Folia histochemica et cytobiologica最新文献

Introduction: Regulatory T cells (Tregs) are a unique CD4+ T cell subset involved in the regulation of immune responses. The traditional immunophenotype used to define Tregs includes CD4+CD25high and the expression of the transcription factor Forkhead box protein 3 (FoxP3). A complex technique of intracellular staining, transient upregulation of FoxP3 in activated conventional T lymphocytes (Tcons), and the omission of naïve CD45RA+ Tregs with downregulated FoxP3 activity but a demethylated FOXP3 promoter region may lead to inaccurate quantification. In an attempt to meet the need for a reliable and simplified enumeration strategy, we investigated different membrane markers to capture the entire Treg compartment and to identify subpopulations of Tregs.

Material and methods: Analyses were performed on whole blood. Tested gating strategies were based on the expression of the following membrane antigens: CD45, CD3, CD4, CD25, CD127, CD26, CD6, CD39, CD71, HLA-DR, CD45RA and CD31. Double controls with FoxP3 were performed.

Results: The final enumeration panel consisted of the membrane markers CD45, CD3, CD4, CD25, CD127, CD26, CD39, CD45RA and CD31. A deep analysis of T cells with the CD4+CD25+CD127low/-CD26low/-CD45RAimmunophenotype revealed high expression of FoxP3 and/or CD39, while cells with the naïve immunophenotype, CD4+CD25+CD127low/-CD26low/-CD45RA+, presented lower expression of suppressor markers. Antigen CD31 is considered to be a valuable membrane marker of thymus-derived Tregs.

Conclusions: The presented 9-color panel that can be easily applied in laboratories enables reliable enumeration of Tregs with additional information about the functionality, maturity and origin of T regulatory cells.

Introduction: A recent study has shown a close neuroanatomical relationship between the enkephalinergic (methionine-enkephalin) and tachykininergic (substance P) systems in the alpaca diencephalon. In this study, our aim is to show this relationship in the alpaca brainstem.

Material and methods: Using an immunohistochemical technique, the distribution of immunoreactive (Ir) fibers and cell bodies containing substance P (SP) or methionine-enkephalin (MET) has been studied in the alpaca brainstem. Five adult males were used; brain tissue was fixed and processed by standard methods.

Results: SP- and MET-Ir fibers showed a widespread and similar distribution in the mesencephalon, pons and medulla oblongata. The co-localization of fibers containing SP or MET was found in most of the nuclei/tracts of the alpaca brainstem. This close neuroanatomical relationship suggests multiple physiological interactions between both neuropeptides. The distribution of the cell bodies containing SP was very restricted (cell bodies were only observed in a few nuclei located in the mesencephalon and medulla oblongata), whereas MET-Ir perikarya showed a moderately widespread distribution in the mesencephalon, pons and medulla oblongata.

Conclusions: This study increases the knowledge on the neuroanatomical distribution/relationship of the tachykininergic (SP) and enkephalinergic (MET) systems in the alpaca central nervous system.

Introduction: Urokinase plasminogen activator (uPA) is a serine protease and it also demonstrates proinflammatory properties. We thus hypothesized that uPA is involved in immunity of Sertoli cells in the rat testis.

Materials and methods: The uPA gene(PLAU) promoter was cloned by RT-PCR and the transcriptional activation of core domain was screened by using Dual-Luciferase Reporter Assay System. The Sertoli cells were harvested from 20-day-old Sprague-Dawley male rats and total RNA was isolated. The uPA mRNA levels and MyD88 pathway were tested by qPCR.

Results: We successfully cloned the 1517-bp rat uPA gene and screened the core domain (-455/+40) in five different fragments of uPA promoter. The uPA expression and uPA promoter activity were upregulated in lipopolysaccharide (LPS)-stimulated Sertoli cells. Furthermore, the uPA expression was regulated through the MyD88-independent pathway by interdicting IRF3 and interferon b.

Conclusion: uPA expression is likely induced by LPS through the core promoter domain of uPA. This finding implied that uPA played a role in the immune function of Sertoli cells in rat testis, which might provide the development of new treatments for male infertility.

Introduction: Cardiac papillary fibroelastomas (CPFs) are rare benign cardiac tumors typically found on the heart valves. The previously published data on the CPF focused on its clinical presentation, optimal management, and prognosis. However, histogenesis of these lesions remains controversial. Accordingly, the aim of this study was to establish the role of endocardial endothelium (EE) in CPF formation.

Materials and methods: Four CPF tumors removed from the right atrioventricular valves were analyzed using hematoxylin & eosin, orcein, and Masson trichrome staining together with immunochemistry for CD-34, CD-68, vimentin, vWF and a-SMA. Moreover, conventional transmission electron microscopy was used for morphological analysis and a-SMA presence confirmation.

Results: Ultrastructural morphology, immunohisto- and immunocytochemical analyses indicated that cells covering collagenous core have an endothelial origin. Some endocardial endothelium cells have the potential to undergo a transition to mesenchymal cells. Moreover, the abundant presence of extracellular vesicles may indicate an active intercellular communication. Within the intermediate translucent zone, amorphous substances with monocytes/macrophage-like cells and fibroblastic cells were found. Finally, within collagenous core activated (myo)fibroblasts were observed.

