Prevalent Cell Type Research Articles

P112 Deeper phenotyping of non CF bronchiectasis through sputum differential counts

Introduction and ObjectivesNon CF Bronchiectasis has diverse aetiologies. This includes idiopathic, systemic disease related and as a complication of asthma. Such diversity may be important in determining therapeutic strategies (personalised...

Prognostic impact of tumour-infiltrating immune cells on biliary tract cancer

Background:Biliary tract cancers (BTC) are relatively rare malignant tumours with poor prognosis. It is known from other solid neoplasms that antitumour inflammatory response has an impact on tumour behaviour and patient outcome. The aim of this study was to provide a comprehensive characterisation of antitumour inflammatory response in human BTC.Methods:Tumour-infiltrating T lymphocytes (CD4+, CD8+, and Foxp3+), natural killer cells (perforin+), B lymphocytes (CD20+), macrophages (CD68+) as well as mast cells (CD117+) were assessed by immunohistochemistry in 375 BTC including extrahepatic (ECC; n=157), intrahepatic (ICC; n=149), and gallbladder (GBAC; n=69) adenocarcinomas. Overall and intraepithelial quantity of tumour-infiltrating immune cells was analysed. Data were correlated with clinicopathological variables and patient survival.Results:The most prevalent inflammatory cell type in BTC was the T lymphocyte. Components of the adaptive immune response decreased, whereas innate immune response components increased significantly in the biliary intraepithelial neoplasia – primary carcinoma – metastasis sequence. BTC patients with intraepithelial tumour-infiltrating CD4+, CD8+, and Foxp3+ T lymphocytes showed a significantly longer overall survival. Number of total intraepithelial tumour-infiltrating Foxp3+ regulatory T lymphocytes (HR: 0.492, P=0.002) and CD4+ T lymphocytes (HR: 0.595, P=0.008) were tumour grade- and UICC-stage-independent prognosticators. The subtype-specific evaluation revealed that the tumour-infiltrating lymphocytic infiltrate is a positive outcome predictor in ECC and GBAC but not in ICC.Conclusion:Our findings characterise the immune response in cholangiocarcinogenesis and identify inflammatory cell types that influence the outcome of BTC patients. Further, we show that BTC subtypes show relevant differences with respect to density, quality of inflammation, and impact on patient survival.

GW24-e2948 Small interfering RNA transfection in cultured cardiac fibroblasts

ObjectivesSmall interfering RNA (siRNA) methodology suppresses specific gene expression, thus mimicking loss-of-function mutation and enabling in vitro and in vivo gene function analysis. siRNA technology is also a promising tool...

Transient Receptor Potential (TRP) Channels and Cardiac Fibrosis

Cardiac fibrosis is associated with most cardiac diseases. Fibrosis is an accumulation of excessive extracellular matrix proteins (ECM) synthesized by cardiac fibroblasts and myofibroblasts. Fibroblasts are the most prevalent cell type in the heart, comprising 75% of cardiac cells. Myofibroblasts are hardly present in healthy normal heart tissue, but appear abundantly in diseased hearts. Cardiac fibroblasts are activated by a variety of pathological stimuli, such as myocardial injury, oxidative stress, mechanical stretch, and elevated autocrine-paracrine mediators, thereby undergoing proliferation, differentiation to myofibroblasts, and production of various cytokines and ECM proteins. A number of signaling pathways and bioactive molecules are involved and work in concert to activate fibroblasts and myofibroblasts in the fibrogenesis cascade. Fibroblasts and myofibroblasts are not only principal ECM producers, but also play a central role in fibrogenesis and myocardial remodeling in fibrotic heart disease. Thus, understanding the biological processes of cardiac fibroblasts will provide novel insights into the underlying mechanisms of fibrosis and provide potential targets for developing antifibrotic drugs. Recent studies demonstrate that Ca2+ signal is essential for fibroblast proliferation, differentiation, and ECM-protein production. This review focuses on the recent advances in understanding molecular mechanisms of Ca2+ signaling in cardiac fibrogenesis, and potential role of Ca(2+)-permeable channels, in particular, the transient potential (TRP) channels in fibrotic heart disease. TRP channels are highly expressed in cardiac fibroblasts. TRPM7 has been shown to be essential in TGFβ1 mediated fibrogenesis, and TRPC3 has been demonstrated to play an essential role in regulating fibroblast function. Thus, the Ca2+-permeable TRP channels may serve as potential novel targets for developing anti-fibrotic drugs.

