Dynamics of telopodes (telocyte prolongations) in cell culture depends on extracellular matrix protein.

Telocytes (TC) are cells with telopodes (Tp), very long prolongations (up to 100 µm) with an uneven caliber ( www.telocytes.com ). Factors determining the dynamics of cellular prolongations are still unknown, although previous studies showed telopode motility in TC cultures. We comparatively investigated, by time-lapse videomicroscopy, the dynamics of Tp of mouse heart TC seeded on collagen, fibronectin, and laminin. Under our experimental conditions, TC and fibroblasts (cell line L929) behaved differently in terms of adherence, spreading, and prolongation extension. Fibroblasts showed lower spreading on the matrix proteins used. The time needed for spreading was 2-4 h for TC, versus 8-10 h for fibroblasts. The values for final cell surface area after spreading were between 200 and 400 µm(2) for fibroblasts and 800-2,000 µm(2) for TC. TC showed a more than three times higher ability to spread on the tested matrix proteins. An extremely low capacity to extend prolongations with lengths shorter than cell bodies was noted for fibroblasts, while TC extended prolongations longer than the cell body length, with a moniliform appearance. The stronger adherence and spreading were noted for TC seeded on fibronectin, while the lowest were on laminin. Collagen determined an intermediate adherence and spreading for TC, but the highest dynamics in Tp extensions. In conclusion, TC behave differently than fibroblasts in terms of adherence, spreading, and cell prolongation extension when seeded on various matrix proteins in cell culture.

Epicardium: interstitial Cajal-like cells (ICLC) highlighted by immunofluorescence.

During the last few years, there is an increasing interest in the role of the epicardium in cardiac development, myocardial remodelling or repair and regeneration. Several types of cells were described in the subepicardial loose connective tissue, beneath the epicardial mesothe-lium. We showed previously (repeatedly) the existence of interstitial Cajal-like cells (ICLCs) in human and mammalian myocardium, either in atria or in ventricles. Here, we describe ICLCs in adult mice epicardium and primary culture as well as in situ using frozen sections. The identification of ICLCs was based on phase contrast microscopy and immunophenotyping. We found cells with characteristic morphologic aspects: spindle-shaped, triangular or polygonal cell body and typical very long (tens to hundreds micrometres) and very thin cyto-plasmic processes, with a distinctive 'beads-on-a-string' appearance. The dilations contain mitochondria, as demonstrated by MitoTracker Green FM labelling of living cells. Epicardial ICLCs were found positive for c-kit/CD117 and/or CD34. However, we also observed ICLCs positive for c-kit and vimentin. In conclusion, ICLCs represent a distinct cell type in the subendocardium, presumably comprising at least two subpopulations: (i) c-kit/CD34-positive and (ii) only c-kit-positive. ICLCs might be essential as progenitor (or promoter) cells for developing cardiomyocyte lineages in normal and/or injured heart.

Insights into the interstitium of ventricular myocardium: interstitial Cajal-like cells (ICLC).

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.

Cajal-type cells from human mammary gland stroma: phenotype characteristics in cell culture.

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.

C-kit immunopositive interstitial cells (Cajal-type) in human myometrium.

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.

Early and late effect of infliximab on circulating dendritic cells phenotype in rheumatoid arthritis patients.

The aim of this study was to analyze the phenotype of circulating dendritic cells (DCc) in rheumatoid arthritis (RA) patients before and after treatment with infliximab (at 24 h and 6 months) and the correlation between these changes and the clinical response to treatment. Sixteen patients with RA were recruited and clinical status was determined using the Disease Activity Score 28 (DAS28). All patients had active disease (mean DAS28 = 5.96) and were suitable for treatment with infliximab. Samples of peripheral venous blood were obtained before administration of the first dose of infliximab and again at 24 h and 6 months after treatment. DCc populations were analyzed by flow cytometry. At 24 h, there were no differences in the clinical status of the patients. However, we found a decrease in CD11c+ and, to a lesser extent, CD123+ DCc percentages. The expression of CD83, the most important activation marker for DC, was also shown to be decreased 24 h after infliximab therapy. After 6 months of treatment, all patients showed significant clinical improvement (mean DAS28 = 3.64, p < 0.001) and expression of the activation marker on DCc remained low. In conclusion, this study supports the role of tumor necrosis factor (TNF)-alpha blockade in preventing the maturation of DCc and in reducing the expression of their activation markers. Although the clinical response to infliximab was not observed after 24 h, DCc activation was strongly reduced by anti-TNF-alpha therapy. After 6 months of treatment, current data show a less active phenotype of DCc associated with clinical improvement in all patients in the study.

[Expression of stem cell markers on fetal and tumoral human liver cells in primary culture]. / Expresia de markeri ai celulelor stem pe celulele în cultura primara izolate din ficatul fetal si ficatul tumoral uman.