Conclusions: Our study demonstrated that the endocardial endothelium of the CPF was "double-sided", i.e., it presented both endothelial and mesenchymal cell characteristics. Another finding was the presence of monocytes, and macrophages which were integrated into CPF core and displayed features of a fibroblast that have been shown to contribute to extracellular matrix production. This could be interpreted as being attributed to the CPF histogenesis.

Introduction: Vascular smooth muscle cells (VSMCs)-based foam cell formation is a crucial factor in the atherosclerosis process. We aimed to explore the mechanism of Golgi a-mannosidase II (GMII) effects on the VSMCs-based foam cell formation.

Material and methods: VSMCs were exposed to different concentrations of low-density lipoproteins (LDLs), lipopolysaccharide (LPS), and/or GMII inhibitor (swainsonine). The qRT-PCR and western blot were used for expression analysis. Oil Red O staining was used to verify changes of lipid droplets in VSMCs. The translocation of the SCAP from the endoplasmic reticulum (ER) to Golgi was detected by immunofluorescence (IF).

Results: LPS disrupted the LDLs-mediated regulation of LDL receptor (LDLr) and increased intracellular cholesterol ester, which was inversely inhibited by swainsonine. The activity of a-mannosidase II and GMII expression were decreased by LDLs but increased by the addition of LPS. Conversely, LPS-induced enhancement was reversed by swainsonine. Additionally, swainsonine reversed the LPS-induced increase of intracellular lipid droplets in the presence of LDLs. Expression analysis demonstrated that LDLr, SCAP, and SREBP2 were up-regulated by LPS, but reversed by swainsonine in LDLs-treated cells. IF staining revealed that swainsonine inhibited the translocation of SCAP to Golgi under inflammatory stress.

Conclusions: Collectively, swainsonine restrained LDLr expression to suppress the formation of VSMCs-based foam cells by reducing SREBP2 and SCAP under inflammatory stress conditions, suggesting that GMII contributes to the formation of VSMCs-based foam cells under inflammatory stress.

Introduction: The heart innervation is made up of plexo-ganglionic formation containing sympathetic, parasympathetic, and sensory components. We examined the distribution and neurochemical coding of the ganglia and nerve fibers in the chinchilla's heart.

Material and methods: The heart sections of 10 male and 10 female adult chinchillas were processed in accordance with the thiocholine method for acetylcholine esterase (AChE), and the SPG method for detecting the presence of adrenergic fibers was applied. The routine technique of immunohistochemical (IHC) staining with primary antibodies directed against ChAT, VAChT, DbH, TH, CART, NPY, VIP, GAL and SOM was used. The secondary antibodies were conjugated with Alexa Fluor 488 and Alexa Fluor 555 fluorophores.

Results: The epicardium contained ganglia and nerve fibers, the myocardium had a few ganglion neurocytes and nerve fibers, and the endocardium contained only nerve fibers. In the epicardium, AChE-positive fibers were more prevalent than SPG-positive fibers. All the ganglion cells were immunopositive for ChAT and VAChT. Some cells also had a positive reaction to DbH and TH. Fibers containing cholinergic and adrenergic markers were numerous, while many of them were ChAT/DbH- and VAChT/TH-positive. CART/NPY and CART/VIP, as well as CART and GAL, were observed to be colocalized in ganglion neurocytes, as well as in individual cells. The nerve fibers were found to contain all the neurotransmitters we tested for, as well as the following co-occurrences: ChAT/DbH, VAChT/TH, CART/NPY, CART/VIP, CART/GAL, and CART/SOM.

Conclusions: Our analysis of the neurochemical profile of the nerve structures in chinchilla's heart showed that, despite interspecies differences, the general pattern of the distribution of autonomic nervous system structures is similar to that of other mammals' species, including humans.

Introduction: Bladder cancer (BCa) is one the most common urinary system malignancies and approximately one quarter of diagnosis is invasive muscle-invasive BCa. Accumulating evidence revealed that keratin 17 (KRT17) is closely related to the prognosis and progression of various tumors including a recent study also implying the potential role of KRT17 in the diagnosis of BCa. However, the specific role of KRT17 in BCa remains to be elucidated.

Material and methods: The expression of KRT17 in 5637 BCa cells and SV-HUC-1 normal human urothelial cells was detected using quantitative real-time PCR (qRT-PCR) and western blot. Short hairpin RNA targeting KRT17 was used to knockdown KRT17 in BCa cells. The colony formation was assessed and the proliferation of cells was studied by Cell Counting Kit-8 (CCK-8). Invasion and epithelial-mesenchymal transition (EMT) capacity of BCa cells were assessed using transwell assay and western blot, respectively. Cisplatin sensitivity of cancer cells was measured by evaluating the cell viability using CCK-8 assay. The downstream pathway of KRT17 was explored by western blot.

