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Purpose: To determine the effectiveness of the basal calcitonin (CTb) determination in the early postoperative period to predict the possible recurrence (persistence) of medullary thyroid cancer (MTC). Material and Methods: A retrospective study of the treatment results in 194 patients with MTC (148 (76.3%) primary - group 1 and 46 (23.8%) recurrent - group 2) according to the levels of CTb in the first week after surgery and one year later. All groups were analyzed by staging, the level of preoperative and postoperative basal calcitonin 5 days and 1 year after the primary surgery. Findings: Among all patients, women prevailed - 144 (74.2%), the average age was (48.7±15.2), the average follow-up period was 67.5 months. Basal calcitonin was studied in patients of all groups in the preoperative and serially in the postoperative periods: 5 days and 1 year after the most radical surgical volumes. To test the hypothesis about the possibility of using CTb indicators in the early postoperative period, the degree of compliance with normal calcitonin indicators (≤18 pg/mL) was assessed by observation groups 5 days and 1 year after surgery. Conclusions: 1. The CTb value 5 days after surgery is no less a reliable marker of the result of surgical treatment of MTC than the currently recommended CTb measurement 2-3 months after surgery. 2. The technique is applicable for both primary and reoperations used for recurrent forms of medullary thyroid cancer.
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The aim of this study is to analyze and identify the main predictors that may indicate multifocal growth of PTC. Materials and methods: The main and control groups included patients with the category of malignant multifocal process T1-3mN0Mx (n=109) and unifocal T1-T3N0Mx (n=50) respectively, who underwent thyroidectomy with lymphadenectomy.Ultrasound characteristics of the nodes, tissue changes of the thyroid gland were taken into account. Results: Fibrous changes can be considered as one of the risk factors of the presence of additional PTC lesion. Discussion: There is no unambiguity in the definition of predictors of multifocal PTC growth. Conclusions: No clear predictors of multifocal PTC have been identified. It is advisable to improve the quality of ultrasound, to focus on single-focus PTC in patients with fibrinous changes in the thyroid gland at normal levels of TSH.
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CONTEXT: Diabetes insipidus (DI) is rare in the neonatal period but of great importance due to increased renal risk and mental retardation despite treatment. OBJECTIVE: This report describes the case of a patient with congenital nephrogenic diabetes insipidus (NDI). Detection of this pathology during the neonatal period, especially in premature newborns, is difficult because of the electrolyte variations that occur as a result of the immature kidney function. SUBJECTS AND METHODS: The subject was a preterm infant with very low birth weight (VLBW) and persistent hypernatremic hyperosmolarity that developed polyuria and polydipsia in the first weeks of life. RESULTS: Taking into account blood and urine laboratory tests, vasopressin levels, as well as family history, the infant was diagnosed with congenital NDI. Early treatment allowed a good development, proving that the prevention of long-term complications is possible through multidisciplinary care and frequent monitoring. The particularity of this case was the presence of persistently elevated presepsin levels. This association prompted the investigation into underlying renal hypernatremia. CONCLUSIONS: NDI is a rare condition and the onset in the neonatal period is a sign of severity and hereditary causality. Early diagnosis, symptomatic treatment and multidisciplinary monitoring may decrease the risk of long-term complications.
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Stromal cells/telocytes (SCs/TCs) were recently described in the human adult trigeminal ganglion (TG). As some markers are equally expressed in SCs/TCs and endothelial cells, we hypothesized that a subset of the TG SCs/TCs is in fact represented by endothelial progenitor cells of a myelomonocytic origin. This study aimed to evaluate whether the interstitial cells of the human adult TG correlate with the myelomonocytic lineage. We used primary antibodies for c-erbB2/HER-2, CD31, nestin, CD10, CD117/c-kit, von Willebrand factor (vWF), CD34, Stro-1, CD146, α-smooth muscle actin (α-SMA), CD68, VEGFR-2 and cytokeratin 7 (CK7). The TG pial mesothelium and subpial vascular microstroma expressed c-erbB2/HER-2, CK7 and VEGFR-2. SCs/TCs neighbouring the neuronoglial units (NGUs) also expressed HER-2, which suggests a pial origin. These cells were also positive for CD10, CD31, CD34, CD68 and nestin. Endothelial cells expressed CD10, CD31, CD34, CD146, nestin and vWF. We also found vasculogenic networks with spindle-shaped and stellate endothelial progenitors expressing CD10, CD31, CD34, CD68, CD146 and VEGFR-2. Isolated mesenchymal stromal cells expressed Stro-1, CD146, CK7, c-kit and nestin. Pericytes expressed α-SMA and CD146. Using transmission electron microscopy (TEM), we found endothelial-specific Weibel-Palade bodies in spindle-shaped stromal progenitors. Our study supports the hypothesis that an intrinsic vasculogenic niche potentially involved in microvascular maintenance and repair might be present in the human adult trigeminal ganglion and that it might be supplied by either the pial mesothelium or the bone marrow niche.