Bone Marrow-Derived Adipocytes Promote Differentiation, Proliferation and Survival of Acute Monoblastic Leukemia Cells

Abstract 1501Acute monoblastic leukemia occurs most commonly in young individuals, whereas acute monocytic leukemia is common in adults (WHO Classification of Tumours of Haematopoietic and Lymphoid Tissues, 2008). Adipocytes are the prevalent stromal cell type in adult bone marrow (BM) and play an important role in the leukemic bone marrow microenvironment (Tabe et al., Blood 2004 103: 1815–22). BM stromal cells (MSCs) from elderly subjects have a reduced capacity to differentiate into osteoblasts and an increased capacity to differentiate into adipocytes, which leads to progressive accumulation of fat in the BM space with increasing age. In this study, we examined whether BM-derived adipocytes participate in the molecular events associated with proliferation, differentiation and apoptosis of monoblastic leukemia cells. To this end, we performed gene expression microarray analysis of 339 genes associated with lipid metabolism (GeneSQUARE, Kurabo, Japan) using RNA from monoblastic leukemia cell line U937 co-cultured with MSC or MSC-derived adipocytes. Five genes were upregulated (>2.0 fold) and 2 genes down-regulated in U937 cells co-cultured with adipocytes compared to U937 cultured alone. The up-regulated genes included monocyte/macrophage differentiation specific genes, such as scavenger receptor CD36, adipsin or fatty acid binding protein 4 (FABP4). CD36 and FABP4 are classical target genes of PPARγ which was also upregulated in U937 co-cultured with adipocytes. Upregulation of the inflammation-related haptoglobin gene was observed in U937 cells co-cultured with either adipocytes or MSC. These findings were confirmed by quantitative RT-PCR assay with concordant results. Down-regulated genes included monocyte chemoattractant protein-1 (MCP-1) (0.1 fold) and glycolytic enzyme hexokinase 2 (0.4 fold). In turn, U937 cells co-cultured with premature or mature adipocytes accumulated in G2 cell cycle phase compared to U937 cultured alone or co-cultured with MSC (% G2/M-phase fraction; U937 cultured alone, 14.6, co-culture with MSC 25.1, co-culture with preadipocyte 32.0, with mature adipocyte 31.9) and were protected from spontaneous cell death under serum-starved conditions (% subG1 fraction: U937 cultured alone 23.9%, co-cultured with MSC 15.7%, with preadipocytes 3.9% and with mature adipocytes 11.6%). In summary, results suggest that BM adipocytes, abundant in adult BM space, promote monocytic differentiation, proliferation and survival of monoblastic leukemia cells. We propose that these cells represent an essential component of the BM microenvironment that contributes to malignant phenotypes of adults with monocytic leukemia. Disclosures:No relevant conflicts of interest to declare.

The response to valve injury. A paradigm to understand the pathogenesis of heart valve disease

Human heart valve diseases have become an important topic in cardiovascular pathology and medicine. These diseases have different etiologies and manifestations. However, the most common ones including calcific aortic stenosis have histopathological features that are best characterized as a "response to tissue injury" similar to ones seen in numerous tissues and organs. The valve interstitial cell is the prevalent cell type in the valve and is likely the master cell which ultimately regulates cell and molecular repair processes within the valve that involve autocrine and paracrine processes as well as interactions with the matrix components of the valve. This presentation explores the concept of "response to tissue injury" in understanding the pathogenesis of calcific aortic stenosis.

The Context‐dependent Effect of Transforming Growth Factor Beta on Valve Interstitial Cell Growth in Vitro

BackgroundValve interstitial cells (VICs) are the most prevalent cell type in heart valves. We showed that in the early stages of VIC wound repair transforming growth factor beta (TGF‐β) promotes VIC proliferation and repair (A. Liu et al. Am J Pathol 2008; 173:1275–1285). However, the effect of TGF‐β on VIC growth independent of injury is poorly understood.Materials and MethodsWe used normal subconfluent VIC monolayer cultures to test the hypothesis that TGF‐β enhances VIC proliferation. Control and TGF‐β treated VICs were plated at low density. Cell number and proliferation (bromodeoxyuridine) were quantified. Cell morphology was observed and the extent of VIC adhesion to the substratum was quantified using a trypsin adhesion assay.ResultsTGF‐β significantly decreased cell number and altered cell morphology as cells were mostly rhomboid‐shaped as opposed to spindle‐shaped. Cell proliferation was significantly inhibited and treated cells showed significantly more adhesion to the substratum, which may contribute to the observed growth inhibition.ConclusionThe effect of TGF‐β on VIC growth is context dependent, allowing it to play a dual role by preventing excessive heart valve growth in normal adult conditions and promoting cell proliferation in the early stages of repair, as we reported previously.Supported by a grant from the Heart and Stroke Foundation of Ontario (grant NA6204).

