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Background/aim As a result of activation of transcription factors engaged in epithelial-mesenchymal transition (EMT), such as Twist, inhibition of epithelial markers and an increased expression of mesenchymal markers are observed. One of the specific markers of cancer-associated fibroblasts is podoplanin (PDPN) - a mucin-type membrane glycoprotein. The aim of this work was to study the localisation and intensity of expression of Twist and PDPN on the mRNA and protein level in cases of invasive ductal breast carcinoma (IDC), and its association with patients' clinico-pathological data. Materials and methods The study included archival material in a form of 80 paraffin IDC blocks and 11 IDC fragments frozen in liquid nitrogen. Immunohistochemical expression of Twist and PDPN was evaluated using light microscope and semiquantitative scale for evaluation of nuclear expression or immunoreactive scale (IRS) for evaluation of cytoplasmic expression. Material was isolated from frozen IDC fragments using laser micro-dissection (from cancer and stromal cells, separately) and was used to perform real-time PCR. Results Twist expression was higher in stromal cells in comparison to cancer cells. Analysis of patients' survival rate showed, that higher expression of Twist in cancer cells was associated with shorter overall survival time and shorter event-free survival time. The expression of PDPN was also higher in stromal cells in comparison with cancer cells. In addition, positive correlation was observed between expression of Twist and PDPN in stromal cells of IDC (r=0.267; p Conclusion The relationship between the higher expression of Twist in both cancer and stromal cells and shorter patients' survival indicates Twist as a potential useful prognostic marker in IDC. Positive correlation of Twist and PDPN expression may indicate the role of PDPN in EMT in IDC.

BACKGROUND Metallothioneins (MTs) are involved in numerous cell processes such as binding and transport of zinc and copper ions, differentiation, proliferation and apoptosis, therefore contributing to carcinogenesis. Scarce data exist on their expression in benign and malignant lesions of the thyroid. MATERIALS AND METHODS mRNA expression of functional isoforms of MT genes (MT1A, MT1B, MT1E, MT1F, MT1G, MT1H, MT1X, MT2A, MT4) was studied in 17 nodular goiters (NG), 12 follicular adenomas (FA) and 26 papillary thyroid carcinomas (PTC). RESULTS One-way ANOVA revealed significant differences in mRNA expression levels of MT1A (p

In our previous studies we showed that in breast cancer podoplanin-positive cancer-associated fibroblasts correlated positively with tumor size, grade of malignancy, lymph node metastasis, lymphovascular invasion and poor patients' outcome. Therefore, the present study was undertaken to assess if podoplanin expressed by fibroblasts can affect malignancy-associated properties of breast cancer cells. Human fibroblastic cell lines (MSU1.1 and Hs 578Bst) overexpressing podoplanin and control fibroblasts were co-cultured with breast cancer MDA-MB-231 and MCF7 cells and the impact of podoplanin expressed by fibroblasts on migration and invasiveness of breast cancer cells were studied in vitro. Migratory and invasive properties of breast cancer cells were not affected by the presence of podoplanin on the surface of fibroblasts. However, ectopic expression of podoplanin highly increases the migration of MSU1.1 and Hs 578Bst fibroblasts. The present study also revealed for the first time, that podoplanin expression affects the formation of pseudo tubes by endothelial cells. When human HSkMEC cells were co-cultured with podoplanin-rich fibroblasts the endothelial cell capillary-like network was characterized by significantly lower numbers of nodes and meshes than in co-cultures of endothelial cells with podoplanin-negative fibroblasts. The question remains as to how our experimental data can be correlated with previous clinical data showing an association between the presence of podoplanin-positive cancer-associated fibroblasts and progression of breast cancer. Therefore, we propose that expression of podoplanin by fibroblasts facilitates their movement into the tumor stroma, which creates a favorable microenvironment for tumor progression by increasing the number of cancer-associated fibroblasts, which produce numerous factors affecting proliferation, survival and invasion of cancer cells. In accordance with this, the present study revealed for the first time, that such podoplanin-mediated effects can affect tube formation by endothelial cells and participate in their pathological properties in the tumor context. Our experimental data were supported by clinical studies. First, when IDC and DCIS were analyzed by immunohistochemistry according to the presence of podoplanin-expressing cells, the numbers of cancer-associated fibroblasts with high expression of this glycoprotein were significantly higher in IDC than in DCIS cases. Second, using immunofluorescence, the co-localization of PDPN-positive CAFs with blood vessels stained with antibody directed against CD34 was observed in tumor stroma of IDC samples.

Nestin is an intermediate filament protein and a stem cell marker expressed in several tumours. There is growing evidence of an association between the expression level of nestin and the pathogenesis of triple-negative breast cancer (TNBC). Nestin is also expressed in newly forming tumour vessels and is a valuable marker of ongoing angiogenesis. In this study, we aimed to evaluate the prognostic value of nestin expression in breast tumour cells and to determine whether this expression influences angiogenesis. Immunohistochemical (IHC) analyses were carried out on 124 cases of invasive ductal carcinoma (IDC) of the breast with a panel of murine monoclonal antibodies against nestin, CD31, CD34, SOX-18 and Ki‑67. We evaluated nestin expression in tumour and endothelial cells, Ki‑67 in tumour cells, and CD31, CD34 and SOX-18 in endothelial cells. Our results demonstrated that nestin expression in tumour cells correlated with the area and number of vessels expressing nestin, CD31, CD34 and SOX-18. We also found a positive correlation between nestin-expressing vessels and SOX-18-expressing vessels. Our results are consistent with those of previous studies, in which nestin expression in endothelial cells was shown to be strongly associated with triple-negative subtype, poorly differentiated G3 tumours, a higher proliferation index and a shorter overall survival. Nestin expression was also examined in human breast cancer cell lines (MCF-7, SK-BR-3, MDA‑MB‑231 and BO2 cells) representing a different level of tumour aggressiveness and reflecting histological grade. A higher nestin protein level was observed in more aggressive MDA‑MB‑231 and BO2 cells than in MCF-7 and SK-BR-3 cells.

Human papillomavirus (HPV) belongs to the Papillomaviridae family and infects squamous cells and mucous membranes of humans. Various studies conducted over the last years have shown a correlation between HPV infection and carcinogenesis process. The DNA of the virus is detected in approximately 20% of cancers of the upper respiratory tract. The presence of HPV in cancerous lesion of the larynx varies depending on the procedure applied for sample collection and the viral DNA detection method. The high variance in the frequency of HPV detection is observed even among results obtained with the use of PCR reaction. It varies between 3 and 85%. HPV is also the etiological factor of laryngeal papillomas in both children and adults. However, a considerable amount of research demonstrates that 1-7% of the larynx papillomas in adults undergo transformation into squamous cell carcinoma. The aim of the study was to summarize the current state of knowledge regarding the presence of the HPV virus in the larynx as well as its participation in malignant transformation.

