as well as between expression of the two proteins and EBERs in B-cell NHLs. Our studies have shown mainly latency III pattern of EBV. We have also demonstrated a novel form of EBV latency with no EBERs expression. The high detectability of EBV-positive cases both in the group of B-cell NHLs (77%), and in the group with non-neoplastic lesions (64%) suggested that only more pronounced tissue expression of EBV markers in B-cell NHLs as compared to the non-neoplastic material may point to a potential role of EBV in pathogenesis of lymphoma in this group of population in our country