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BACKGROUND: Individuals with congenital solitary functioning kidney (SFK) are at an increased risk of kidney damage. According to some studies, the risk is higher in unilateral kidney agenesis (UKA) than in unilateral multicystic dysplastic kidney (UMCDK). We hypothesized that with early detection of children with UKA and UMCDK, there would be no difference in the presence of hypertension, proteinuria, and reduced glomerular filtration rate (GFR) between UKA and UMCDK. METHODS: Based on a long-term follow-up protocol, we evaluated a cohort of 160 children followed from birth for SFK (84 with UKA and 76 with UMCDK) detected by prenatal or routine neonatal ultrasound screening. Hypertension, proteinuria, and reduced GFR were monitored as markers of kidney damage. We compared the characteristics and outcomes of the subgroups of children with UKA and UMCDK. RESULTS: GFR was reduced in 42 (26.2%) children, of whom 41 showed only mild reduction. Hypertension and proteinuria were found in 22 (13.8%) and 14 (8.8%) children, respectively. Combined kidney damage was present in 57 (35.6%) children. The UMCDK and UKA subgroups differed in GFR at final examination, with UMCDK patients being significantly more likely to have normal GFR compared to UKA patients (82% vs. 67%; p = 0.039). CONCLUSIONS: One third of the children showed signs of SFK damage, albeit mild. Patients with UKA had reduced GFR significantly more often than those with UMCDK, but did not differ in the rates of hyperfiltration injury or congenital anomalies of the kidneys and urinary tract (CAKUT) in SFK.
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Diagnóstico Precoz , Tasa de Filtración Glomerular , Riñón , Riñón Displástico Multiquístico , Proteinuria , Riñón Único , Humanos , Femenino , Riñón Displástico Multiquístico/diagnóstico , Riñón Displástico Multiquístico/complicaciones , Riñón Displástico Multiquístico/fisiopatología , Masculino , Riñón Único/complicaciones , Riñón Único/diagnóstico , Riñón Único/fisiopatología , Riñón/anomalías , Riñón/fisiopatología , Riñón/diagnóstico por imagen , Recién Nacido , Pronóstico , Proteinuria/etiología , Proteinuria/diagnóstico , Lactante , Preescolar , Niño , Hipertensión/diagnóstico , Hipertensión/etiología , Hipertensión/epidemiología , Hipertensión/fisiopatología , Estudios de Seguimiento , Anomalías Congénitas/diagnóstico , Anomalías Congénitas/diagnóstico por imagen , Tamizaje Neonatal/métodos , Enfermedades Renales/congénitoRESUMEN
Introduction: Inflammation of the placenta is harmful to both the fetus and the mother. Inflammation is strongly associated with diabetes, a common complication of pregnancy. Hofbauer cells (HBCs), unique immune system cells of fetal origin in the placenta, play complex roles, including growth of placental villi and their branching, stromal remodelling, and angiogenesis. Methods: Our study investigated the expression of IL-1ß, IL-10, CYP2C8, CYP2C9, CYP2J2 and sEH in HBCs from patients with type 1 diabetes mellitus (T1DM) and gestational diabetes mellitus (GDM) compared to healthy controls using immunohistochemistry. We also assessed the structure of the villus stroma using Masson´s trichrome. Results: In T1DM, HBCs showed inflammatory activation characterised by increased IL-1ß and decreased CYP epoxygenase expression compared to normal placentas. Conversely, significant inflammation in HBCs appeared less likely in GDM, as levels of IL-1ß and CYP epoxygenases remained stable compared to normal placentas. However, GDM showed a significant increase in sEH expression. Both types of diabetes showed delayed placental villous maturation and hypovascularisation, with GDM showing a more pronounced effect. Conclusion: The expression profiles of IL-1ß, CYP epoxygenases and sEH significantlly differ between controls and diabetic placentas and between T1DM and GDM. These facts suggest an association of the CYP epoxygenase-EETs-sEH axis with IL-1ß expression as well as villous stromal hypovascularisation. Given the stable high expression of IL-10 in both controls and both types of diabetes, it appears that immune tolerance is maintained in HBCs.