Results: The expression of KRT17 was elevated in BCa cells in comparison with the normal human urothelial cell at the mRNA and protein levels. The in vitro assays demonstrated that KRT17 interference affected the proliferation, colony formation and invasion capacity of BCa cells, as well as EMT. Furthermore, knockdown of KRT17 enhanced cisplatin sensitivity in BCa cells. Mechanically, KRT17 ablation led to the inactivation of both AKT and ERK pathways.

Conclusions: Our results elucidate the vital role of KRT17 in the development of malignancy of BCa cells, probably by the activation of AKT and ERK pathways and suggest that it may represent a novel therapeutic target for BCa.

Introduction: In endochondral ossification septoclasts and osteoclasts (also called chondroclasts) release growth factors deposited in non-calcified and calcified zones of the growth plate. They stimulate, within the metaphysis, initial stages of the bone formation. We have recently reported quantitation of several growth factors in calcified cartilage from calf costochondral junction. Data from the analogous human cartilage could possibly help to choose efficient combinations of growth factors for clinical applications, but the amount of the calcified cartilage needed for analysis of numerous growth factors would be difficult to collect. The estimation of growth factors expression in endochondral chondrocytes may, indirectly, indicate which of them play a leading role in the stimulation of osteoprogenitor cells in metaphysis. To test this hypothesis, we used rat chondrocytes to evaluate mRNA levels of several growth factors.

Materials and methods: Chondrocytes were isolated from proliferative and hypertrophic zones of the epiphyseal cartilage forming costochondral junctions of inbred Lewis rats. The total RNA was isolated from chondrocytes and the level of mRNA for bone morphogenetic proteins 1-7 (BMP-1-7), vascular endothelial growth factor A (VEGF-A), basic fibroblast growth factor (bFGF), growth/differentiation factor 5 (GDF-5), NEL-like protein 1 (NELL-1), transforming growth factor beta 1 (TGF-b1), mesencephalic astrocyte-derived neurotrophic factor (MANF), connective tissue growth factor (CTGF), osteoclast-stimulating factor 1 (OSTF-1) and insulin-like growth factor 1 (IGF-1) was evaluated using real-time PCR method.

Results: All studied factors were expressed. The highest level of mRNA was detected for CTGF, MANF, VEGF-A and TGF-b1. Expression was also quite high for BMP-1, BMP-2, BMP-5, BMP-6, BMP-7, IGF-1, GDF-5 and OSTF-1. Very low level of mRNA was detected for BMP-3, BMP-4 and NELL-1.

Conclusions: Chondrocytes from the proliferative and hypertrophic zones of the growth plate produce factors involved in the cartilage metabolism and bone formation. The determination of these growth factors in humans could help to choose their optimal composition necessary for stimulation of bone formation in clinical practice. In rat the best stimulation of bone formation would presumably be achieved with a mixture of BMP-2, BMP-5, BMP-6 and BMP-7.

Introduction: Obesity and type 2 diabetes mellitus (T2DM) are leading causes of morbidity and mortality worldwide. Ghrelin is implicated in the pathophysiology of both disease states. Laparoscopic sleeve gastrectomy is an emerging safe therapeutic technique for patients with morbid obesity. Since the removal of ghrelin-secreting cells by sleeve gastrectomy may be associated with diminished hunger sensation the aim of the study was to: (i) compare body weight and body mass index (BMI) in both obese non-diabetic and obese diabetic patient groups, (ii) determine the ghrelin expression in the resected gastric tissue in both groups, (iii) evaluate relationships between ghrelin cell expression and pre- and post-operative serum ghrelin concentration and glucose levels, and (iv) assess the influence of sleeve gastrectomy on serum glycaemic parameters in this patient population.

Material and methods: Twenty morbidly obese female patients from Saudi Arabia, of whom ten suffered from T2DM participated in the study. All subjects underwent laparoscopic sleeve gastrectomy. The removed fundus, body and antrum were biopsied and underwent immunohistochemical staining to detect ghrelin cell expression. Serum samples were assayed for ghrelin concentration and indicators of glycaemic status at the baseline and three months after sleeve gastrectomy.

Results: BMI (p < 0.05) and body weight (p < 0.001) were significantly lower in non-diabetic obese patients compared with diabetic patients before and 3 months after the surgery. Also, pre-operative serum ghrelin level was higher in non-diabetic patients compared with diabetic patients group, and postoperative plasma ghrelin level was reduced in diabetic patients (p < 0.001) compared with non-diabetic patients. Gastric fundic mucosa of the diabetic patients exhibited lower number of ghrelin-positive cells (p < 0.05) compared with non-diabetic patients. There were significant negative correlations between pre- and post-operative ghrelin serum level and blood glucose (r = -0.736, p = 0.0002 and r = -0.656, p = 0.0007, respectively) in all patient populations.

Conclusions: The results of this study suggest that the diabetic status of obese female patients may affect the incidence of ghrelin cells in three major stomach's regions and this novel observation warrants further studies.