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Células Endoteliais/ultraestrutura , Células-Tronco/ultraestrutura , Células Estromais/ultraestrutura , Telócitos/ultraestrutura , Gânglio Trigeminal/ultraestrutura , Biomarcadores/análise , Células Endoteliais/química , Humanos , Imuno-Histoquímica , Microscopia Eletrônica de Transmissão , Receptor ErbB-2/química , Células-Tronco/química , Células Estromais/química , Telócitos/química , Gânglio Trigeminal/anatomia & histologia , Gânglio Trigeminal/química , Nervo Trigêmeo/química , Nervo Trigêmeo/ultraestrutura , Corpos de Weibel-Palade/química , Corpos de Weibel-Palade/ultraestruturaRESUMO
Telocyte (TC) is a newly identified type of cell in the cardiac interstitium (www.telocytes.com). TCs are described by classical transmission electron microscopy as cells with very thin and long telopodes (Tps; cellular prolongations) having podoms (dilations) and podomers (very thin segments). TCs' three-dimensional (3D) morphology is still unknown. Cardiac TCs seem to be particularly involved in long and short distance intercellular signalling and, therefore, their 3D architecture is important for understanding their spatial connections. Using focused ion beam scanning electron microscopy (FIB-SEM) we show, for the first time, the whole ultrastructural anatomy of cardiac TCs. 3D reconstruction of cardiac TCs by FIB-SEM tomography confirms that they have long, narrow but flattened (ribbon-like) telopodes, with humps generated by the podoms. FIB-SEM tomography also confirms the network made by TCs in the cardiac interstitium through adherens junctions. This study provides the first FIB-SEM tomography of a human cell type.
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Diagnóstico por Imagem , Coração/fisiopatologia , Microscopia Eletrônica de Transmissão , Miocárdio/ultraestrutura , Humanos , Tomografia/métodosRESUMO
Conventionally, cells described in the stroma of the intestinal wall are fibroblasts/fibrocytes, mast cells, plasma cells, eosinophils, macrophages and, interstitial cells of Cajal (ICCs), the latter being considered as the pacemakers of gastrointestinal rhythmicity. Recently, a new type of stromal cell called telocyte (TCs) was found in various cavitary and non-cavitary organs (www.telocytes.com). We show here direct electron microscopical evidence for the presence of TCs in the lamina propria of rat jejunum just beneath the epithelial layer of the mucosal crypts and in between the smooth muscle cells (SMCs) of muscularis mucosae. TCs are characterized by: several very long (tens to hundreds of µm) prolongations called telopodes (Tps). Tps (with caliber below the resolving power of light microscopy) display podomeres (thin segments ≤ 0.2 µm) and podoms (dilations accommodating caveolae, mitochondria, and endoplasmic reticulum). Tps present dichotomous branching and form a three dimensional network close to immune cells, SMCs or nerve bundles. TCs could play a role in intercellular signaling and control of local tissue homeostasis.
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Células Intersticiais de Cajal/citologia , Jejuno/citologia , Mucosa/citologia , Células Estromais/citologia , Animais , Extensões da Superfície Celular/ultraestrutura , Homeostase/fisiologia , Imageamento Tridimensional , Células Intersticiais de Cajal/ultraestrutura , Masculino , Mucosa/ultraestrutura , Organelas/ultraestrutura , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia , Células Estromais/ultraestruturaRESUMO
Human myometrium includes two important cell populations involved in its contractility: smooth muscle fibers and interstitial cells. The pacemaking mechanism is not yet identified, but it is possible that myometrial smooth muscle cells contract in response to a signal generated by c-kit positive interstitial cells. The aim of this study was to investigate the effects of imatinib as a c-kit receptor antagonist on the spontaneous or oxytocin (OT) induced contractions of human non-pregnant myometrium in vitro. Myometrial strips were obtained from non-pregnant women (reproductive age) undergoing hysterectomy for benign indications. The strips were suspended in organ baths for recording of isometric tension. Imatinib effects were assessed on spontaneous contraction and after preexposure to OT.Direct exposure of myometrial strips to imatinib inhibits both amplitude and frequency of contractions (80-320 µM) in a dose dependent manner. Amplitude reverted back to 90% of the baseline amplitude by consequent addition of imatinib (until 480 µM). Total inhibition of myometrial contraction was obtained after addition of OT 60 nM. If myometrium was pre-exposed to OT (320 nM), imatinib 80-160 µm increased amplitude, while decreasing frequency. These data provide evidence that telocytes may be involved as modulators of the spontaneous contractions of the non-pregnant human uterus, via a tyrosine-kinase independent signaling pathway.