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Diabetes Mellitus Tipo 1 , Diabetes Gestacional , Embarazo , Humanos , Femenino , Placenta/metabolismo , Interleucina-10/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Inflamación/metabolismoRESUMEN
Intestinal epithelial differentiation is a highly organised process. It is influenced by a variety of signalling pathways and enzymes, such as the PI3K pathway and soluble epoxide hydrolase (sEH) from arachidonic acid metabolism. We investigated the changes in the expression of enzymes and lipid messenger from the PI3K pathway, including PTEN, during intestinal cell differentiation in vitro using HT-29 and Caco2 cells and compared them with immunohistochemical patterns of these proteins in human colon. To investigate the possible crosstalk between the PI3K pathway and sEH, we treated HT-29 and Caco2 cells with the sEH inhibitor TPPU. Administration of TPPU to differentiated cells decreased the expression of PTEN, thus reversing the change in its expression observed during cell differentiation. In addition, multiplex immunofluorescence staining confirmed the relationship between the expression of PTEN and villin, a marker of intestinal cell differentiation, ranging from a moderate correlation in undifferentiated cells to a very strong correlation in differentiated cells treated with TPPU. Furthermore, we confirm that PTEN and sEH mirrored their expression patterns in samples of prenatal and adult human intestine compared to tumours using immunohistochemical staining. Taken together, it appears that PTEN and sEH cooperate in the process of intestinal cell differentiation. A better understanding of the crosstalk between the PI3K pathway and sEH and its consequences for cell differentiation is highly desirable, as several sEH inhibitors are under clinical investigation for the treatment of various diseases.
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Epóxido Hidrolasas , Fosfatidilinositol 3-Quinasas , Humanos , Células CACO-2 , Fosfatidilinositol 3-Quinasas/metabolismo , Intestinos , Diferenciación Celular , Fosfohidrolasa PTENRESUMEN
Fibrates are widely used hypolipidaemic agents that act as ligands of the peroxisome proliferator-activated receptor α (PPARα). p38 is a protein kinase that is mainly activated by environmental and genotoxic stress. We investigated the effect of the PPARα activators fenofibrate and WY-14643 and the PPARα inhibitor GW6471 on the levels of activated p38 (p-p38) in the colorectal cancer cell lines HT-29 and Caco2 in relation to their differentiation status. Fibrates increased p-p38 in undifferentiated HT-29 cells, whereas in other cases p-p38 expression was decreased. HT-29 cells showed p-p38 predominantly in the cytoplasm, whereas Caco2 cells showed higher nuclear positivity. The effect of fibrates may depend on the differentiation status of the cell, as differentiated HT-29 and undifferentiated Caco2 cells share similar characteristics in terms of villin, CYP2J2, and soluble epoxide hydrolase (sEH) expression. In human colorectal carcinoma, higher levels of p-p38 were detected in the cytoplasm, whereas in normal colonic surface epithelium, p-p38 showed nuclear positivity. The decrease in p-p38 positivity was associated with a decrease in sEH, consistent with in vitro results. In conclusion, fibrates affect the level of p-p38, but its exact role in the process of carcinogenesis remains unclear and further research is needed in this area.
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Hipolipemiantes , PPAR alfa , Humanos , Ácidos Fíbricos/farmacología , PPAR alfa/metabolismo , Células CACO-2 , Hipolipemiantes/farmacología , Diferenciación CelularRESUMEN
We investigated the effects of PPARα activators fenofibrate and WY-14643 as well as the PPARα inhibitor GW6471 on the PI3K/Akt/PTEN pathway of intestinal cell differentiation. Our previous study showed that all these compounds increased the expression of villin, a specific marker of intestinal cell differentiation in HT-29 and Caco2 cells. Our current results confirmed the central role of lipid messenger phosphatidylinositol-4,5-bisphosphate (PIP2), a known player in brush border formation, in mediating the effects of tested PPARα ligands. Although all tested compounds increased its levels, surprisingly, each of them affected different PIP2-metabolizing enzymes, especially the levels of PIP5K1C and PTEN. Moreover, we found a positive relationship between the expression of PPARα itself and PIP2 as well as PIP5K1C. By contrast, PPARα was negatively correlated with PTEN. However, the expression of antigens of interest was independent of PPARα subcellular localization, suggesting that it is not directly involved in their regulation. In colorectal carcinoma tissues we found a decrease in PTEN expression, which was accompanied by a change in its subcellular localization. This change was also observed for the regulatory subunit of PI3K. Taken together, our data revealed that fenofibrate, WY-14643, and GW6471 affected different members of the PI3K/Akt/PTEN pathway. However, these effects were PPARα-independent.