Pro-Apoptotic and Proliferative Effects of Bone Marrow Adipocytes on Myeloid Leukemia Cells.

Abstract 4572Bone marrow stromal cells (MSCs) from elderly subjects have a reduced capacity to differentiate into osteoblasts and an increased capacity to differentiate into adipocytes, which leads to progressive accumulation of fat in the bone marrow space with increasing age. Adipocytes are the prevalent stromal cell type in adult BM that play an important role in the leukemic bone marrow microenvironment (Tabe et al., Blood 2004 103:1815-22). In this study, we examined the role of BM-derived adipocytes at different stages of differentiation on proliferation and apoptosis of AML cells. U937 cells were co-cultured with BM-derived MSC, MSC-differentiated pre-adipocytes (containing few small lipid vesicles), and mature adipocytes (with multiple hypertrophic lipid vesicules). Under serum-starved conditions, MSC and premature/mature adipocytes induced cell cycle progression of U937 cells with increase in the proportion of cells in S- and G2/M-phase fractions, and inhibited spontaneous cell death with decrease in subG1 fractions. However, only pre-adipocytes inhibited Ara-C-induced cell killing (Table 1). [Display omitted] We next focused on lepin and plasminogen activator inhibitor 1(PAI-1) as potential mediators of these effects by adipocytes. Leptin mRNA and protein levels were upregulated during adipocytic differentiation (mRNA relative expression to GAPDH (PCR): MSC 0, premature adipocyte 2.0±0.5, mature adipocyte 123.3± 35.0; leptin secretion: MSC 23.1±2.9, premature adipocyte 49.3±11.3, mature adipocyte 110.0±4.6 pg/mL (ELISA). PAI-1 mRNA levels were increased in mature adipocyte (relative expression to GAPDH: MSC 314.9±46.5, premature adipocyte 215.1, mature adipocyte 3766.1±656.2). Since PPARÿ activation is known to promote maturation and re-generation of fat-derived adipocytes, we next examined the potential of the synthetic triterpenoid 2-cyano-3,12-dioxooleana-1,9-dien-28-oic acid (CDDO) on the BM adipocytes, leptin and PAI-1 production and the survival of the leukemic cells. CDDO induced adipocyte re-generation with significant increase of the number of Oil-Red(+) small sized lipid vesicles without apoptosis induction. This resulted in a markedly enhanced leptin release from adipocytes (10-fold and 23-fold increase at 0.5 μM and 1.0 μM CDDO, respectively, at 72 hrs) without change in leptin mRNA transcription. On the contrary, PAI-1 mRNA levels were significantly decreased by CDDO (6 fold decrease in MSC, 4 fold decrease in premature adipocyte, 6 fold decrease in mature adipocyte). Co-culture of U937 cells with CDDO-primed premature adipocytes and mature adipocytes resulted in increased spontaneous apoptosis of U937 cells compared to adipocytes not exposed to CDDO (% specific apoptosis, U937 co-cultured with CDDO-primed premature adipocytes 26.9 %, mature adipocytes 20.9 %). At the same time, CDDO-primed premature adipocytes induced significant cell cycle progression with decreased proportion of G0/G1-phase and increase in S-phase fractions in U937 cells (Table 2). Co-culture with CDDO-primed premature adipocytes or with premature adipocytes co-treated with recombinant leptin increased subG1- and S-phase fractions in Ara-C-treated U937 cells compared to U937 cells co-cultured with premature adipocytes (Table 3). [Display omitted] In mature adipocytes, which already produce high levels of leptin, CDDO or leptin treatment failed to modulate anti-apoptotic or proliferative effects of AraC on U937 cells. In contrast, human recombinant PAI-1 effectively inhibited spontaneous and Ara-C induced apoptosis of U937 cells (decreased % of Annexin V: spontaneous apoptosis 11.2±1.1%, Ara-C induced apoptosis 15.1± 1.7%).In summary, these results suggest that BM pre-adipocytes support proliferation and survival of myeloid leukemia cells in part through complementary effects of leptin and PAI-1. Our findings indicate that secretion of leptin during MSC differentiation or through PPARg ligation promotes cell cycle progression, while PAI-1 primarily inhibits apoptosis of AML cells. It is conceivable that increased adipocyte content of BM in elderly AML patients may negatively affect the responsiveness of AML cells to chemotherapy. Disclosures:No relevant conflicts of interest to declare.