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Miométrio/efeitos dos fármacos , Ocitócicos/farmacologia , Ocitocina/farmacologia , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Pirimidinas/farmacologia , Contração Uterina/efeitos dos fármacos , Benzamidas , Relação Dose-Resposta a Droga , Feminino , Humanos , Mesilato de Imatinib , Técnicas In Vitro , Miométrio/metabolismo , Proteínas Proto-Oncogênicas c-kit/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
BACKGROUND: Recent findings suggest a higher prevalence of hepatic steatosis in patients with chronic hepatitis C, estimated at 50%. Both host and viral factors contribute to the development of steatosis in chronic hepatitis C. Steatosis is an initial stage, which promotes hepatic fibrosis through oxidative stress. AIM: To assess the pathogenic mechanism of genotype 1 hepatitis C virus in steatosis and to evaluate the correlation between the degree of steatosis and the level of oxidative stress. PATIENTS AND METHODS: The study was carried out on 50 patients (29 males, 21 females) with genotype 1 HCV and liver biopsy proven chronic hepatitis C. Patients with other etiology of chronic liver disease were excluded. We statistically correlated the degree of steatosis with clinical (age, sex, waist circumferences) and biological parameters (alaninaminotransferase, gammaglutamyltranspeptidase - GGT, insulin, ferritin, serum viral load, oxidative stress). Insulin resistance (IR) was determined by the homeostasis model assessment (HOMA) method. The oxidative stress was estimated by serum malondialdehyde (MDA) and glutathione (GSH). RESULTS: 27 patients presented steatosis (57%): 14 out of 29 men (48%) and 14 out of 21 women (66%); in two thirds of them, steatosis was moderate. Univariate analysis identified five parameters that significantly influenced steatosis: age >45 years, sex - female, IR (HOMA>2.5), BMI, central adiposity (as reflected by waist circumferences and high GGT-values). Multivariate analysis identified four significant parameters: sex - female, insulin resistance (HOMA>2.5), BMI>30 kg/m(2) and GGT>2N. No relationship was found between steatosis and viral replication. The study demonstrated a significant correlation between steatosis and IR on the one hand and between steatosis and liver fibrosis on the other hand (p<0.05). Liver fibrosis was significant correlated with the increase levels of free radicals (MDA>250 nmol/dL). CONCLUSIONS: The pathogenic mechanism of genotype 1 HCV in steatosis is independent from viral replication and it may be linked to virus induced metabolic abnormalities such as IR. More women (66%) than men (48%) developed steatosis. Increased levels of free radicals, correlated with moderate and severe steatosis suggest the intervention of oxidative stress in determining the hepatic lesions associated with steatosis.
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Fígado Gorduroso/virologia , Hepacivirus/genética , Hepatite C Crônica/complicações , Fígado Gorduroso/metabolismo , Feminino , Genótipo , Hepatite C Crônica/metabolismo , Hepatite C Crônica/virologia , Humanos , Masculino , Pessoa de Meia-Idade , Estresse OxidativoRESUMO
A strict regulation of contractility in the uterus and fallopian tube is essential for various reproductive functions. The uterus contributes, through either increased contractility or periods of relative quiescence, to: (i) expulsion of menstrual debris, (ii) sperm transport, (iii) adequate embryo placement during implantation, (iv) enlarging its capacity during pregnancy and (v) parturition. The dominant cell population of the uterine wall consists of smooth muscle cells that contain the contractile apparatus responsible for the generation of contractile force. Recent interest has focused on a new population of cells located throughout the myometrium on the borders of smooth muscle bundles. These cells are similar to interstitial cells of Cajal (ICC) in the gut that are responsible for the generation of electrical slow waves that control peristalsis. A precise role for myometrial Cajal-like interstitial cells (m-ICLC) has not been identified. m-ICLC express the c-kit receptor, involved in creating and maintaining the ICC phenotype in the gastrointestinal tract. However, both acute and prolonged inhibition of this receptor with the c-kit antagonist imatinib mesylate does not appear to affect the spontaneous contractility of myometrium. Calcium imaging of live tissue slices suggests that contractile signalling starts on the borders of smooth muscle bundles where m-ICLC are located and recently the possible role of extracellular ATP signalling from m-ICLC has been studied. This manuscript reviews the evidence regarding tissue-level signalling in the myometrium with a particular emphasis on the anatomical and possible functional aspects of m-ICLC as new elements of the contractile mechanisms in the uterus.