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Gene inactivation of the cyclindependent kinase inhibitors p16INK4a, p15INK4b and p21WAF is frequently mediated by promoter gene methylation, whereas histone deacetylases (HDACs) control gene expression through their ability to deacetylate proteins. The effect of suberohydroxamic acid (SBHA) and 5Aza2'deoxycytidine (Decitabine) (DAC) treatments on the transcription of CDKN2A, CDKN2B and CDKN1A genes, and their effects on molecular biological behavior were examined in two myeloma cell lines, RPMI8226 and U266, which differ in p53functionality and IL6 expression. In both tested myeloma cell lines, a nonmethylated state of the CDKN2B gene promoter region was detected with normal gene expression, and the same level of p15INK4b protein was detected by immunocytochemical staining. Furthermore, in myeloma cells treated with SBHA and DAC alone, the expression of both p15INK4b and p21WAF was significantly upregulated in RPMI8226 cells (p53functional, without IL6 expression), whereas in the U266 cell line (p53 deleted, expressing IL6) only p21WAF expression was significantly increased. Moreover, the analysis revealed that treatment with DAC induced DNMT3B enhancement in U266 cells. In conclusion, in myeloma cells with IL6 expression, significantly increased DNMT3B expression indicated the tumorigenic consequences of 5Aza2'deoxycytidine treatment, which requires careful use in diseases involving epigenetic dysregulation, such as multiple myeloma (MM).
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ADN (Citosina-5-)-Metiltransferasas , Decitabina , Epigénesis Genética , Mieloma Múltiple , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Línea Celular Tumoral , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p15 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Decitabina/farmacología , Silenciador del Gen , Humanos , Interleucina-6/genética , Interleucina-6/metabolismo , Mieloma Múltiple/genética , Mieloma Múltiple/metabolismo , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , ADN Metiltransferasa 3BRESUMEN
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-1ß, 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-1ß 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-1ß 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.
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Corioamnionitis , Citocromo P-450 CYP2C8 , Epóxido Hidrolasas , Antiinflamatorios , Citocromo P-450 CYP2C8/metabolismo , Epóxido Hidrolasas/metabolismo , Femenino , Humanos , Inmunomodulación , Interleucina-10 , Placenta/metabolismo , EmbarazoRESUMEN
The ultraviolet (UV) part of solar radiation can permanently affect skin tissue. UVA photons represent the most abundant UV component and stimulate the formation of intracellular reactive oxygen species (ROS), leading to oxidative damage to various biomolecules. Several plant-derived polyphenols are known as effective photoprotective agents. This study evaluated the potential of quercetin (QE) and its structurally related flavonoid taxifolin (TA) to reduce UVA-caused damage to human primary dermal fibroblasts (NHDF) and epidermal keratinocytes (NHEK) obtained from identical donors. Cells pre-treated with QE or TA (1 h) were then exposed to UVA light using a solar simulator. Both flavonoids effectively prevented oxidative damage, such as ROS generation, glutathione depletion, single-strand breaks formation and caspase-3 activation in NHDF. These protective effects were accompanied by stimulation of Nrf2 nuclear translocation, found in non-irradiated and irradiated NHDF and NHEK, and expression of antioxidant proteins, such as heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1 and catalase. For most parameters, QE was more potent than TA. On the other hand, TA demonstrated protection within the whole concentration range, while QE lost its protective ability at the highest concentration tested (75 µM), suggesting its pro-oxidative potential. In summary, QE and TA demonstrated UVA-protective properties in NHEK and NHDF obtained from identical donors. However, due to the in vitro phototoxic potential of QE, published elsewhere and discussed herein, further studies are needed to evaluate QE safety in dermatological application for humans as well as to confirm our results on human skin ex vivo and in clinical trials.
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Flavonoides , Quercetina , Fibroblastos , Flavonoides/metabolismo , Humanos , Queratinocitos , Estrés Oxidativo , Quercetina/análogos & derivados , Quercetina/farmacología , Piel/metabolismo , Rayos UltravioletaRESUMEN
Peroxisome proliferator-activated receptor α (PPARα) is a ligand-dependent transcription factor that plays a role in various processes including differentiation of several cell types. We investigated the role of PPARα in the differentiation of intestinal cells using HT-29 and Caco2 cell lines as a model as well as human normal colon and colorectal carcinoma tissues. We detected a significant increase in PPARα expression in differentiated HT-29 cells as well as in normal surface colon epithelium where differentiated cells are localised. Thus, it seems that PPARα may play a role in differentiation of intestinal cells. Interestingly, we found that both PPARα activators (fenofibrate and WY-14643) as well as its inhibitor (GW6471) regulated proliferation and differentiation of HT-29 cells in vitro in the same way. Both compounds led to a decrease in proliferation accompanied by a significant increase in expression of villin, intestinal alkaline phosphatase (differentiation markers). Moreover, the same trend in villin expression was observed in Caco2 cells. Furthermore, villin expression was independent of subcellular localisation of PPARα. In addition, we found similar levels of PPARα expression in colorectal carcinomas in comparison to adjacent normal epithelium. All these findings support the hypothesis that differentiation of intestinal epithelium is PPARα-independent.