BACKGROUND/AIM One of the current hypotheses assumes that brain tumors exert an immunosuppressive influence on the surrounding cellular environment. Interleukin-10 (IL-10) is one of the immunosuppressive cytokines modifying the biological activity of cancer. The aim of this study was to assess the expression of IL10R in CD45+ cells within primary brain tumors and metastases and establish its association with tumor basic immunophenotype. PATIENTS AND METHODS Tissue samples were obtained intraoperatively during surgeries of 32 patients suffering from meningiomas (n=9), gliomas (n=12) and metastatic tumors (n=11). Expression was assessed with flow cytometry and immunohistochemical reactions. RESULTS Expression of IL10R subunits within the leukocyte population (CD45+ cells) was significantly higher in primary tumors than in metastases. CONCLUSION To the best of our knowledge, this is the first study describing a correlation between the IL10R expression on leukocytes and histological types of brain tumors.

BACKGROUND/AIM The risk of breast cancer is related to duration of exposure to sex hormones, especially estrogen. The aim of this study was to assess the impact of physical training (PT) on estrogen and progesterone levels and expression of their receptors during carcinogenesis induced by N-methyl-N-nitrosourea (MNU) in rats. MATERIALS AND METHODS Fifty female Sprague-Dawley rats were intraperitoneally administered MNU and divided into four groups: low-, moderate-, and high-intensity PT, and no PT (control). Plasma levels of sex hormones and tissue expression of their receptors were quantified and statistically analyzed. RESULTS In the group of rats subjected to PT, a significantly higher progesterone level was observed. The highest progesterone level was noted in the low-intensity PT group. An increase in apoptosis of MNU-induced tumor cells was also demonstrated in the PT groups. CONCLUSION PT stimulates apoptosis of tumor cells without an increase in their proliferative activity. The increase in apoptosis of tumor cells correlates positively with the progesterone level.

The molecular pathogenesis of the development of non-small cell lung carcinomas (NSCLCs) is extremely complex. Understanding the molecular basis of the development of this malignant tumor may enable the use of targeted therapy, which may result in a better treatment outome for these patients. Adenocarcinoma (AC) is the most common NSCLC subtype, equally common among smokers and non-smokers, and its pathogenesis remains unknown. The SOX18 protein is an important protein that plays a role in the development of blood and lymphatic vessels during the process of embryogenesis. Recent studies have also shown that the SOX18 protein may play a significant role in tumors, including lung cancers. In the present study, we analyzed the expression of the SOX18 protein and the mRNA level in postoperative samples of AC and non-malignant lung tissues (NMLTs), and a disparity in both levels was observed. Based on our previous observations that miR-7a and miR-24-3p are able to modulate SOX18 expression in NSCLC, the main aim of this study was to verify the miRNA modulation of the SOX18 transcript with the use of the MirTrap System in established lung cancer cell lines NCI-H1703, NCI-H522 and A549. The SOX18 mRNA expression level was significantly lower in AC than that noted in the NMLTs (P

Multi-drug resistance (MDR) is the main cause of low effectiveness of cancer chemotherapy. P-glycoprotein (P-gp) is one of the main factors determining MDR. Some studies indicate the potential role of melatonin (MLT) in MDR. In this study, we examined the effect of MLT on colon cancer cell’s resistance to doxorubicin (DOX). Using the sulforhodamine B (SRB), method the effect of tested substances on the survival of LoVo (colon cancer cells sensitive to DOX) and LoVoDX (colon cancer cells resistant to DOX) was rated. Using immunocytochemistry (ICC), the expression of P-gp in the LoVo and LoVoDX was determined. With the real-time PCR (RT-PCR) technique, the ABCB1 expression in LoVoDX was evaluated. Based on the results, it was found that MLT in some concentrations intensified the cytotoxicity effect of DOX in the LoVoDX cells. In the ICC studies, it was demonstrated that certain concentrations of MLT and DOX cause an increase in the percentage of cells expressing P-gp, which correlates positively with ABCB1 expression (RT-PCR). The mechanism of overcoming resistance by MLT is probably not only associated with the expression of P-gp. It seems appropriate to carry out further research on the use of MLT as the substance supporting cancer chemotherapy.

Background/aim Melatonin (MLT), through the interaction with membrane melatonin receptors MT1, can improve the effectiveness of cytostatic agents, including cisplatin (CP). The aim of this study was to examine the synergistic effect of MLT and CP in three cell lines: IOSE 364, SK-OV-3 and OVCAR-3, as well as to assess the role of MT1 receptors in this mechanism. Materials and methods Using the SRB assay we investigated the effect of different concentrations of CP and MLT on cell viability. Tests, using luzindole - MT1 inhibitor, allowed us to assess the potential involvement of MT1 in the mechanism of MLT action. Results MLT at certain concentrations demonstrated a synergistic effect in combination with CP. The addition of luzindole did not affect the action of MLT in combination with CP. Conclusion In summary, the synergistic effect of MLT with CP seems to be independent of membrane MT1 receptors.

The risk of breast cancer is related to duration of exposure to sex hormones, especially estrogen. The aim of this study was to assess the impact of physical training (PT) on estrogen and progesterone levels and expression of their receptors during carcinogenesis induced by N-methyl-N-nitrosourea (MNU) in rats.Fifty female Sprague-Dawley rats were intraperitoneally administered MNU and divided into four groups: low-, moderate-, and high-intensity PT, and no PT (control). Plasma levels of sex hormones and tissue expression of their receptors were quantified and statistically analyzed.In the group of rats subjected to PT, a significantly higher progesterone level was observed. The highest progesterone level was noted in the low-intensity PT group. An increase in apoptosis of MNU-induced tumor cells was also demonstrated in the PT groups.PT stimulates apoptosis of tumor cells without an increase in their proliferative activity. The increase in apoptosis of tumor cells correlates positively with the progesterone level.

Major depressive disorder (MDD) is not a single disease, but a number of various ailments that form one entity. Psychomotor retardation, anhedonia, sleep disorders, an increased suicide risk, and anxiety are the main symptoms that often define the clinical diagnosis of depression. Interleukin-6 (IL-6), as one of the proinflammatory cytokines, seems to be overexpressed during certain mental disorders, including MDD. Overexpression of IL-6 in depression is thought to be a factor associated with bad prognosis and worse disease course. IL-6 may directly affect brain functioning and production of neurotransmitters; moreover, its concentration is correlated with certain clinical symptoms within the wide range of depressive symptomatology. Furthermore, there is a strong correlation between IL-6 synthesis and psychosomatic functioning of the patient. This article discusses potential sources and significance of IL-6 in the pathogenesis of depression.