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Células Intersticiais de Cajal/citologia , Miométrio/citologia , Miométrio/fisiologia , Contração Uterina/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Feminino , Humanos , Modelos Biológicos , Proteínas Proto-Oncogênicas c-kit/metabolismoRESUMO
UNLABELLED: Oxidative stress is an important pathophysiological mechanism in chronic hepatitis with C-virus infection (CHC). Steatosis is frequently observed in CHC and seems to have a significant impact on the natural history of the disease with respect to development of fibrosis. The aim of this study was to investigate the relationship between systemic parameters of oxidative stress, insulin resistance, steatosis degree, and fibrosis in CHC. Fifty patients with chronic hepatitis C (29 men and 21 women with the average age 45 years), with or without steatosis, were tested for: oxidative stress and antioxidant status by measuring serum malondialdehyde (MDA), total blood non-proteic thiols concentration (GSH), gamma glutamyl transpeptidase (GGT) activity, lipid parameters, and liver function tests. Our results show that the prevalence of insulin resistance (IR) in chronic hepatitis with C-virus genotype 1 was 32% and the association with hepatic steatosis was in a proportion of 48%, IR is mediated by both metabolic factors as well as viral factors. Hepatic steatosis was associated with an increase of MDA, correlated with its severity, and secondary with a decrease of GSH. The activity of serum GGT was net superior, in patients with steatosis, proportional with its degree. CONCLUSIONS: In patients infected by HCV genotype 1, oxidative stress and insulin resistance contribute to steatosis, which in turn exacerbates both insulin resistance and oxidative stress and accelerates the progression of fibrosis. The induction of GGT is an adaptive response against oxidative damage elicited by lipid peroxidation and it may be critical in the progression of the disease.
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Hepatite C Crônica/etiologia , Hepatite C Crônica/metabolismo , Estresse Oxidativo , Fígado Gorduroso/complicações , Fígado Gorduroso/patologia , Feminino , Hepatite C Crônica/complicações , Humanos , Cirrose Hepática/complicações , Cirrose Hepática/patologia , Masculino , Pessoa de Meia-Idade , Análise de RegressãoRESUMO
Cells of the female reproductive tract are subject to hormonal control via sex steroid genomic receptors expressed at nuclear level. We previously showed that interstitial Cajal-like cells (ICLC) of human myometrium expressed estrogen and progesterone receptors (ER/PR). Our aim, based on these results, was to see if ER and/or PR could be found also in tubal ICLC. Indeed, we present here immunohistochemical evidence that ICLC of human Fallopian tube (isthmic region) have such receptors. Stromal ICLC, as well as ICLC among smooth muscle layers, were identified in tissue sections by their morphological features (e.g. several very long, moniliform, prolongations of cell body) as well as by c-kit positivity, vital staining with methylene blue or silver impregnation. Additional evidence was provided by sequential staining for c-kit and for PR on the same cell, by 'sandwich method'. In vitro, the 4th passage cell cultures from Fallopian tube muscularis exhibiting ICLC morphology showed the presence of ER-alpha and/or PR-A by immunofluorescence. In conclusion, our data suggest that ICLC could function as steroid sensors, and might be implicated in Fallopian tube motility (via gap junctions or juxta- and/or paracrine mechanisms).