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Software based analyses of immunohistochemical staining are designed for obtaining quantitative, reproducible, and objective data. However, often times only a certain type of positive cells or structures need to be quantified thus whole image analysis cannot be performed. Such an example is Hofbauer placental cells, which show positivity of some antigens together with trophoblast, but only Hofbauer cells represent the regions of interest (ROIs). Two independent observers evaluated the immunohistochemical staining intensity of Hofbauer cells in placenta samples stained for cytoplasmic antigens by ImageJ, QuPath and light microscopy. Thus, the precise manual determination of ROIs, i.e. Hofbauer cells, was necessary. We detected low inter-observer variability in staining intensity. Almost perfect agreement between observers was reached for ImageJ and QuPath whilst substantial agreement was reached for light microscopy evaluation. As for the comparison of ImageJ, QuPath and light microscopy, the agreement of all three methods (identical immunohistochemical intensity) was achieved for 38.1% samples. The almost perfect agreement of staining intensities was reached between ImageJ and QuPath, and moderate agreement for comparison of the light microscopy to both software. Software analyses are much more time-consuming, thus their utilization is at least questionable to evaluate ROIs with selection.
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INTRODUCTION: The success of pregnancy depends on the regulation of immunological processes in the placenta. Important mediators of an immune response include pro- and anti-inflammatory interleukins which may be regulated by CYP epoxygenases and their metabolites. The relation between interleukins and CYP epoxygenases expression in human placenta has not yet been studied vastly. MATERIAL AND METHODS: We investigated the expression patterns of IL-1ß and IL-10 in embryonic (n=8), early foetal (n=16) and term (n=7) human placenta tissue by an immunohistochemical method and evaluated the results by Kruskal-Wallis test. The obtained data was correlated using Spearman's correlation coefficient to our previously published data of CYP epoxygenases expression in the same samples. To confirm that Hofbauer cells express IL-10 and IL-1ß as well as CYP2C8 and IL-10 together, and thus there is a relation between proteins of interests, we used multiplex immunofluorescent staining. RESULTS: The expression of IL-1ß decreased with gestational age in cytotrophoblast, syncytiotrophoblast, as well as in Hofbauer cells whilst IL-10 decreased in cytotrophoblast, remained at the same levels in syncytiotrophoblast and increased in Hofbauer cells. In trophoblast cells, we found a statistically significant positive correlation between the expression of CYP2J2 and CYP2C9 with IL-1ß, whereas there was no relation between IL-10 and any of the tested CYP epoxygenases. In Hofbauer cells, we found a significant positive correlation between CYP2C8 and IL-10 and a significant negative correlation between CYP2C8 and IL-1ß. CONCLUSION: Our results showed that the exact role and relation of interleukins and CYP epoxygenases and their metabolites is dependent on their respective cellular context. Because of IL-10, IL-1ß, as well as HBCs play a role in various pathological conditions, further investigation of the exact role of CYP epoxygenase, interleukins and their relations is needed.
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Interleucina-10 , Placenta , Citocromo P-450 CYP2C8 , Femenino , Humanos , Interleucina-1 , Embarazo , TrofoblastosRESUMEN
Hofbauer cells are macrophages residing in the stroma of placental villi and play a number of roles during normal pregnancy, as well as pathological conditions. A morphometric analysis of Hofbauer cells, in particular to investigate the number of cells, their size and shape in samples of normal human placenta from 1st trimester, term and with chorioamnionitis was performed. Tissue samples were immunostained for CD206 antigen and evaluated using ImageJ software. We detected significant changes in number and morphology of HBCs between normal placenta and placenta with chorioamnionitis samples. In chorioamnionitis, the cells were unevenly distributed within the villi, generally present in higher numbers, larger and more elongated than those in normal 1st trimester and term placenta.
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Corioamnionitis , Animales , Corioamnionitis/veterinaria , Vellosidades Coriónicas , Femenino , Humanos , Macrófagos , Placenta , EmbarazoRESUMEN
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.