The global epidemic of cardiovascular diseases leads to increased morbidity and mortality caused mainly by myocardial infarction and stroke. Atherosclerosis is the major pathological process behind this epidemic. We designed a novel model of atherosclerosis in swine. Briefly, the first group (11 pigs) received normal pig feed (balanced diet group-BDG) for 12 months, the second group (9 pigs) was fed a Western high-calorie diet (unbalanced diet group-UDG) for 12 months, the third group (8 pigs) received a Western type high-calorie diet for 9 months later replaced by a normal diet for 3 months (regression group-RG). Clinical measurements included zoometric data, arterial blood pressure, heart rate and ultrasonographic evaluation of femoral arteries. Then, the animals were sacrificed and the blood serum, urine and skeletal muscle tissue were collected and 1H NMR based metabolomics studies with the application of fingerprinting PLS-DA and univariate analysis were done. Our results have shown that the molecular disturbances might overlap with other diseases such as onset of diabetes, sleep apnea and other obesity accompanied diseases. Moreover, we revealed that once initiated, molecular changes did not return to homeostatic equilibrium, at least for the duration of this experiment.

Metallothionein-3 (MT-3) has been shown to be expressed in several malignancies and to have an impact on patients’ survival in breast and urinary bladder cancer cases. However, its expression has not been determined in normal skin or in its malignant lesions. MT-3 expression was studied using immunohistochemistry in 17 cases of normal skin, 18 of actinic keratosis (AK), 39 of squamous cell carcinoma (SCC), and 23 of basal cell carcinoma (BCC). Low MT-3 expression was observed in normal skin epidermis with faint or no expression in the epidermis basal layer. Significantly higher MT-3 expression was noted in AK (P = 0.007) and SCC (P

The study aimed to demonstrate the expression of hypoxia-inducible factor (HIF-1α) in soft tissue mesenchymal tumours (fibroma and fibrosarcoma) in dogs. An attempt was made to correlate the obtained results with density of blood vessels (expression of von Willebrand Factor, vWF), expression of Ki-67 proliferation antigen, and with intensity of apoptosis in studied tumours. The study was performed on paraffin sections of 15 fibromas and 40 fibrosarcomas sampled from 55 female dogs aged 6 to 16 years. Immunohistochemical staining against HIF-1α, vWF, and Ki-67 was performed. Apoptosis was detected with the use of TUNEL reaction. A significantly higher HIF-1α expression was noted in fibrosarcomas in comparison to fibromas (P < 0.0001). HIF-1α expression in fibromas manifested strong positive correlation with tumour vascularity (r = 0.67, P = 0.007). Moreover, HIF-1α expression in fibrosarcomas manifested a moderate positive correlation with tumour malignancy grade (r = 0.44, P = 0.004), tumour vascularity (r = 0.52, P < 0.001), Ki-67 antigen expression (r = 0.42; P = 0.007), and TUNELpositive cells (r = 0.37, P = 0.017). Expression of HIF-1α was detected in 86.7% of fibroma type tumours and in 100% of fibrosarcomas. In all studied tumours expression of HIF-1α manifested positive correlation with the density of blood vessels, and in fibrosarcomas it correlated also with malignancy grade, intensity of Ki-67 expression, and with intensity of apoptosis in tumour cells.

Special AT-rich sequence-binding protein 1 (SATB1) is a nuclear matrix protein which interacts with specific regions of DNA, ensuring its proper organization and function in the cell. The expression of SATB1 was primarily found in thymocytes, but its increased levels were observed in various types of cancers. However, the knowledge of the function and application possibilities of this protein is still limited. The aim of this study was to investigate the expression of SATB1 protein using immunohistochemistry and tissue microarray (TMA) technique and determine its possible relationship with the proliferative marker Ki-67, estrogen a (ER) and pro- gesterone (PR) receptors as well as grade of histological malignancy (G). The study was performed on material of 48 archival invasive ductal breast cancers (IDC). The TMAs were prepared with the use of 0.6 mm diameter punches. Immunohistochemical reactions were carried out using antibodies against Ki-67, ER, PR and SATB1 proteins. The intensity of the nuclear reaction was evaluated using a light microscope and computer-assisted image analysis. Expression of Ki-67 and SATB1 protein was observed in 89.58% and 31.25% of cancer cases, respec- tively. 62.5% of tumors were classified as ER-positive, and 47.92% as PR-positive. Statistical analysis showed a moderate positive correlation between Ki-67 and SATB1 expression (r = 0.291, p = 0.045 independently on the receptor status, and r = 0.392, p = 0.032 in ER-negative tumors). The expression of the Ki-67 antigen increased with higher grade of histological malignancy (G). The results suggest that SATB1 protein may play an indirect role in the cell proliferation and should be evaluated in relation to the other markers. Further studies concern- ing determination of its role in cancer progression and metastasis, in terms of application as therapeutic target and prognostic marker, are recommended. (Folia Histochemica et Cytobiologica 2013, Vol. 51, No. 4, 333-338)

Physical exercise and massage are regarded as key factors in regulating tendon structure. However, information on the mechanism through which massage influences the structure and biology of a tendon is scarce. In this study, we attempted to define the impact of these two activities on rat tendons by using morphological and molecular techniques, determining the expression of VEGF-A, FGF-2, and CD34 in the tendons of rats subjected to 10 weeks of physical exercise (running) with massage of varied duration. The group of rats that was trained and massaged during the entire study was characterized by the highest expression of these markers, compared to the rats subjected to massage before training and to the control group subjected to physical exercises only. The greatest significant differences, compared to the control, were noted in the expression of all the studied markers at mRNA level, and in the case of VEGF-A, at protein level, in the third and fifth weeks of the experiment. The results of this study could point to the synergistic impact of simultaneous massage and physical exercise on the expression of angiogenesis markers in rat tendons.

Objectives : We have previously shown that 12 months (mo.) high-fat diet (HFD) in pigs led to pathophysiological alterations, incl. fattening and increased femoral artery intima-media-thickness, which were partly reversed after 3 mo. return to control diet (Zabek et al., PLoS One 2017). The aim of this study was to decipher underlying mechanism of action of these dietary interventions on the arteries by nutrigenomics analyses of intima and media of aorta. Methods : 32 female pigs were divided into 3 groups: Control diet (CD) for 12 mo.; HFD for 12 mo.; 3) Reversal diet group (RD): HFD for 9 mo. followed by CD for 3 mo. After 12 mo. animals were killed and abdominal aorta collected. RNA was isolated from aorta intima and media for whole genome microarray analyses followed by bioinformatics analyses. Results : HFD compared to CD group significantly affected gene expression profile in intima with genes belonging to the chemotaxis, inflammation or endothelial permeability. RD induced gene expression profile was distinct from the CD group. This suggests that 3 mo. of reversal to CD is not sufficient to correct gene expression changes induced by HFD. Comparison of RD profile with that of HFD group revealed a group of genes with opposite expression, e.g. genes regulating inflammation, toll-like cell signaling pathway or cytoskeleton organization involved in the regulation of cell permeability. This suggests that return to the RD only partly restored gene expression alterations due to the HFD. Significant changes in expression of genes in media following HFD were also observed, such as genes involved in cytoskeleton organization and migration MAPK signaling. As for intima, the expression profile of media of pigs on RD was different on that of these on CD diet. Compared to HFD, a group of genes involved in PI3K or MAPK pathways presented opposite expression suggesting that RD can partly correct the changes in genomic effect induced by HFD. Conclusions : This study revealed genomic modifications induced by long-term HFD consumption on arterial intima and media. The return to normal diet for 3 mo. was not sufficient to counteract the genomic effect of long-term HFD consumption. Funding Sources : WROVASC Integrated Cardiovascular Centre, co-financed by the European Regional Development Fund.

