Piotr Dziegiel

Expression of melatonin receptor MT1 in cells of human invasive ductal breast carcinoma

In humans, two main types of membrane melatonin receptors have been identified, MT1 and MT2. Expression of MT1 in neoplastic cells seems to increase the efficacy of melatonins oncostatic activity. The purpose of this study was to determine the distribution and the intensity of MT1 expression in breast cancer cells and to correlate it with clinicopathological factors. Immunohistochemical studies (IHC) were conducted on 190 cases of invasive ductal breast carcinomas (IDC) and molecular studies were performed on 29 cases of frozen tumor fragments and selected breast cancer cell lines. Most of the studied tumors manifested a membranous/cytoplasmic IHC expression of MT1. In IDC, the MT1 expression was higher than in fibrocystic breast disease. MT1 expression was higher in estrogen receptor positive (ER+) and HER2 positive (HER2+) tumors. Triple negative tumors (TN) manifested the lowest MT1 expression level. The lowest MT1 protein expression level was noted in the TN breast cancer cell line MDA‐MB‐231 compared with ER+ cell lines MCF‐7 and SK‐BR‐3. MT1 mRNA expression was negatively correlated with the malignancy grade of the studied IDC cases. Moreover, higher MT1 expression was associated with patients longer overall survival (OS) in the group of ER+ breast cancers and treated with tamoxifen. Multivariate analysis indicated that MT1 was an independent prognostic factor in the ER+ tumors for OS and event‐free survival in the ER+ tumors. The results of this study may point to a potential prognostic and therapeutic significance of MT1 in IDC.

Development of porcine model of chronic tachycardia-induced cardiomyopathy

BACKGROUNDnThere 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.nnnMETHODSnHomogenous 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.nnnRESULTSnIn 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 p<0.001). 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.nnnCONCLUSIONSnIn 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.

Impact of SOX18 expression in cancer cells and vessels on the outcome of invasive ductal breast carcinoma.

PurposeSOX18 is a transcription factor known to be involved in hair follicle, blood and lymphatic vessel development, as well as wound healing processes (together with SOX7 and SOX17). In addition, it has been reported that SOX18 may affect the growth of cancer cells in vitro. Until now, the exact role of SOX18 expression in invasive ductal breast carcinoma (IDC) has remained unknown.MethodsIn this study, we have investigated SOX18 expression in cancer cells and endothelial cells in 122 IDC samples using immunohistochemistry (IHC). SOX18 expression was also determined using real-time PCR and Western blotting in a series of breast cancer-derived cell lines (i.e., MCF-7, BT-474, SK-BR-3, MDA-MB-231, BO2).ResultsUsing IHC, we observed SOX18 nuclear expression in cancer cells, as well as in blood and lymphatic vessels of the IDC samples tested. SOX18 expression in the IDC samples correlated with a higher malignancy grade (Grade 2 and Grade 3 versus Grade 1; pu2009=u20090.02 and pu2009=u20090.009, respectively) and VEGF-D expression (ru2009=u20090.27, pu2009=u20090.007). SOX18 expression was also associated with HER2 positivity (pu2009=u20090.02). A significantly higher SOX18 expression was found in the HER2-positive cell line BT-474, and a significantly lower expression in the triple negative cell lines MDA-MB-231 and BO2. Laser capture microdissection of IDC samples revealed significantly higher mRNA SOX7, SOX17 and SOX18 expression levels in the vessels as compared to the cancer cells (pu2009=u20090.02 and pu2009=u20090.0002, pu2009<u20090.0001, respectively). SOX18 positive intratumoral and peritumoral microvessel counts (MVC) were associated with higher malignancy grades (pu2009=u20090.04 and pu2009=u20090.02, respectively). Moreover, peritumoral SOX18 positive MVC were found to act as an independent marker for a poor prognosis (pu2009=u20090.04).ConclusionSOX18 expression may serve as a marker for a poor prognosis in IDC.

Expression of the MT1 Melatonin Receptor in Ovarian Cancer Cells

Ovarian cancer (OC) is the leading cause of death among women with genital tract disorders. Melatonin exhibits oncostatic properties which it may effect through binding to its membrane receptor, MT1. The aim of this study was to determine the expression of MT1 in OC cells and to correlate this with clinical and pathological data. Immunohistochemistry was performed on 84 cases of OC. Normal ovarian epithelial (IOSE 364) and OC (SK-OV-3, OVCAR-3) cell lines were used to examine the MT1 expression at protein level using the western blot and immunofluorescence technique. The expression of MT1 was observed as cytoplasmic-membrane (MT1CM) and membrane (MT1M) reactions. A positive correlation between MT1CM and MT1M was found in all the studied cases. There were no significant differences between the expression of MT1CM, MT1M, and histological type, staging, grading, presence of residual disease, or overall survival time. Immunofluorescence showed both MT1M and MT1CM expression in all the tested cell lines. Western blot illustrated the highest protein level of MT1 in IOSE 364 and the lowest in the OVCAR-3. The results indicate the limited prognostic significance of MT1 in OC cells.

Hsp-27 Expression in Invasive Ductal Breast Carcinoma

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<0.01) and G3 cases (p<0.0001) as compared to FC. HER-2 positive cases had higher Hsp-27 expression (p=0.0153), than HER-2 negative cases. Our research showed that Hsp-27 could have a impact on tumour malignancy. Moreover, the positive correlation between expression of Hsp-27 and HER-2 positive cases was demonstrated.