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Tubas Uterinas/citologia , Células Intersticiais de Cajal/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Adulto , Células Cultivadas , Feminino , Humanos , Imuno-Histoquímica , Células Intersticiais de Cajal/citologia , Transporte ProteicoRESUMO
Smooth muscle cell (SMC) caveolae have been investigated by quantitative and qualitative analysis of transmission electron microscopy (TEM) images of rat stomach, bladder and myometrium, guinea pig taenia coli, human ileum, and rat aortic SMCs. Ultrathin (below 30 nm) serial sections were used for examination of caveolar morphology and their connections with SMC organelles. Average caveolar diameter was smaller in vascular SMCs (70 nm, n=50) than in visceral SMCs (77 nm, n=100), but with the same morphology. Most of the caveolae, featured as flask-shaped plasma membrane (PM) invaginations, opened to the extracellular space through a 20 nm stoma (21 +/- 3 nm) having a 7 nm thick diaphragm. A small percentage of caveolae (3%), gathered as grape-like clusters, did not open directly to the extracellular space, but to irregular PM pockets having a 20-30 nm opening to the extracellular space. In visceral SMCs, caveolae were disposed in 4-6 rows, parallel to myofilaments, whilst aortic SMCs caveolae were arranged as clusters. This caveolar organization in rows or clusters minimizes the occupied volume, providing more space for the contractile compartment. The morphometric analysis of relative volumes (% of cell volume) showed that caveolae were more conspicuous in visceral than in vascular SMCs (myometrium - 2.40%; bladder - 3.66%, stomach - 2.61%, aorta - 1.43%). We also observed a higher number of caveolae per length unit of cellular membrane in most visceral SMCs compared to vascular SMCs (myometrium - 1.06/microm, bladder - 0.74/microm, aorta - 0.57/microm, stomach - 0.48/microm). Caveolae increase the cellular perimeter up to 15% and enlarge the surface area of the plasma membrane about 80% in SMCs. Threedimensional reconstructions (15micro(3)) showed that most caveolae, in both visceral and vascular SMCs, have nanocontacts with SR (87%), other with mitochondria (10%) and 3% apparently have no contact with these organelles. Usually, 15 nm wide junctional spaces exist between caveolae and SR, some of them with nanostructural links between each other or with mitochondria: direct contacts (space <2 nm or none) and molecular links, so called 'feet' (about 12 nm electron dense structures between organellar membranes). Direct contacts possibly allow molecular translocation between the two membranes. Electron-dense 'feet'-like structures suggest a molecular link between these organelles responsible for intracellular Ca(2+) homeostasis (excitation-contraction coupling or pharmaco-mechanical coupling). Close appositions (approximately 15 nm) have also been observed between caveolae and perinuclear SR cisternae, suggesting that caveolae might be directly implicated in excitation-transcription coupling.
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Cavéolas/ultraestrutura , Músculo Liso/ultraestrutura , Animais , Aorta/ultraestrutura , Núcleo Celular/ultraestrutura , Colo/ultraestrutura , Feminino , Cobaias , Humanos , Íleo/ultraestrutura , Imageamento Tridimensional , Microscopia Eletrônica de Transmissão , Mitocôndrias/ultraestrutura , Miométrio/ultraestrutura , Ratos , Ratos Wistar , Sarcolema/ultraestrutura , Estômago/ultraestrutura , Bexiga Urinária/ultraestruturaRESUMO
Expression of estrogen (ER) and progesterone (PR) receptors was investigated in cultured human normal myometrial cells (non-pregnant uterus, fertile period). The ER and PR expression was studied by immunohistochemistry and immunofluorescence on either myocytes or interstitial Cajal-like cells (ICLC). Only those cells double immunostained for c-kit and steroid receptors were considered as ICLC. ER and/or PR immunoreactivity was localized in ICLC, primarily concentrated at the nucleus level, but it was also observed in the cell body (cytoplasm) and processes. Stronger immunopositive reaction in the ICLC nucleus for PR than for ER was noted. Under our experimental conditions, a clear positive repeatable reaction for steroid receptors could not be detected in myocytes. In conclusion, these data suggest that ICLC could be true hormonal 'sensors', possibly participating in the regulation of human myometrial contractions (via gap junctions with myocytes and/or by paracrine signaling).