Background: The latest immunotherapy, used in the treatment of non-small cell lung cancer (NSCLC), uses monoclonal antibodies directed against programmed death ligand 1 (PD-L1) to inhibit its interaction with the PD-1 receptor. Elevated levels of PD-L1 expression were observed on NSCLC cells. The association between PD-L1 expression and clinicopathological features is still unclear. Therefore, we examined this relationship and also compare PD-L1 expression levels with Ki-67, p63 and TTF-1. Methods: 866 samples of NSCLCs were used to prepare tissue microarrays (TMAs) on which immunohistochemical (IHC) reactions were performed. Changes in the level of CD274 (PD-L1) gene expression in 62 NSCLC tumors were tested in relation to 14 normal lung tissues by real-time PCR reactions (RT-PCR). Results: PD-L1 expression was observed in 32.6% of NSCLCs. PD-L1 expression was increased in higher malignancy grades (G) (p &lt, 0.0001) and in higher lymph node status (pN) (p = 0.0428). The patients with low PD-L1 expression had longer overall survival compared to the group with high expression (p = 0.0332) in adenocarcinoma (AC) only. Conclusions: PD-L1 expression seems to be associated with increased tumor proliferation and aggressiveness as well as shorter patient survival in NSCLC, predominantly in the AC group.

Nogo-B (Reticulon 4B) is considered to be a novel vascular marker, which may have a protective role in injury-induced neointima formation and atherosclerosis. Nogo A/B is found to be crucial for monocyte/macrophage recruitment in acute inflammation and it is expressed in CD68 + macrophages. We hypothesize that macrophage infiltration in atherosclerosis is not dependent on Nogo-B expression in arterial wall. We have assessed Nogo-B expression and macrophage accumulation in the iliac arteries of healthy organ donors and organ donors with cardiovascular risk factors. Paraffin sections of 66 iliac arteries, from 44 deceased organ donors (17 women and 27 men), were studied. The healthy and cardiovascular risk (CVR) subgroups were created. With regard to staging of the atherosclerotic process, the thickness of arterial intima was measured in digitalized images of H+E stained tissue sections. Immunohistochemical reactions (Nogo-B and CD68) were carried out in all arteries (66 samples). Western blotting (WB-19 samples) and real-time PCR (27 samples) were performed on selected arteries. Significantly higher Nogo-B expression was demonstrated in the intima of the healthy subjects' subgroup, using immunohistochemistry. WB and real-time PCR revealed a trend toward lower Nogo-B expression in the adventitia of the CVR subgroup. Furthermore, the thickness of the intima was found to negatively correlate with the expression of Nogo-B in the intima and media (r = -0.32; p < 0.05; r = -0.32; p < 0.05). Macrophage infiltrates were more prominent in intima of CVR subjects (0.65 vs 3.52 a.u.; p < 0.01). Macrophage density in intima increased with atherosclerosis progression (r = 0.37; p < 0.01). CD68 macrophages density in adventitia was lower in CVR arteries than in healthy arteries. The expression of Nogo-B, in arterial intima, is impeded in the early stages of atherosclerosis. Accumulation of arterial intimal CD68 macrophages has been shown to progress; however, the overall macrophage density in the adventitia is reduced in arteries shown to have intimal thickening. Macrophage infiltration is not accompanied by Nogo-B expression in atherosclerotic arteries.

Thirty-two canine mammary tubulopapillary carcinomas and 14 simple adenomas were studied by immunohistochemistry for the expression of UDP-galactose:ceramide galactosyltransferase (UGT8). The majority of tissue specimens (57%) representing adenomas had no or weak reaction with anti-UGT8 antibodies (0-2 pts according to IRS scale) in comparison to the majority of carcinomas (90%) which stained with high intensities (3-9 pts according to IRS scale). When the average values of the reaction intensities (IRS) for malignant and benign tumors were compared, using the Mann-Whitney U-test, significant differences in UGT8 expression between them were found (P < 0.001). Mammary tubulopapillary carcinomas were further analyzed by IHC and the same rabbit polyclonal antibody directed against UGT8 according to their malignancy grade. It was found that the level of UGT8 increased in tumor specimens together with their grading. A comparison of the average values of the reaction intensity (IRS scale) revealed a significant difference (Mann-Whitney U-test, P < 0.05) in UGT8 expression between tumors representing malignancy grades G3 and G1. Based on the obtained results, it is proposed that UGT8 is associated with malignancy of canine mammary gland cells and may have a potential value as a diagnostic marker.

Vaults are cytoplasmic ribonucleoprotein particles composed of three proteins (MVP, TEP1, vPARP) and vault‑associated RNAs (vRNAs). Although the cellular functions of vaults remain unclear, vaults are strongly linked to the development of multidrug resistance (MDR), the major obstacle to the efficient treatment of cancers. Available published data suggest that vaults and their components are frequently upregulated in broad variety of multidrug-resistant cancer cell lines and tumors of different histological origin. Here, we provide detailed analysis of vault protein expression in post-surgery ovarian cancer samples from patients that were not exposed to chemotherapy. Our analysis suggests that vault proteins are expressed in the ovaries of healthy individuals but their expression in cancer patients is changed. Specifically, MVP, TEP1 and vPARP mRNA levels are significantly decreased in cancer samples with tendency of lower expression in higher-grade tumors. The pattern of vault protein mRNA expression is strongly correlated with the expression of other MDR-associated proteins such as MDR1, MRP1 and BCRP. Surprisingly, the protein levels of MVP, TEP1 and vPARP are actually increased in the higher‑grade tumors suggesting existence of post-transcriptional regulation of vault component production.

