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INTRODUCTION There are only sporadic references in literature regarding general medicine and dentistry student´s preparedness for Histology, study resources and how students might use them in the era of virtual microscopy. METHODS A structured questionnaire was used to evaluate students´ opinion, with 192 students of general medicine and 82 students of dentistry responding. RESULTS The dentistry students evaluate their previous knowledge of basic high school disciplines as less helpful when compared to their general medicine colleagues, but this difference diminishes during the first year of medical school studies. Students of dentistry display a better orientation in the amount of study resources (electronic vs printed) and also the ways of their use (practical vs theoretical preparation). The main problems surfacing in the study of Histology have been: the lack of time due to the high demands of Anatomy, problems with correct identification of structures in specimens and correct orientation in a large number of available study resources. Students indicate that they would appreciate the introduction of interactive exercise tests to verify practical and theoretical knowledge. CONCLUSION We revealed significant differences between students of general medicine and dentistry in terms of student´s preparedness and learning habits. According to our findings, it is still necessary to further develop teaching methods utilising virtual microscopy, taking into account the needs of both general medicine and dental school students.

Aim of the study: The aim was to perform a morphometric analysis of subcutaneous and visceral adipose tissue of morbidly obese women and to determine the relationship between adipocyte size and the development of type 2 diabetes (T2D). Materials and methods: White adipose tissue of morbidly obese women was obtained from subcutaneous and omental adipose tissue during bariatric surgery. The same tissues were obtained at judicial autopsy in non-obese (lean) non-diabetic patients. The harvested tissue was embedded in paraffin and 5 μm thick hematoxylin-eosin stained sections were analyzed by the Olympus cellSens system. Statistical evaluation was performed by GraphPad Prism 6.1 software. Results: We found a relationship between adipocyte size and the presence of T2D. The most pronounced changes were seen in visceral adipocytes (cell diameter increased from 61.9 μm in controls to 79.5 μm in patients with T2D). Also, the size of the subcutaneous adipocytes increased against the control. A statistically significant difference between diabetic and non-diabetic patients was not proven in subcutaneous adipocytes. We also observed differences in the distribution of adipocyte mean diameters. Whilst in the control group there was a normal (Gaussian) distribution, in the morbidly obese we found an asymmetric distribution with a positive skewness to the right. Conclusion: We have demonstrated that in morbidly obese women a significant increase in visceral adipocyte size is associated with the development of both insulin resistance and T2D.

Aims Hofbauer cells (HBCs) are placental macrophages playing various roles during normal and complicated pregnancies, and of the latter, chorioamnionitis is the most frequent. Methods In placenta with chorioamnionitis, we examined immunohistochemical expression profiles of IL-1β, IL-10, and their potential regulators, CYP2C8 and soluble epoxide hydrolase (sEH), in Hofbauer cells and compared the results with our previously published data for normal placenta. Results We found that the expression profiles of the studied proteins in Hofbauer cells in chorioamnionitis differs from normal placenta. In chorioamnionitis, HBCs showed a moderate expression of IL-1β together with a weak expression of IL-10 and CYP2C8. Contrary to normal placenta, HBCs in chorioamnionitis express sEH. We demonstrated a moderate positive correlation between the expression of CYP2C8 and sEH in chorioamnionitis (Spearman r = 0.5654), suggesting enhanced degradation of anti-inflammatory epoxyeicosatrienoic acids. Moreover, the relations of IL-1β and IL-10 to CYP2C8, previously described in normal placenta, disappeared. Furthermore, a weak expression of anti-inflammatory IL-10 in chorioamnionitis was accompanied by change in circularity of HBCs (Spearman r = 0.8193). Conclusion Taken together, these findings suggest a possible alteration of the anti-inflammatory role of HBCs and its regulation in chorioamnionitis.

INTRODUCTION Virtual microscopy, used as a method to teach histology, has many undeniable advantages. However, the usefulness of this method is somewhat limited by the difficulties students face in finding their way through huge amounts of digital data, compounded by decreased interaction between students and teachers. We describe the results of a recent pilot project which combined the modern teaching methods of active learning, where students themselves present histological slides and make use of the virtual microscopy system. METHODS Students' responses to a structured questionnaire and examination results were evaluated. RESULTS We found that a combination of both electronic materials and textbooks was commonly used by students to prepare for practical teaching sessions, with electronic resources being used regularly by the majority of students. No statistically relevant differences were found between the approaches of dentistry vs general medicine students. Cooperation between students' groups during the preparation for individual presentations was seen to be beneficial by a majority of dentistry students; they reported that the introduction of student-led presentations improved their quality of preparation for practical lessons, as well as increasing their participation and activity level in the lessons themselves. These different approaches and motivations between students of dentistry and general medicine are reflected in the test results where dentistry students are more successful. CONCLUSION We confirm that there are differences in motivation, approaches and examination results between both groups of students, which should be taken into account and which could lead to differentiation of future curricula for both study courses.