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Imuno-Histoquímica/métodos , Miométrio/citologia , Miométrio/patologia , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Células Cultivadas , Feminino , Humanos , Azul de Metileno/farmacologia , Microscopia de Fluorescência , Miométrio/metabolismo , Proteínas Proto-Oncogênicas c-kit/biossínteseRESUMO
We have previously described interstitial Cajal-like cells (ICLC) in human atrial myocardium. Several complementary approaches were used to verify the existence of ICLC in the interstitium of rat or human ventricular myocardium: primary cell cultures, vital stainings (e.g.: methylene blue), traditional stainings (including silver impregnation), phase contrast and non-conventional light microscopy (Epon-embedded semithin sections), transmission electron microscopy (TEM) (serial ultrathin sections), stereology, immunohistochemistry (IHC) and immunofluorescence (IF) with molecular probes. Cardiomyocytes occupy about 75% of rat ventricular myocardium volume. ICLC represent approximately 32% of the number of interstitial cells and the ratio cardiomyocytes/ICLC is about 70/1. In the interstitium, ICLC establish close contacts with nerve fibers, myocytes, blood capillaries and with immunoreactive cells (stromal synapses). ICLC show characteristic cytoplasmic processes, frequently two or three, which are very long (tens up to hundreds of microm), very thin (0.1-0.5 microm thick), with uneven caliber, having dilations, resulting in a moniliform aspect. Gap junctions between such processes can be found. Usually, the dilations are occupied by mitochondria (as revealed by Janus green B and MitoTracker Green FM) and elements of endoplasmic reticulum. Characteristically, some prolongations are flat, with a veil-like appearance, forming a labyrinthic system. ICLC display caveolae (about 1 caveola/ 1 microm cell membrane length, or 2-4% of the relative cytoplasmic volume). Mitochondria and endoplasmic reticulum (rough and smooth) occupy 5-10% and 1-2% of cytoplasmic volume, respectively. IHC revealed positive staining for CD34, EGFR and vimentin and, only in a few cases for CD117. IHC was negative for: desmin, CD57, tau, chymase, tryptase and CD13. IF showed that ventricular ICLC expressed connexin 43. We may speculate that possible ICLC roles might be: intercellular signaling (neurons, myocytes, capillaries etc.) and/or chemomechanical sensors. For pathology, it seems attractive to think that ICLC might participate in the process of cardiac repair/remodeling, arrhythmogenesis and, eventually, sudden death.
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Corpos Enovelados/metabolismo , Corpos Enovelados/ultraestrutura , Ventrículos do Coração/citologia , Ventrículos do Coração/ultraestrutura , Miocárdio/citologia , Miocárdio/ultraestrutura , Animais , Células Cultivadas , Conexina 43/metabolismo , Humanos , Imuno-Histoquímica , Masculino , Modelos Biológicos , Miócitos Cardíacos/ultraestrutura , Ratos , Ratos WistarRESUMO
We present transmission electron microscope (TEM) evidence that ICC and ICC-like cells frequently establish close contacts (synapses) with several types of immunoreactive cells (IRC): lymphocytes, plasma cells, eosinophils, basophils, macrophages and mast cells. Such synapses were found in various organs: human mammary gland and myometrium, as well as rat stomach, gut, bladder and uterus. Specimens were observed by conventional TEM on ultrathin sections. Based on morphometric analyses and computer-aided 3-D reconstructions from serial sections, we propose an operational definition of ICC-IRC synapses: cell-to-cell close contacts where the two cells are separated by only approximately 15 nm, equivalent to twice the plasmalemmal thickness. Two types of such synapses were found: (i) uniform ('plain') synapses (PS). close contact extending for >200 nm, and (ii) multi-contact ('kiss and run') synapses (MS)--with multiple, focal, close-contact points alternating with regions of wider intermembrane distance. For instance, a typical PS between a rat bladder ICC-like cell and an eosinophil was 2.48 microm long and 11+/-4 nm wide. By contrast, a MS synapse in rat myometrium (between an ICC-like cell and an eosinophil) was 8.64 microm long and had 13 contact points. The synaptic cleft measured 15+/-8 nm at contact points and approximately 100 nm or more in wider areas. These synapses are different from gap junctions usually seen between ICC and between ICC and smooth muscle cells. We previously proposed that ICC-like cells might represent stromal progenitor cells, participate in juxtacrine/paracrine signaling and play a role in immune surveillance. The nanoscopic distances between the two contiguous membranes suggest a juxtacrine cell-to-cell signaling (chemical synapse), via juxtacrinins, a specific case of phenomenins. However, the (micro)vesicles found in the synaptic cleft may correspond to an exosome-based mechanism.
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Músculo Liso/inervação , Músculo Liso/ultraestrutura , Estômago/inervação , Sinapses/ultraestrutura , Animais , Humanos , Mucosa Intestinal/inervação , Mucosa Intestinal/ultraestrutura , Leucócitos/citologia , Leucócitos/ultraestrutura , Microscopia Eletrônica , Ratos , Estômago/citologia , Estômago/ultraestruturaRESUMO
We report here the in vitro isolation of Cajal-like interstitial cells from human inactive mammary-gland stroma. Primary cell cultures examined in phase-contrast microscopy or after vital methylene-blue staining revealed a cell population with characteristic morphological phenotype: fusiform, triangular or polygonal cell body and the corresponding (very) long, slender, moniliform cytoplasmic processes. Giemsa staining pointed out the typical knobbed aspect of cell prolongations. Immunofluorescence (IF) showed, like in situ immunohistochemistry, that Cajal-type cells in vitro (primary cultures), expressed c-kit/CD117 and vimentin. In conclusion, the images presented here reinforce our previous hypothesis that human mammary glands have a distinct population of Cajal-like cells in non-epithelial tissue compartments.