Metallothioneins (MT) are intracellular, low molecular weight proteins (6–7 kDa) involved in binding of metal ions, scavenging of free radicals, cell proliferation and apoptosis and resistance to certain chemotherapeutics. Four basic families of MT proteins are distinguished: MT-I, MT-II, MT-III, MT-IV, within each of them different isoforms occur. The study aimed at examining the expression level of nine MT isoforms: MT-1A, -1B, -1E, -1F, -1G, -1H, -1X, MT-2A and MT-IV by using real-time PCR and MT-I/II expression by immunohistochemical (IHC) technique in 69 cases of non-small cell lung cancer (NSCLC) and 12 non-malignant lung tissues (NMLT) and to correlate them with patients clinicopathological data and Ki-67 antigen expression. Out of all the analyzed cases, 62 (89.9%) demonstrated an increased MT-I/II expression. MT-1B, 1F, -1G, -1H and MT-1X were significantly up-regulated, whereas MT-1E was significantly down-regulated in NSCLC as compared to NMLT. Only in two cases MT-IV mRNA expression was noted. Significant positive correlations were observed between each particular MT isoform expressions. Higher MT-1F and MT-1A mRNA expression was associated with larger primary tumor size (P = 0.0362 and P < 0.0001, respectively). Moreover, up-regulated MT-1F mRNA expression was associated with higher grade of malignancy of NSCLC (P = 0.0085). Higher MT-1B mRNA expression was associated with squamocellular and adenocarcinoma subtype of NSCLC (P = 0.0358). Univariate analysis showed, that up-regulated MT-1F and MT-2A mRNA predicted poor patients' survival (P = 0.0206 and P = 0.0097, respectively). The levels of MT-1F and MT-2A mRNA could be considered as new markers of poor prognosis of NSCLC patients.

The aim of this study was to determine the intensity of Hsp-27 protein expression in fibrocystic breast changes (FC) and invasive ductal breast carcinoma (IDC) and to examine its impact on patients’ clinico-pathological characteristics and overall survival. Immunohistochemical reactions were conducted on archival samples of 20 cases of FC and 101 cases of IDC treated in the years 1999-2002. Nuclear-cytoplasmic Hsp-27 expression was observed in 92 (92.1%) of the examined cases of IDC and all the cases of FC. Significantly higher Hsp-27 expression was observed in G2 (p

Background One of the most important prognostic indicators in gastric cancer is the presence of metastases in lymph nodes. Even now, little is known about lymphangiogenesis in neoplastic tissue, and little is also known about the transmission of a neoplastic cell from the tumor mass into a lymphatic vessel. Methods This study examined the relationships between the density of lymphatic vessels (LVD) stained immunohistochemically with lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and D2-40 (podoplanin) antibodies, the expression of vascular endothelial growth factor (VEGF)-C/D, selected clinical and pathomorphological factors, and the 5-year overall survival of gastric cancer patients. Results Statistical analysis showed no impact of increased intratumoral or peritumoral LVD on gastric cancer patient survival, irrespective of the protein used to stain lymphatic vessels. Analysis showed that the probability of overall survival was decreased in the cases with enhanced VEGF-D immunoreactivity (P = 0.0045). Conclusion The study showed that the studied markers cannot be used to determine the required extent of the surgical procedure, as they have no statistically significant correlation with the degree of progression of the cancer, the stage of the disease assessed according to the TNM 5th classification of malignant tumors, clinicopathological features, and patient survival. VEGF-D is the only marker that can be regarded as an unfavorable prognostic indicator for patients with advanced gastric cancer.

Kidney surface cooling was used during implantation to assess the effect of warm ischemia elimination on allograft function, histological changes and immune-related gene expression. 23 recipients were randomly assigned to a group operated on with kidney surface cooling during implantation (ice bag technique, IBT group), and the other 23 recipients receiving the contralateral kidney from the same donor were operated on with a standard technique. Three consecutive kidney core biopsies were obtained during the transplantation procedure: after organ recovery, after cold ischemia and after reperfusion. Gene expression levels were determined using low-density arrays (Format 32, TaqMan). The IBT group showed a significantly lower rate of detrimental events (delayed graft function and/or acute rejection, p = 0.015) as well as higher glomerular filtration rate on day 14 (p = 0.026). A greater decrease of MMP9 and LCN2 gene expression was seen in the IBT group during total ischemia (p = 0.003 and p = 0.018). Elimination of second warm ischemia reduced the number of detrimental events after kidney transplantation, and thus had influence on the short-term but not long-term allograft function. Surface cooling of the kidney during vascular anastomosis may reduce some detrimental effects of immune activation resulting from both brain death and ischemia-reperfusion injury.

The aim of this study was to investigate the role of lymphangiogenesis in the clinical progression and outcome of mycosis fungoides. Immunohistochemistry and Western blot techniques were used to assess the expression of podoplanin and vascular endothelial growth factor C in mycosis fungoides. Expression of vascular endothelial growth factor C measured by immunohistochemistry was significantly higher in mycosis fungoides samples in comparison with control cases (chronic benign dermatoses) (p = 0.0012). Increased expression of podoplanin was found in advanced vs. early mycosis fungoides (p

UNLABELLED: Long-term potentiation (LTP) is widely perceived as a memory substrate and in the hippocampal CA3-CA1 pathway, distinct forms of LTP depend on NMDA receptors (nmdaLTP) or L-type voltage-gated calcium channels (vdccLTP). LTP is also known to be effectively regulated by extracellular proteolysis that is mediated by various enzymes. Herein, we investigated whether in mice hippocampal slices these distinct forms of LTP are specifically regulated by different metalloproteinases (MMPs). We found that MMP-3 inhibition or knock-out impaired late-phase LTP in the CA3-CA1 pathway. Interestingly, late-phase LTP was also decreased by MMP-9 blockade. When both MMP-3 and MMP-9 were inhibited, both early- and late-phase LTP was impaired. Using immunoblotting, in situ zymography, and immunofluorescence, we found that LTP induction was associated with an increase in MMP-3 expression and activity in CA1 stratum radiatum. MMP-3 inhibition and knock-out prevented the induction of vdccLTP, with no effect on nmdaLTP. L-type channel-dependent LTP is known to be impaired by hyaluronic acid digestion. We found that slice treatment with hyaluronidase occluded the effect of MMP-3 blockade on LTP, further confirming a critical role for MMP-3 in this form of LTP. In contrast to the CA3-CA1 pathway, LTP in the mossy fiber-CA3 projection did not depend on MMP-3, indicating the pathway specificity of the actions of MMPs. Overall, our study indicates that the activation of perisynaptic MMP-3 supports L-type channel-dependent LTP in the CA1 region, whereas nmdaLTP depends solely on MMP-9. SIGNIFICANCE STATEMENT: Various types of long-term potentiation (LTP) are correlated with distinct phases of memory formation and retrieval, but the underlying molecular signaling pathways remain poorly understood. Extracellular proteases have emerged as key players in neuroplasticity phenomena. The present study found that L-type calcium channel-dependent LTP in the CA3-CA1 hippocampal projection is critically regulated by the activity of matrix metalloprotease 3 (MMP-3), in contrast to NMDAR-dependent LTP regulated by MMP-9. Moreover, the induction of LTP was associated with an increase in MMP-3 expression and activity. Finally, we found that the digestion of hyaluronan, a principal extracellular matrix component, disrupted the MMP-3-dependent component of LTP. These results indicate that distinct MMPs might act as molecular switches for specific types of LTP. ispartof: Journal of Neuroscience vol:37 issue:5 pages:1240-1256 ispartof: location:United States status: published