Hepatic stem cells can be identified by the expression of putative markers such as CD117 (c-kit), CD90 (Thy-1), CD34, and HLA-DR. We have identified populations expressing these markers in both fetal and tumoral human liver by flow cytometry, using monoclonal antibodies against CD90, CD117, CD34, and HLA-DR. In tumoral liver CD117+/CD90+ cells were found in decreasing number from the neoplastic (2.48 +/- 0.67) and peritumoral region (0.88 +/- 0.12) to the area of para-tumoral (normal) parenchyma (0.13 +/- 0.04). The CD117+/CD34+ cells showed the following distribution: 0.35 +/- 0.05% in the tumoral region, 1.01 +/- 0.23% in the peritumoral region and 0.35 +/- 0.01 in the para-tumoral region. Using the same markers on fetal liver cells we have also identified small populations of CD117+/CD90+ cells (0.28 +/- 0.07%) and CD117+/CD34+ cells (1.13 +/- 0.24%), presumably resident stem cells or hematopoietic stem cells. Immunomagnetic negative separation was then performed on fetal liver cells using monoclonal antibodies against specific markers of hematopoietic lineages such as CD3, 14, 16, 19, 22, and CD56 to eliminate this population. The remaining cells were then incubated with fluorescently labeled monoclonal antibodies against CD90 and CD117 and analyzed using fluorescence microscopy. As expected these markers were expressed on the majority of the selected cells (89.28 +/- 9.56%). Isolation using appropriate markers and initiation of primary cultures is a first step to the therapeutic use of fetal stem cells and for the study of adult liver stem cells involvement in carcinogenesis.

Correlation between the immunophenotypical presentation of dendritic cells and the clinical response to anti-rheumatic treatment in rheumatoid arthritis.

BACKGROUND: Dendritic cells (DCs) are potent antigen-presenting cells (APC) that are deeply implicated in the initiation and exacerbation of rheumatoid arthritis (RA). Active RA is associated with an activated DCs population as demonstrated by high expression of adhesion and co-stimulatory molecules. PURPOSE: To compare the expression of adhesion and co-stimulatory molecules on DCs from synovial tissue (ST) in patients (pts) with RA and the clinical status before and after treatment with disease modifying antirheumatic drugs (DMARDs). METHODS: Samples of ST were obtained from RA patients at the time of hip or knee replacement or arthroscopy. Clinical status (assessed by the American College of Rheumatology - ACR - core set and the DAS28) and co-stimulatory and adhesion molecules on DCs were evaluated before and after treatment. Immunophenotype of DCs was analyzed by two-color immunofluorescence microscopy. RESULTS: In patients with active RA we found a highly differentiated subpopulation of DCs that expressed an activated phenotype. After treatment, the expression of adhesion and co-stimulatory molecules on ST DCs was correlated with the ACR and DAS28 clinical response. CONCLUSION: Clinical outcome and the immunophenotypical presentation of ST DCs after DMARDs treatment are closely correlated in pts with RA. The co-stimulatory activity in the synovium is important in determining the course of the disease and provide new therapeutic targets for immune intervention.

Co-stimulatory and adhesion molecules of dendritic cells in rheumatoid arthritis.

Dendritic cells (DCs) in the rheumatoid arthritis (RA) joint mediate the immunopathological process and act as a potent antigen presenting cell. We compared the expression of co-stimulatory and adhesion molecules on DCs in RA patients versus controls with traumatic joint lesions and evaluated the correlation between the immunophenotypical presentation of DCs and the clinical status of the disease. Samples of peripheral venous blood, synovial fluid (SF) and synovial tissue (ST) were obtained from 10 patients with RA at the time of hip or knee replacement and from 9 control patients with knee arthroscopy for traumatic lesions. Clinical status was appreciated using the DAS28 score. Blood, SF and dissociated ST cell populations were separated by centrifugation and analyzed by flow cytometry. Cells phenotypes were identified using three-color flow cytometry analysis for the following receptors HLA-DR, CD80, CD83, CD86, CD11c, CD18, CD54, CD58, CD3, CD4, CD8, CD19, CD20, CD14, CD16, CD56. HLA-DR molecules, co-stimulatory receptors CD80, CD86, CD83 and adhesion molecules CD18, CD11c, CD54, CD58, were analyzed by two-color immunofluorescence microscopy on ST serial sections. In patients with active RA (DAS28>5.1) we found a highly differentiated subpopulation of DCs in the ST and SF that expressed an activated phenotype (HLA-DR, CD86+, CD80+, CD83+, CD11c+, CD54+, CD58+). No differences were found between circulating DCs from RA patients and control patients. Our data suggest an interrelationship between clinical outcome and the immunophenotypical presentation of DCs. Clinical active RA (DAS28>5.1) is associated with high incidence of activated DCs population in the ST and SF as demonstrated by expression of adhesion and co-stimulatory molecules.

Apoptosis in the immune system: 1. Fas-induced apoptosis in monocytes-derived human dendritic cells.