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Mama/citologia , Células Estromais/citologia , Técnicas de Cultura de Células , Feminino , Humanos , Microscopia de Fluorescência , Microscopia de Contraste de FaseRESUMO
Previous reports describing Cajal-like interstitial cells in human uterus are contradictory in terms of c-kit immunoreactivity: either negative (but vimentin-positive) in pregnant myometrium, or positive, presumably in the endometrium. The aim of this study was to verify the existence of human myometrial Cajal-like interstitial cells (m-CLIC). Six different, complementary approaches were used: 1) methylene-blue supravital staining of tissue samples (cryosections), 2) methylene blue and Janus green B vital staining (m-CLIC and mitochondrial markers, respectively), and 3) extracellular single-unit electrophysiological recordings in cell cultures, 4) non-conventional light microscopy on glutaraldehyde/osmium fixed, Epon-embedded semi-thin sections (less than 1 microm) stained with toluidine blue (TSM), 5) transmission electron microscopy (TEM), and 6) immunofluorescence (IF). We found m-CLIC in myometrial cryosections and in cell cultures. In vitro, m-CLIC represented approximately 7% of the total cell number. m-CLIC had 2-3 characteristic processes which were very long (approximately 60 microm), very thin (< or =0.5 microm) and moniliform. The dilated portions of processes usually accommodated mitochondria. In vitro, m-CLIC exhibited spontaneous electrical activity (62.4+/-7.22 mV membrane potentials, short duration: 1.197+/-0.04 ms). Moreover, m-CLIC fulfilled the usual TEM criteria, the so-called 'gold' or 'platinum' standards (e.g. the presence of discontinuous basal lamina, caveolae, endoplasmic reticulum, and close contacts between each other, with myocytes, nerve fibers and/or capillaries etc.). IF showed that m-CLIC express CD117/c-kit, sometimes associated with CD34, with vimentin along their processes. In conclusion, we describe myometrial Cajal-like interstitial cells that have affinity for methylene blue and Janus green B vital dyes, fulfill (all) TEM criteria, express CD117/c-kit and have spontaneous electric activity.
Assuntos
Células do Tecido Conjuntivo/citologia , Miométrio/citologia , Proteínas Proto-Oncogênicas c-kit/análise , Actinas/análise , Antígenos CD34/análise , Células Cultivadas , Células do Tecido Conjuntivo/química , Células do Tecido Conjuntivo/ultraestrutura , Eletrofisiologia , Feminino , Imunofluorescência , Junções Comunicantes/ultraestrutura , Humanos , Azul de Metileno/química , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Miócitos de Músculo Liso/química , Miócitos de Músculo Liso/citologia , Miócitos de Músculo Liso/ultraestrutura , Miométrio/química , Miométrio/ultraestrutura , Gravidez , Vimentina/análiseRESUMO
We describe here--presumably for the first time--a Cajal-like type of tubal interstitial cells (t-ICC), resembling the archetypal enteric ICC. t-ICC were demonstrated in situ and in vitro on fresh preparations (tissue cryosections and primary cell cultures) using methylene-blue, crystal-violet, Janus-Green B or MitoTracker-Green FM Probe vital stainings. Also, t-ICC were identified in fixed specimens by light microscopy (methylene-blue, Giemsa, trichrome stainings, Gomori silver-impregnation) or transmission electron microscopy (TEM). The positive diagnosis of t-ICC was strengthened by immunohistochemistry (IHC; CD117/c-kit+ and other 14 antigens) and immunofluorescence (IF; CD117/c-kit+ and other 7 antigens). The spatial density of t-ICC (ampullar-segment cryosections) was 100-150 cells/mm2. Non-conventional light microscopy (NCLM) of Epon semithin-sections revealed a network-like distribution of t-ICC in lamina propria and smooth muscle meshwork. t-ICC appeared located beneath of epithelium, in a 10-15 microm thick 'belt', where 18+/-2% of cells were t-ICC. In the whole lamina propria, t-ICC were about 9%, and in muscularis approximately 7%. In toto, t-ICC represent ~8% of subepithelial cells, as counted by NCLM. In vitro, t-ICC were 9.9+/-0.9% of total cell population. TEM showed that the diagnostic 'gold standard' (Huizinga et al., 1997) is fulfilled by 'our' t-ICC. However, we suggest a 'platinum standard', adding a new defining criterion- characteristic cytoplasmic processes (number: 1-5; length: tens of microm; thickness: < or =0.5 microm; aspect: moniliform; branching: dichotomous; organization: network, labyrinthic-system). Quantitatively, the ultrastructural architecture of t-ICC is: nucleus, 23.6+/-3.2% of cell volume, with heterochromatin 49.1+/-3.8%; mitochondria, 4.8+/-1.7%; rough and smooth endoplasmic-reticulum (1.1+/-0.6%, 1.0+/-0.2%, respectively); caveolae, 3.4+/-0.5%. We found more caveolae on the surface of cell processes versus cell body, as confirmed by IF for caveolins. Occasionally, the so-called 'Ca2+-release units' (subplasmalemmal close associations of caveolae+endoplasmic reticulum+mitochondria) were detected in the dilations of cell processes. Electrophysiological single unit recordings of t-ICC in primary cultures indicated sustained spontaneous electrical activity (amplitude of membrane potentials: 57.26+/-6.56 mV). Besides the CD117/c-kit marker, t-ICC expressed variously CD34, caveolins 1&2, alpha-SMA, S-100, vimentin, nestin, desmin, NK-1. t-ICC were negative for: CD68, CD1a, CD62P, NSE, GFAP, chromogranin-A, PGP9.5, but IHC showed the possible existence of (neuro)endocrine cells in tubal interstitium. We call them 'JF cells'. In conclusion, the identification of t-ICC might open the door for understanding some tubal functions, e.g. pace-making/peristaltism, secretion (auto-, juxta- and/or paracrine), regulation of neurotransmission (nitrergic/purinergic) and intercellular signaling, via the very long processes. Furthermore, t-ICC might even be uncommitted bipotential progenitor cells.
Assuntos
Células do Tecido Conjuntivo/citologia , Tubas Uterinas/citologia , Actinas/análise , Antígenos CD34/análise , Membrana Basal/citologia , Membrana Basal/ultraestrutura , Vasos Sanguíneos/ultraestrutura , Antígenos CD57/análise , Cavéolas/ultraestrutura , Caveolinas/análise , Contagem de Células , Núcleo Celular/ultraestrutura , Extensões da Superfície Celular/ultraestrutura , Células Cultivadas , Cromogranina A , Cromograninas/análise , Células do Tecido Conjuntivo/química , Células do Tecido Conjuntivo/ultraestrutura , Citoplasma/ultraestrutura , Eletrofisiologia , Tubas Uterinas/química , Tubas Uterinas/ultraestrutura , Feminino , Histocitoquímica , Humanos , Junções Intercelulares/ultraestrutura , Proteínas de Filamentos Intermediários/análise , Microscopia Eletrônica de Transmissão , Microscopia de Fluorescência , Mitocôndrias/ultraestrutura , Mucosa/citologia , Músculo Liso/citologia , Músculo Liso/ultraestrutura , Fibras Nervosas/ultraestrutura , Proteínas Proto-Oncogênicas c-kit/análise , Proteínas S100/análise , Coloração e Rotulagem , Ubiquitina Tiolesterase/análiseRESUMO
We show here (presumably for the first time) a special type of cell in the human and rat exocrine pancreas. These cells have phenotypic characteristics of the enteric interstitial cells of Cajal (ICC). To identify pancreatic interstitial cells of Cajal (pICC) we used routine light microscopy, non-conventional light microscopy (less than 1 mum semi-thin sections of Epon-embedded specimens cut by ultramicrotomy and stained with Toluidine blue), transmission electron microscopy (TEM), and immunocytochemistry. The results showed that pICC can be recognized easily by light microscopy, particularly on semi-thin sections, as well as by TEM. Two-dimensional reconstructions from serial photos suggest a network-like spatial distribution of pICC. pICC represent 3.3+/-0.5% of all pancreatic cells, and seem to establish close spatial relationships with: capillaries (43%), acini (40%), stellate cells (14%), nerve fibres (3%). Most of pICC (88%) have 2 or 3 long processes (tens of mum) emerging from the cell body. TEM data show that pICC meet the criteria for positive diagnosis as ICC (e.g. numerous mitochondria, 8.7+/-0.8% of cytoplasm). Immunocytochemistry revealed that pICC are CD117/c-kit and CD34 positive. We found pICC positive (40-50%) for smooth muscle alpha-actin or S-100, and, occasionally, for CD68, NK1 neurokinin receptor and vimentin. The reactions for desmin and chromogranin A were negative in pICC. At present, only hypotheses and speculations can be formulated on the possible role of the pICC (e.g., juxtacrine and/or paracrine roles). In conclusion, the quite-established dogma: "ICC only in cavitary organs" is overpassed.