Immunohistochemistry and immunocytochemistry (ICC) play an irreplaceable role in research and diagnostics. It is well known that antigen retrieval (AR) can, as a technique, have beneficial outcomes on immunohistochemistry results when using formalin-fixed, paraffin-embedded tissue samples. The main purpose of AR is to break protein crosslinks which are formed during formalin fixation. Although AR was originally designed for formalin-fixed, paraffin-embedded samples, the usefulness of AR in ICC has been described in previous studies. Cytologic samples are often fixed in alcohol-based fixatives which does not lead to the formation of crosslinks. Therefore, alcohol-fixed samples can be successfully immunostained without AR. We investigated the effect of heat-induced antigen retrieval (HIAR) on alcohol-fixed HEK293 cell line samples and patient cytologic samples from thyroid gland obtained by fine needle aspiration technique. We compared indirect 2-step ICC staining results performed according to the protocol with or without HIAR in citrate buffer pH 6 for several antibodies. Utilizing HIAR against intracellular antigens has beneficial effects. Therefore, more diluted antibodies can be used for satisfactory results. However, surface antigens were probably damaged by HIAR treatment. We demonstrated evident changes in cell surface topography after HIAR treatment by atomic force microscopy. Staining specificity of patient samples improves and background staining is reduced, allowing higher dilutions of primary antibody. Improving staining specificity is necessary for accurate diagnostics. Although we have shown the beneficial effect of HIAR for immunostaining intracellular antigens, proper staining protocol should be tested on appropriate controls for individual antibodies.

Heat-induced antigen retrieval (HIAR) treatment improves the antigen immunodetection in formalin-fixed, paraffin-embedded tissue samples and it can also improve the detection of intracellular antigens in alcohol-fixed cytological samples, although it could deleteriously impact immunodetection, particularly that of membranous antigens. We examined the differences in cell surface topography on MCF7 cells fixed in methanol/acetone (M/A) or 4% paraformaldehyde (4% PFA), as well as the changes caused by HIAR treatment at three different temperatures (60, 90, and 120°C), using atomic force microscopy. Furthermore, the consequences for immunostaining of five membranous antigens [epidermal growth factor receptor (EGFR), E-cadherin, CD9, CD24, and CD44] were examined. Our results illustrate that while there was no one single optimal immunostaining condition for the tested antibodies, the surface topography could be an important factor in successful staining. Generally, the best conditions for successful immunostaining were M/A fixation with no HIAR treatment, whereas in 4% PFA-fixed cells, HIAR treatment at 120°C was optimal. These conditions showed similarity in cell surface skewness. A correlation factor between successful immunocytochemical staining and the skewness parameter was 0.8000. Our results indicate that the presence of valleys, depressions, scratches, and pits on the cell surface is unfavorable for the successful immunodetection of cell surface antigens.

Fibrates belong to a group of ligands of peroxisome proliferator-activated receptor alpha (PPARα), which play a role in the regulation of CYP epoxygenases and soluble epoxide hydrolase (sEH), key enzymes in the metabolism of biologically highly active epoxyeicosatrienoic acids (EETs). We demonstrated that low doses of fibrates stimulate proliferation of the MCF7 cell line, while high doses suppress it. The increase in cell proliferation was accompanied by an increase in CYP epoxygenases and decrease in sEH levels. The overall level of PPARα remained same after low-dose fibrate stimulation; however, there was a significant shift of the receptor to the cell nucleus. PPARα expression was further demonstrated by immunohistochemistry in both carcinoma and healthy breast tissue samples both in the cytoplasm and in the nuclei. We have also observed higher nuclear PPARα positivity in tumor tissues. Although our results obtained for MCF7 cells suggest the potential role of PPARα in cell proliferation, we did not find an association between nuclear localization of PPARα and the expression of proliferation marker Ki-67 in tumor tissues. The exact role of PPARα in carcinogenesis still remains unclear.

Aims: To determine the frequency of pregnancy terminations due to prenatal congenital heart defect (CHD) and assess the agreement fetal echocardiography (FECHO) and autopsy findings. Methods: The data were retrospectively assessed between 2008 and 2017 in a population of 116 698 live births. The correlations between the FECHO and autopsy findings were classified into five levels of agreement: complete, partial, altered diagnosis, disagreement, and unfeasible autopsy. Results: Totally, 293 CHDs were identified and 49% of families (143/293) decided to terminate the pregnancy. In 1% (2/143) of cases, the autopsy could not be performed, for the other 99% (141/143), the pathologist confirmed the presence of CHDs. Complete agreement between FECHO and autopsy was achieved in 85% (122/143). In 10% (14/143) of cases, the pathologist found minor findings, which were not described in the FECHO. In 4% (5/143) of cases, the pathologist changed the main diagnosis. Conclusion: Altogether, the results indicated that FECHO is a highly sensitive method for the prenatal detection of CHD but is incapable of detecting the complete spectrum of cardiac defects. Autopsies verified the diagnosis, confirmed the overall impairment in the fetus, and provided data for further counselling of the affected family.