Dendritic cells (DC) are cells of the hematopoietic system specialized in capturing antigens and initiating T cell-mediated immune responses. We show here that human DC generated from adherent peripheral blood mononuclear cells (PBMC) after in vitro stimulation with granulocyte macrophage colony stimulating factor (GM-CSF) and interleukin-4 (IL-4) express Fas antigen (APO-1, CD95) and can undergo apoptosis upon triggering of Fas by monoclonal antibodies. Immature monocytes-derived dendritic cells (MDDC) upregulate CD86 and HLA-DR expression and develop dendrites and veiled processes. Flow cytometry analysis revealed CD95 expression in approx. 40% of these MDDC and incubation with anti-CD95 mAb (0.5 microg/ml) induced apoptosis when compared to untreated controls. The extent of apoptosis induced by the agonist anti-Fas antibody strongly related to the percentage of cells expressing CD 95. Upon tumor necrosis factor alpha (TNF-alpha) additional stimulation, MDDC assumed a characteristic mature dendritic cells morphology showing prolonged veils, CD83 expression, and high levels of HLA-DR. These cells have downregulated their Fas receptors (to approx. 20%) and undergo apoptosis to a lesser extent when treated with anti-CD 95, as demonstrated by the hardly noticeable effect of this antibody on the viability of cultured cells as compared to controls. Thus, upon TNF-alpha induced maturation, MDDC became resistant to Fas-induced apoptosis. The apoptotic episodes surrounding the earlier stage of DC differentiation appeared to be mediated by Fas. In contrast, a Fas independent pathway is probably responsible for the apoptotic events associated with terminally differentiated DC.

Stem cells (p63(+)) in keratinocyte cultures from human adult skin.

Epidermal stem cells (ESC) are responsible for maintaining skin cellular homeostasis, as they give rise to fast-dividing transit amplifying cells committed to terminal differentiation, while retaining their self-renewal capacity. However, no pure ESC cultures are available and no highly specific cytochemical marker was identified. We report here the experimental conditions allowing the selective enrichment in ESC, using cultured adult human keratinocytes. The main step was the selection of cells able to rapidly adhere to human collagen type IV in vitro. Thus, an increased proportion of putative ESC of about 65% was obtained, as demonstrated by p63 expression.

Apoptosis in human embryo development: 3. Fas-induced apoptosis in brain primary cultures.

Fas (APO-1/CD95) is an important apoptotic mediator for both immune and nervous systems. In the present study, we have investigated the expression and function of Fas in human embryonic/fetal brain primary cultures from 12 human embryos and fetuses with gestational ages between 5 to 22 weeks. Anti-Fas fluorescent antibody was used for labeling of Fas positive cells and for quantitation of Fas expression in brain cultures. To demonstrate that Fas receptor is functional in human embryonic/fetal brain cells, anti-Human-Fas monoclonal antibody (0.5 microg/ml) was used to induce apoptosis in brain primary cultures. Apoptosis was investigated by flow-cytometry and fluorescent microscopy using TUNEL and annexin V labeling. Fas was found to be expressed in the embryonic/fetal human primary brain cultures, on neuronal and glial cells or their precursors, varying with gestational ages. Cross-linking of Fas induced apoptosis in brain cultures indicating that Fas receptor functions as a death receptor. We also showed that cell death triggered through Fas receptor was caspase dependent, hence it was blocked by a selective caspase-8 inhibitor (IETD-fmk). These results suggest that Fas is involved in neuronal apoptosis in the developing human brain.

Lymphocyte subset reference ranges in Romanian adult Caucasians.

The paper reports the distribution of lymphocyte subpopulations in the Caucasian population of Romania. Investigations were carried out in cells bearing the following antigens: CD3 (T cells), CD19 (B cells), CD4 (T helper/inducer cells), CD8 (T suppressor/cytotoxic and some NK cells), and CD16 CD56 (NK cells). Reference values for the lymphocyte subpopulation were obtained from over 100 healthy Caucasian adult volunteers. Blood from these donors was analyzed using FACScan flow cytometer, Leuco-GATE, Simultest and FACS Lysing Solution, and SimulSET software. As an internal quality control, it was verified that %T+%B+%NK approximates 100% in all samples. The results presented here, obtained on healthy donors (51 males and 49 females), showed that there are no statistically significant variations of CD4/CD8 ratio related to sex or age, the mean value of this ratio (2.0 +/- 0.02) being similar to that reported by the West-European countries. Additional similarities were found when the relative percentage, mean +/- standard deviation (CD3 = 74.2 +/- 2.8; CD19 = 10.8 +/- 1.6; CD4 = 42.0 +/- 2.5; CD8 = 28.9 +/- 5.7; CD56 = 15.2 +/- 0.4) and the absolute cell number of the major peripheral blood mononuclear subsets established in our study were compared with other published results. This study was entirely supported by Becton Dickinson--Europe (Division Heidelberg, Germany).