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Histología , Facultades de Odontología , Educación en Odontología , Hábitos , Histología/educación , Humanos , Aprendizaje , EstudiantesRESUMEN
The harmful effects of low energy UVA photons (315-400â¯nm) are associated with the massive production of reactive oxygen species resulting in oxidative stress. In response to oxidative damage, NF-E2-related factor 2 (Nrf2) is translocated to the nucleus and drives the expression of detoxication and antioxidant enzymes. UVA's effect on Nrf2 has been quite well characterised in dermal fibroblasts. However, there is a dearth of such information for keratinocytes. This study aimed to evaluate and compare the effect of UVA radiation on the Nrf2 pathway and oxidative stress related proteins in primary human dermal fibroblasts (NHDF), epidermal keratinocytes (NHEK) and human keratinocyte cell line HaCaT. NHDF were exposed to doses of 2.5-7.5â¯J/cm2, NHEK and HaCaT to 10-20â¯J/cm2 using a solar simulator. Effects on Nrf2 translocation were evaluated after 1, 3 and 6â¯h and Nrf2-controlled proteins (heme oxygenase 1 (HO-1), NAD(P)H:quinone oxidoreductase 1 (NQO1), glutathione reductase (GSR), glutathione-S-transferase (GST), interleukine-6 (IL-6), and matrix metalloproteinases (MMP-1, MMP-2)) after 3, 6 and 24â¯h. The results showed the fastest Nrf2 translocation was in UVA-irradiated HaCaT (1â¯h), persisting until the subsequent time interval (3â¯h), while in primary keratinocytes the effect of radiation was minimal. In NHDF, UVA-stimulated Nrf2 translocation was conspicuous 3â¯h after UVA treatment. In NHDF, most of the studied proteins (NQO1, HO-1, GSR, GSTM1 and MMP-1) showed the highest level 24â¯h after UVA exposure, except for MMP-2 and IL-6 which had their highest level at a shorter time incubation interval (3â¯h). In NHEK, NQO1, HO-1 and GST were increased 6â¯h after UVA exposure, GSR and MMP-2 level was slightly below or above the control level, and MMP-1 and IL-6 increased at shorter time intervals. When comparing NHEK and HaCaT, these cells displayed contrary responses in most of the Nrf2-controlled proteins. Thus, primary keratinocytes cannot be replaced with HaCaT when studying cell signalling such as the Nrf2 driven pathway and Nrf2-controlled proteins.
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Factor 2 Relacionado con NF-E2/metabolismo , Transducción de Señal/efectos de la radiación , Piel/efectos de la radiación , Rayos Ultravioleta , Supervivencia Celular/efectos de la radiación , Células Cultivadas , Humanos , Queratinocitos/metabolismo , Queratinocitos/efectos de la radiación , Transporte de Proteínas , Piel/citología , Piel/metabolismoRESUMEN
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.
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Antígenos/química , Etanol/química , Calor , Inmunohistoquímica , Adhesión en Parafina , Glándula Tiroides/química , Fijación del Tejido , Adulto , Anciano , Femenino , Células HEK293 , Humanos , Masculino , Persona de Mediana Edad , Estudios RetrospectivosRESUMEN
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.
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Diabetes Mellitus Tipo 2/inmunología , Grasa Intraabdominal/inmunología , Macrófagos/inmunología , Obesidad/inmunología , Grasa Subcutánea/inmunología , Tejido Adiposo Blanco/inmunología , Tejido Adiposo Blanco/patología , Adulto , Anciano , Antígenos CD , Antígenos de Diferenciación Mielomonocítica , Cirugía Bariátrica , Estudios de Casos y Controles , Femenino , Humanos , Inmunohistoquímica , Inmunofenotipificación , Grasa Intraabdominal/patología , Macrófagos/patología , Masculino , Persona de Mediana Edad , Obesidad/patología , Obesidad/cirugía , Epiplón , Receptores Depuradores de Clase A , Grasa Subcutánea/patología , Adulto JovenRESUMEN
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.
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Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Sistema Enzimático del Citocromo P-450/metabolismo , Epóxido Hidrolasas/metabolismo , Genes erbB-2 , PPAR alfa/metabolismo , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Animales , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citocromo P-450 CYP2J2 , Femenino , Ácidos Fíbricos/farmacología , Humanos , Concentración 50 Inhibidora , Antígeno Ki-67/metabolismo , Células MCF-7 , Transporte de Proteínas/efectos de los fármacos , Pirimidinas/farmacología , Solubilidad , Fracciones Subcelulares/metabolismoRESUMEN
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.
Asunto(s)
Antígenos de Superficie/análisis , Membrana Celular/efectos de la radiación , Membrana Celular/ultraestructura , Calor , Nanoestructuras , Propiedades de Superficie , Humanos , Células MCF-7 , Microscopía de Fuerza AtómicaRESUMEN
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.