Background and Aims: Macrophages are linked to the initiation of the chronic inflammation believed to underlie the changes taking place in the white fatty tissue of obese people. Both the number of macrophages, but their functional status, play an important role in the development of inflammation. Classically, macrophages are divided into two types: pro-inflammatory (M1) and anti-inflammatory (M2) types, and based on current immunological studies, further views on the functional distribution of macrophages are suggested. In this study, we evaluated the M1 and M2 macrophages ratio in obese subjects with, or without diabetes. To identify all macrophages, we used CD68 expression, while CD204 expression is typically used for the M2 macrophage. Materials and Methods: During bariatric surgery, carried out in obese people with and without type 2 diabetes (T2D), we obtained subcutaneous adipose tissue from the navel and omental adipose tissue. We also obtained the same tissue from people with a physiological range of BMI from a judicial autopsy. Applying immunohistochemical staining anti-CD68 and anti-CD204, we carried out a quantitative evaluation of the number of macrophages. Results: We found CD68+ and CD204+ positive macrophages in perivascular spaces and between fat cells, both isolated and in larger infiltrates. They were also present in so-called "crown-like structures" (CLS) around dying adipocytes. Quantitative analysis showed an increased number of macrophages in all obese patients compared to the control group of non-obese, individuals without T2D. The most striking observation was the macrophage increase in the visceral fatty tissue of diabetics. The number of CD68 and CD204 positive macrophages was statistically significantly smaller in patients without T2D. Conclusion: We demonstrated a significantly greater number of macrophages in visceral adipose tissue, especially in patients with T2D. Our results also show a positive correlation between the presence of T2D and the total number of macrophages; a significantly greater number of macrophages were found in visceral adipose tissue, especially in patients with T2D.

The interaction between ABCB1 transporter and its substrates takes place in cell membranes but the available data precludes quantitative analysis of the interaction between transporter and substrate molecules. Further, the amount of transporter is usually expressed as a number of ABCB1 molecules per cell. In contrast, the substrate concentration in cell membranes is estimated by determination of substrate-lipid partition coefficient, as examples. In this study, we demonstrate an approach, which enables us to estimate the concentration of ABCB1 molecules within plasma membranes. For this purpose, human leukemia K562 cells with varying expression levels of ABCB1 were used: drug selected K562/Dox and K562/HHT cells with very high transporter expression, and K562/DoxDR2, K562/DoxDR1, and K562/DoxDR05 cells with gradually decreased expression of ABCB1 derived from K562/Dox cells using RNA interference technology. First, we determined the absolute amount of ABCB1 in cell lysates using immunoblotting and recombinant ABCB1 as a standard. We then determined the relative portion of transporter residing in the plasma membrane using immunohistochemistry in nonpermeabilized and permeabilized cells. These results enabled us to estimate the concentration of ABCB1 in the plasma membrane in resistant cells. The ABCB1 concentrations in the plasma membrane of drug selected K562/Dox and K562/HHT cells containing the highest amount of transporter reached millimolar levels. Concentrations of ABCB1 in the plasma membrane of resistant K562/DoxDR2, K562/DoxDR1, and K562/DoxDR05 cells with lower transporter expression were proportionally decreased.

Background Fibrates are widely used hypolipidemic drugs, which serve as ligand of peroxisome proliferator-activated receptor α (PPARα). Recently, they have also been considered as potential anticancer agents. We studied effect of fibrates treatment on cell proliferation, expression of CYP2J2 and concomitant changes in expression of cell cycle regulatory proteins in three different human cell lines: HEK293, HepG2, and HT-29. Methods We used WST-1 viability test, western blot and immunocytochemistry for detection of proteins of interests and analysis of cell cycle. Results Our results showed that at lower concentrations of all tested fibrates, viability of all tested cell lines is increased, whereas at higher concentrations, repression is apparent. Unfortunately, the viability of tested cells is predominantly increased in a range of concentration which is reached in patient plasma. This phenomenon is accompanyed by elevation of CYP2J2, increased number of cyclin E-positive cells and decreased number of Cdc25A-positive cells in all tested cell lines, and elevated cyclin A expression in HepG2 and HT-29. These changes are concentration-dependent. We suppose that increased level of CYP2J2 could explain enhanced cell proliferation in lower concentration of fibrates. Conclusion Based on our results, we suggested there is no anti-cancer effect of fibrates in tested carcinoma cell lines. Electronic supplementary material The online version of this article (doi:10.1186/s12944-016-0335-z) contains supplementary material, which is available to authorized users.