The metallothionein 1 (MT1) coding sequence of red deer was identified and compared to orthologous sequences from other mammals. Over 90% identity was observed between red deer MT1 amino acid sequence and MT1 sequences of other ruminants. Liver and kidney samples of red deer were collected from the industrial zinc smelting site of Miasteczko Slaskie and from the Masuria Lake District serving as a pollution-free control site. The concentrations of cadmium (Cd), lead (Pb), copper (Cu) and zinc (Zn) were analyzed by the atomic absorption spectrometry technique (AAS). The levels of Cd in the liver of red deer from the metal smelting region was about 8 times higher than for the reference control site. Next, the expression of MT1 mRNA in the liver of red deer was quantified by the reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and the expression of MT1/2 protein in the liver and kidneys was analyzed by immunohistochemistry. Positive correlations were found between expression levels for MT1 mRNA and the concentrations of Cu and Zn in liver of red deer, and with the age of animals. Immunohistochemical staining demonstrated the nuclear and cytoplasmatic expression in both liver and kidney tissues, but with no obvious relationship shown for the expression of MT1/2 protein and tissue metal levels. Our results showed that the analysis of MT expression levels in the red deer could not be used independently as a biomarker for identifying exposure to Cd, but could be co-analyzed with tissue metal levels to give better prognosis for environmental exposure to metals.

Introduction. Cytokeratin-19 (CK19) is recognized as a reliable tumor marker of papillary thyroid carcinoma (PTC). The general prognosis in the classical subtype of PTC (CSTPTC) remains favorable, but some cases can be very aggressive. The aim of this study was to evaluate the localization and intensity of immunohistochemical CK19 expression in CSTPTC and its associations with the clinical and pathological characteristics of patients with CSTPTC in the Silesian region. Material and methods. All the available clinical and histopathological data for 149 patients with CSTPTC from the Silesian region were retrospectively analyzed. The group consisted of 135 (90.6%) women and 14 (9.4%) men (mean age and SD: 52.3 ± 15.0). All these patients with CSTPTC underwent surgery at the same center between 2008 and 2013; the follow-up period was 24 to 90 months (mean and SD: 47 ± 20). Results. In 142 (95.3%) of the patients with CSTPTC, positive cytoplasmic staining of CK19 was found. A higher expression of CK19 was observed in the group of patients without the recurrence of the disease (p = 0.015). CK19 expression was not associated with age, gender, tumor focality, disease stage, tumor size (pT), lymph node involvement (pN), or distant metastases (pM). Conclusions. Decreased CK19 expression in CSTPTC cases with relapse suggests that it plays a role in the carcinoma progression of CSTPTC. The association between lower CK19 expression and patients’ unfavorable postoperative course could suggest its possible role as a marker of CSTPTC poor prognosis.

Metallothioneins (MTs) are low molecular weight proteins, which are present in almost all types of organisms. In mammals, four main MT isoforms designated from MT-1 to MT-4 were identified. Their biological role, according to their characteristic structure, was shown to be mostly associated with cellular metabolism of metal ions, especially zinc. Moreover, the available evidence suggests broad regulatory properties of MTs in the control of cell senescence and various pathological processes including neurodegeneration, cardiovascular pathology, metabolic disorders, and various malignancies. This extensive review provides general in formation on the structure of MT family members and the cellular functions of MT-1, MT-2, and MT-4 isoforms as well as insights into divergent biological roles of MT-3. Due to the involvement of MT molecules in various processes related to carcinogenesis, an organ-specific presentation of current data concerning their potential impact on the progression of various tumors is given. The regulatory role of MT family members in the function of the immune system is also discussed in depth.

Early studies analyzing the expression of MTs in particular organs had already suggested the existence of a tissue-specific repertoire of MT proteins (Chakrabarty and Maiti 1985). MTs were also detected in immune organs, including the spleen, the bone marrow, and the thymus (Savino et al. 1984; Huber and Cousins 1993). In the human thymus, MTs were found to colocalize with thymulin on the cellular level (Savino et al. 1984). The biological activity of thymulin—a metallopeptide known to be one of the main regulators of immune cell development and function (Safieh-Garabedian et al. 2012)—strongly depends on zinc ion binding (Dardenne et al. 1982), which suggests a potential role of MTs as zinc donors in the control of active thymulin secretion. The expression of both the thymic and the bone marrow MT-1 and MT-2 genes has been shown to respond positively to inflammatory stimuli, such as IL-1, and to correlate with increased zinc uptake in these immune organs (Cousins and Leinart 1988). Multiple factors, including metal ions and proinflammatory substances, such as bacterial products (lipopolysaccharide, LPS) and mitogens (concanavalin A, Con A), have been demonstrated to stimulate MT expression in human peripheral blood immune cells, both directly and by inducing the secretion of other inflammatory mediators (Oberbarnscheidt et al. 1988; Vandeghinste et al. 2000). The basal and induced expression of MTs in immune cells has been shown to be dependent on the cell type, with monocytes expressing more MTs than lymphocytes and granulocytes (Harley et al. 1989; Yurkow and DeCoste 1999). In the lymphocytic population, cadmium-induced MT levels determined by a specific antibody and flow cytometry were shown to be higher in CD4+ and CD8+ T cells than in B cells and natural killer (NK) cells (Yurkow and Makhijani 1998). More detailed analyses performed on human peripheral blood lymphocytes have shown the expression of genes of various MT-isoforms, including MT-1A, MT-1E, MT-1F, MT-1G, MT-1H, MT-1X, MT-2A, and MT-3. The presence of MT-1A, MT-1E, MT-1F, MT-1G, MT-1H, MT-1X, MT-1K, and MT-2A has also been confirmed on the protein level (Vandeghinste et al. 2000).