Myocarditis in dogs: etiology, clinical and histopathological features (11 cases: 2007–2013)

BackgroundMyocarditis is a disease caused by numerous etiological factors and characterized by a non-specific course. The only method allowing for precise characterization of inflammatory changes is the histopathological examination of heart muscle specimens. The study was conducted on heart muscle preparations from 11 dogs with ante-mortem diagnosis of cardiac disease. Animals presented with a poor response to an applied treatment or had suspected sudden cardiac death. The heart specimens were taken post-mortem, preserved and stained with haematoxylin and eosin. Subsequently, the presence and intensity of changes, i.e. inflammatory infiltration, the amount of connective tissue and features of cardiomyocyte degeneration were estimated. The specimens from dogs suspected of having a myocarditis of bacteriological etiology underwent additional bacteriological and immunohistochemical examination.ResultsThe examination revealed an inflammatory infiltration of variable intensity combined with the degenerative changes in all dogs. There were vegetative and abnormal cystic forms of Borrelia burgdorferi sensu lato in 6 dogs. A Staphylococcus aureus infection was confirmed in one dog and an acute coronary syndrome with neutrophil infiltration was revealed in another one.ConclusionsAlthough the clinical pattern in patients with myocarditis is diverse, the definitive morphological diagnosis is made based on the histopathological examination. This examination can lead to a better understanding of the pathogenesis of the disease. To the best of our knowledge, this is the first description of myocarditis combined with the presence of spore forms of Borrelia burgdorferi sensu lato in the heart specimens of dogs.

Metallothionein 3 Expression in Normal Skin and Malignant Skin Lesions

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 (Pu2009=u20090.007) and SCC (Pu2009<u20090.0001), as compared with normal skin epidermis. BCC cases were characterized by the lowest MT-3 expression of all the examined groups, which was significantly lower in comparison to normal skin epidermis, AK, and SCC (Pu2009=u20090.009; Pu2009<u20090.0001 and Pu2009<u20090.0001, respectively). In conclusion, MT-3 may be involved in the development of SCC.

Increased skeletal muscle expression of VEGF induced by massage and exercise

INTRODUCTIONnNumerous investigations have been carried out to describe the role of massage in preparing for and restoring efficiency after physical exercise. Furthermore, vascular endothelial growth factor (VEGF) enhances blood vessel growth, and in effect contributes to the regeneration of tissues. Since its expression in active skeletal muscles has not been yet determined, the aim of this study was to investigate whether muscle massage performed before and during running exercise affects the expression of VEGF-A in muscles.nnnMATERIAL AND METHODSnThe study was carried out on 75 adult Buffalo rats subjected to running exercise training for 10 weeks. Rats were massaged prior (group PM) or during exercise (group M) or were not massaged (group C). The massage consisted of spiral movements along the plantar surface of flexor digitorum brevis muscle. After 1, 3, 5, 7 and 10 week of training, five rats from every group were anesthetized and immunohistochemistry, Western blot, and PCR analyses were performed on obtained muscle tissue to determine VEGF-A expression.nnnRESULTSnAfter the first week of training, a significant increase of VEGF-A gene expression analyzed by qPCR in muscle tissue was observed in the PM group, whereas in the third week, the predominant growth of studied marker was seen in the M group. Increased VEGF-A expression on the protein level was observed in both massaged groups following the first week. A moderate positive correlation was found between the expression of the VEGF-A gene and protein in all experimental groups (r = 0.389).nnnCONCLUSIONnShort-term repeated massage may contribute to processes of creation of new and development of already existing vascular networks in the skeletal muscle tissue during increased exercise.

Distribution of subpopulations of dendritic cells in peripheral blood of patients treated with exogenous thyrotropin

BackgroundDendritic cells (DCs) play a major role as regulators of inflammatory events associated with thyroid pathology. The immunoregulatory function of DCs depends strongly on their subtype, as well as maturation and activation status. Numerous hormonal factors modulate the immune properties of DCs, however, little is known about effects exerted by the hypothalamus-pituitary-thyroid-axis. Recently, we have shown a direct regulatory influence of thyroid hormones (TH) on human DCs function. The aim of the present study was to analyze the effect of systemically administered thyrotropin (TSH) on human blood DCs ex vivo.MethodsBlood samples for the cytometric analysis of peripheral blood plasmacytoid and myeloid DCs subtypes were collected from patients subjected to total thyroidectomy because of differentiated thyroid carcinoma at 2 time points: (i) directly before the commencement of TSH administration and (ii) 5u2009days after first TSH injection. The whole blood quantitative and phenotypic analysis of plasmacytoid and myeloid DCs subtypes was performed by flow cytometry.ResultsAdministration of TSH did not influence the percentage of plasmacytoid DCs in peripheral blood of study participants. Also the percentage of the two main myeloid DCs subpopulations – CD1c/BDCA1+ DCs and CD141/BDCA3+ DCs did not change significantly. TSH administration had no effect on the surface expression of CD86 – one of the major costimulatory molecules – neither in the whole peripheral blood mononuclear cell (PBMC) fraction nor in particular DCs subtypes.ConclusionsIn the present study, we demonstrated no influence of systemic TSH administration on human peripheral blood DCs subtypes. These results are in accordance with our previous work suggesting the direct effect of TH on human DCs ex vivo.

Nogo-B expression, in arterial intima, is impeded in the early stages of atherosclerosis in humans.

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.