All metallothioneins (MTs) possess a highly conserved amino acid sequence and present only a few structural changes even when isolated from different animal species. In mammals, a single MT molecule is made up of 61–68 amino acids, depending on the isoform (the MT-1, MT-2, and MT-4 isoforms consist of 61–62 amino acids, whereas the MT-3 isoform comprises 68 amino acids), and the protein sequence is composed of up to 20 cysteine (Cys) residues (Vasak 2005; Vasak and Meloni 2011). Furthermore, in mammals, no aromatic amino acids are found in the MT molecules. Protein sequencing has revealed that the MT molecule is a single polypeptide chain, in which the Cys residues are organized in the sequences Cys-X-Cys, Cys-X-X-Cys, and Cys-Cys, where “X” denotes an amino acid other than Cys (Kojima et al. 1976; Huang and Yoshida 1977). The Cys residues are the metal-binding domains of the MT molecule, in which they are juxtaposed with lysine (Lys) and arginine (Arg) amino acid residues and arranged in two thiol-rich sites designated domains α and β (Fig. 2.1). The two metal-binding domains are separated by a non-cysteine-containing sequence often designated as the spacer or linker (Zangger et al. 2001; Babula et al. 2012). The α-domain consists of amino acids 31–68 and is located on the C-terminal edge, whereas the N-terminal β-domain contains amino acids 1–30 (Zangger et al. 2001; Dziegiel 2004). It has been demonstrated that the α-domain is capable of binding up to four, and the β-domain up to three, bivalent metal ions such as zinc, cadmium, mercury, or lead (Coyle et al. 2002b; Duncan et al. 2006). The part of the protein with no bound metal ions is termed apo-metallothionein (apo-MT) or thionein (Coyle et al. 2002b). Metallothioneins are also capable of reacting with up to 12 univalent metal ions (Palmiter 1998; Coyle et al. 2002b). Zinc ions, which naturally occur in the organism, are regarded as the main binding partner of apo-MT. However, other nonessential metal ions occurring pathologically in the organism—such as lead, copper, cadmium, mercury, platinum, chromate, bismuth, and silver—often possess higher affinity to the apo-MT-binding sites (Nordberg and Nordberg 2000; Ngu and Stillman 2009; Ngu et al. 2010b; Gumulec et al. 2011; Babula et al. 2012). So far, only iron ions (Fe2+) have been identified to possess lower affinity to the metal-binding sites of the apo-MT domains (Foster and Robinson 2011). Interestingly, only a small proportion of MT molecules was found bound to zinc ions in various organisms. In rat tissues, apo-MT has been shown to constitute up to 54 % of the total amount of MT, whereas higher apo-MT levels were detected in rat cancer cells (Yang et al. 2001). Recent studies have also identified small amounts of sulfide ligands bound to recombinant MT-1 and MT-4 proteins overexpressed in Escherichia coli (Capdevila et al. 2005; Tio et al. 2006). Nevertheless, studies analyzing MT proteins in the cytoplasm of mammalian cells have failed to detect sulfide ligands bound to their molecules (Mounicou et al. 2010).

Due to their role in various cellular processes, metallothioneins have been studied in numerous benign and malignant lesions, in regard to carcinogenesis, and in tumor progression (Dziegiel 2004; Krizkova et al. 2009b, 2012; Pedersen et al. 2009; Pula et al. 2012; Fic et al. 2013). Although the role of MTs in tumor initiation and progression has been intensely studied, it is only recently that studies have allowed the elucidation of the tumor suppressory roles of some MT isoforms—such as of MT-1G in papillary thyroid cancer and the large intestine and MT-1E in melanomas (Ferrario et al. 2008; Faller et al. 2010; Arriaga et al. 2014). On the other hand, MT-2A has been shown to promote the progression of breast and non-small cell lung cancer (Jin et al. 2002; Lim et al. 2009; Lai et al. 2010; Werynska et al. 2013b). Lines of evidence indicate that MTs exert divergent expression patterns in normal tissue and tumors; for this reason, more detailed studies concentrated on each isoform, and not on MT-1 and MT-2 collectively, need to be conducted. Nevertheless, the results of many studies have identified the involvement of MTs in cancer cell proliferation (Jin et al. 2002), apoptosis (Krizkova et al. 2009b), invasiveness, and migration (Jin et al. 2001; Kim et al. 2011; Ryu et al. 2012), as well as on cancer cells sensitivity to various anticancer agents (Kondo et al. 1995a, b, 1997). In this chapter, the role of MT isoforms in the development and progression of various tumors, with special emphasis on their prognostic significance, will be summarized in an organ-specific manner.

Renal transplant candidates present immune dysregulation, caused by chronic uremia. The aim of the study was to investigate whether pretransplant peripheral blood gene expression of immune factors affects clinical outcome of renal allograft recipients.Methods. In a prospective study, we analyzed pretransplant peripheral blood gene expression in87 renal transplant candidates with real-time PCR on custom-designed low density arrays (TaqMan).Results. Immediate posttransplant graft function (14-day GFR) was influenced negatively byTGFB1(P=0.039) and positively byIL-2gene expression (P=0.040). Pretransplant blood mRNA expression of apoptosis-related genes (CASP3,FAS,andIL-18) and Th1-derived cytokine geneIFNGcorrelated positively with short- (6-month GFRCASP3:P=0.027,FAS:P=0.021, andIFNG:P=0.029) and long-term graft function (24-month GFRCASP3:P=0.003,FAS:P=0.033,IL-18:P=0.044, andIFNG:P=0.04).Conclusion. Lowered pretransplant Th1-derived cytokine and apoptosis-related gene expressions were a hallmark of subsequent worse kidney function but not of acute rejection rate. The pretransplantIFNGandCASP3andFASandIL-18genes’ expression in the recipients’ peripheral blood is the possible candidate for novel biomarker of short- and long-term allograft function.

The benefits of plant polyphenols as chemotherapeutic agents are of great interest due to their possible anti-cancerogenic activities. Results available up to now suggest that flavonoid quercetin induces lethal effect in many types of tumours and may sensitize resistant cells to drugs. The aim of our study was to examine the effect of quercetin on human gastric carcinoma cells and to determine mode of its action. Parental EPG85-257P cell line and its daunorubicin-resistant variant EPG85-257RDB were used as cell models. Our data revealed that quercetin exerted antiproliferative impact on studied cells (with IC(50) value of 12 μM after 72 h), mainly through induction of apoptosis. In sensitive cells cytostatic drug and flavonoid had synergistic effects, in EPG85-257RDB cells quercetin acted as a chemosensitizer. Its impact on resistance mechanism involved decrease of P-glycoprotein expression, inhibition of drug transport and downregulation of ABCB1 gene expression. The results demonstrate that quercetin may be considered as a prospective drug to overcome classical resistance in gastric cancer cells.

International audience; A good correlation between the expression of mucin1 (MUC1) and T antigen was found in breast cancer tumors and breast cancer cell lines, especially after treatment with neuraminidase. The association between the appearance of T antigen and the overexpression of MUC1 was further confirmed by transfecting MDA-MB-231 cells and murine 4T1 mammary carcinoma cells with cDNA for MUC1 and using an RNAi approach to inhibit the expression of MUC1 gene in T47D cells. Furthermore, we discovered that in 4T1 cells which express the sialyl Le(X) antigen, overexpression of MUC1 caused not only appearance of T antigen, but also loss of the sialyl Le(X) structure. As the observed changes in O-glycan synthesis can be associated with changes in the expression of specific glycosyltransferases, core 1 β1,3-galactosyltransferase, core 2 β1,6-N-acetylglucosaminyltransferase (C2GnT1) and β-galactoside α2,3-sialyltransferase (ST3Gal I), we studied their expression in parental, vector-transfected and MUC1-transfected MDA-MB-231 and 4T1 cells as well as T47D cells transduced with small hairpin RNA targeted MUC1 mRNA. It was found that the expression of C2GnT1 and ST3Gal I is highly decreased in MUC1-expressing MDA-MB-231 and 4T1 cells and increased in T47D cells with suppressed expression of MUC1. Therefore, we found that changes in the structure of O-linked oligosaccharides, resulting in the occurrence of T antigen, are at least partially associated with MUC1 overexpression which down-regulates the expression of C2GnT1 and ST3Gal I. We showed also that the overexpression of MUC1 in 4T1 cells changes their adhesive properties, as MUC1-expressing cells do not adhere to E-selectin, but bind galectin-3.

Background The aim of the study was to demonstrate the immunohistochemical expression of proteins that affect the metastatic potential of a tumour, including matrix metalloproteinase 2 (MMP-2) and E-cadherin. Another objective was to determine their correlation with the expression of the Ki-67 antigen in metastasizing and non-metastasizing mammary carcinomas in female dogs. The study was conducted on 32 canine mammary carcinomas (12 metastatic and 20 non-metastatic), classified as simple tubular and tubulopapillary carcinomas. Immunohistochemistry was performed to evaluate the expression of MMP-2, E-cadherin and Ki-67 antigen. Results MMP-2 was expressed in 85 % of the non-metastatic tumours and in all the metastatic tumours, while E-cadherin was expressed in 85 % of the non-metastatic tumours and in 66 % of the metastatic tumours. The Ki-67 antigen was expressed in 65 % of the non-metastatic tumours and in 91 % of the metastatic tumours. The mean Ki-67 expression was slightly higher in tumours that had metastasized (1.5 ± 0.90 vs 1.1 ± 0.94; p = 0.22). A similar relationship was found in terms of the intensity of the MMP-2 expression (2.9 ± 1.9 vs 2.7 ± 2.4; p = 0.50). A decrease in the expression of E-cadherin (2.8 ± 2.5) was found in metastatic tumours compared to the expression in non-metastatic tumours (3.2 ± 2.3). However, these differences were not statistically significant (p = 0.63). Conclusion We did not show significant differences in MMP-2, E-cadherin and Ki-67 expression between metastatic and non-metastatic tumours due to low number of cases studied, however further experiments are necessary to assess the role of these antigens in the process of canine mammary tumours metastasis.

Pula B, Jethon A, Piotrowska A, Gomulkiewicz A, Owczarek T, Calik J, Wojnar A, Witkiewicz W, Rys J, Ugorski M, Dziegiel P & Podhorska-Okolow M (2011) Histopathology 59, 1249–1260 Podoplanin expression by cancer-associated fibroblasts predicts poor outcome in invasive ductal breast carcinoma Aims: It has recently been shown that podoplanin, a mucin-type glycoprotein, is expressed by cancer cells and cancer-associated fibroblasts (CAFs), and promotes cancer cell migration and invasiveness. The biological role of podoplanin expression in tumour stroma of invasive ductal carcinoma of the breast (IDC) has not been determined. Methods and results: Podoplanin expression was analysed in 117 cases of IDC and 27 cases of fibrocystic change, as well as in breast cancer cell lines, with the use of immunohistochemistry and real-time polymerase chain reaction. In 82.1% of analysed tumours, podoplanin was found only in CAFs. Only two of 117 IDC cases (1.7%) were characterized by expression of this glycoprotein in cancer cells. None of the fibrocystic changes or stroma surrounding normal ducts showed podoplanin expression. Podoplanin-positive CAFs correlated with tumour size (P = 0.0125), grade of malignancy (P = 0.0058), lymph node metastasis (P = 0.0149), lymphovascular invasion (LVI) (P = 0.0486) and Ki67 expression in cancer cells (P = 0.0128). High-level podoplanin expression (>50% of positive stroma) in the tumour stroma was significantly associated with a negative oestrogen status (P = 0.0201). Univariate, but not multivariate, analysis showed that podoplanin expression by CAFs was associated with poor patient outcome (P = 0.0202). Conclusions: Our results suggest that podoplanin expression by CAFs could be an unfavourable prognostic marker for IDC.

There are few experimental models of heart failure (HF) in large animals, despite structural and functional similarities to human myocardium. We have developed a porcine model of chronic tachycardia-induced cardiomyopathy.Homogenous siblings of White Large breed swine (n=6) underwent continuous right ventricular (RV) pacing at 170 bpm; 2 subjects served as controls. In the course of RV pacing, animals developed a clinical picture of HF and were presented for euthanasia at subsequent stages: mild, moderate and end-stage HF. Left ventricle (LV) sections were analyzed histologically and relative ANP, BNP, phospholamban and sarcoplasmic reticulum calcium ATPase 2a transcript levels in LV were quantified by real time RT-PCR.In the course of RV pacing, animals demonstrated reduced exercise capacity (time of running until being dyspnoeic: 6.6 ± 0.5 vs. 2.4 ± 1.4 min), LV dilatation (LVEDD: 4.9 ± 0.4 vs. 6.7 ± 0.4 cm), impaired LV systolic function (LVEF: 69 ± 8 vs. 32 ± 7 %), (all baseline vs. before euthanasia, all p0.001). LV tissues from animals with moderate and end-stage HF demonstrated local foci of interstitial fibrosis, congestion, cardiomyocyte hypertrophy and atrophy, which was not detected in controls and mild HF animals. The up-regulation of ANP and BNP and a reduction in a ratio of sarcoplasmic reticulum calcium ATPase 2a and phospholamban in failing myocardium were observed as compared to controls.In pigs, chronic RV pacing at relatively low rate can be used as an experimental model of HF, as it results in a gradual deterioration of exercise tolerance accompanied by myocardial remodeling confirmed at subcellular level.

Fatalities due to mushroom poisonings are increasing worldwide, with high mortality rate resulting from ingestion of amanitin-producing species. Intoxications caused by amanitin-containing mushrooms represent an unresolved problem in clinical toxicology since no specific and fully efficient antidote is available. The objective of this study was a comparative evaluation of benzylpenicillin (BPCN), acetylcysteine (ACC) and silibinin (SIL) as an antidotes in human hepatocytes intoxicated with alpha-amanitin (alpha-AMA). All experiments were performed on cultured human hepatocytes. Cytotoxicity evaluation of cultured cells using MTT assay and measurement of lactate dehydrogenase (LDH) activity was performed at 12, 24 and 48h of exposure to alpha-AMA and/or antidotes. The significant decline of cell viability and significant increase of LDH activity were observed in all experimental hepatocyte cultures after 12, 24 and 36h exposure to alpha-AMA at concentration 2microM. Exposure of the cells to alpha-AMA resulted also in significant reduction of cell spreading and attachment. However, addition of tested antidotes to experimental cultures significantly stimulated cell proliferation and attachment. In cell cultures exposed simultaneously to alpha-AMA and tested antidotes cytotoxic parameters (MTT and LDH) were not significantly different from control incidences. The cytoprotective effect of all antidotes was not dose-related, which reflects a high efficacy of all these substances. Administration of studied antidotes was not associated with any adverse effects in hepatocytes. The administration of ACC, BPCN or SIL to human hepatocyte cultures showed a similar strong protective effect against cell damage in alpha-AMA toxicity.