GATA4, a new regulator of cardiac fibroblasts, is sensitive to natriuretic peptides

Cardiac fibroblasts are the most prevalent cell type in the heart. These cells exert a critical role in regulating normal myocardial function and in adverse myocardial remodelling that occur with hypertension, myocardial infarction, and heart failure.1 In normal cardiac tissue, fibroblasts are found throughout the cardiac tissue in parallel to the prevailing direction of muscle fibres. In essence, every cardiac myocyte is therefore in direct contact with fibroblasts. Adverse myocardial remodelling includes enlargement of myocytes, proliferation of fibroblasts, and increased procollagen synthesis. Natriuretic peptides, including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), released from myocytes have been shown to act as autocine/paracrine factors that reduce myocyte hypertrophy and exert antifibrotic and antiproliferative effects on cardiac fibroblasts.2,3 Antifibrotic effects are attributed to C-type natriuretic peptide (CNP) released from endothelial cells and cardiac fibroblasts.4 Natriuretic peptides initiate their signalling via guanylyl cyclase receptors (NPR-A, NPR-B) and C-type receptor (NPR-C) all expressed in cardiac fibroblasts.3,4 These effects may be of particular importance during pathological conditions, when the fibrosis and increased deposition of collagen results in increased myocardial stiffness and diastolic dysfunction. Myocardial hypertrophy is also characterized by activation of transcription factors. A number of stimuli that induce myocyte hypertrophy are known to enhance GATA4 transcriptional activity through … *Corresponding author. Tel: +1 514 890 8000; fax: +1 514 412 7204; E-mail address : marek.jankowski{at}umontreal.ca

Fibroblastic reticular cells and their role in viral hemorrhagic fevers

Viral hemorrhagic fevers (VHFs) caused by Ebola, Marburg and Lassa viruses often manifest as multiple organ dysfunction and hemorrhagic shock with high mortality. These viruses target numerous cell types, including monocytes and dendritic cells, which are primary early targets that mediate critical pathogenetic processes. This review focuses on fibroblastic reticular cells (FRCs), another prevalent infected cell type that is known as a key regulator of circulatory and immune functions. Viral infection of FRCs could have debilitating effects in secondary lymphoid organs and various other tissues. FRCs may also contribute to the spread of these deadly viruses throughout the body. Here, we review the salient features of these VHFs and the biology of FRCs, emphasizing the potential role of these cells in VHFs and the rapid deterioration of immune and hemovascular sytems that are characteristic of such acute infections.

Pattern of FGF-2 isoform expression correlated with its biological action in experimental prolactinomas

Fibroblast growth factor-2 (FGF-2) synthesized in the pituitary is involved in the formation and progression of pituitary tumors. The aim of this study was to analyze the pattern expression of two FGF-2 isoforms at different subcellular levels and to determine its correlation with prolactinoma development. Estrogen administration to male rats for 7, 20, and 60 days generated pituitary tumors, with lactotrophs being the prevalent cell type. Ultrastructural immunolabeling showed FGF-2 in the cytosolic and nuclear compartments of somatotrophs, lactotrophs and gonadotrophs, as well as in folliculo-stellate cells of normal rats. Estrogen stimulation increased FGF-2 immunoreactivity in various tumors and enhanced the expression of two FGF-2 isoforms, 18 and 22 kDa, as quantified by western blot. The 18 kDa isoform observed in cytosol extracts reached the highest levels after 60 days of hormonal stimulation and this was related to lactotroph proliferation. However, the 22 kDa FGF-2 isoform was only detected in the nuclear compartment and achieved the maximum expression at 7 days of estrogen treatment, without any correlation with lactotroph proliferation. These results suggest that the 18 kDa FGF-2 may play a role in the modulation of lactotroph proliferation in prolactinomas induced by estrogen. The overproduction of both FGF-2 isoforms appears to be implicated in autocrine-paracrine-intracrine mitogenic loops; this FGF-2 activity could lead to uncontrolled cell growth, angiogenesis, and tumor formation.

Giant Cell Osteosarcoma in the Calvarium of a Cat

Feline primary osteosarcomas involving the skull are extremely rare. When they occur, orbit, mandible, and maxilla are the most common sites. Microscopically, scattered multinucleated giant cells (MGCs) are not an uncommon occurrence in osteosarcoma (OSC), but they are generally in low number. Only in a rare variant, the giant cell-rich OSC, are MGCs the prevalent cell type. Although osteoclast and osteoblast origin have been postulated in human and veterinary literature, the origin of MGCs in osteosarcomas is poorly understood. This report describes a giant cell-rich OSC in the calvarium of a 13-year-old spayed female shorthair cat. The animal exhibited a range of neurologic signs, including left circling, compulsive gait, lack of proprioception, and bilateral absence of menace reaction, with indication of left forebrain involvement. Gross lesions were characterized by a multilobate, spherical mass located in the left calvarium, compressing the left forebrain. Histologically, the tumor was characterized by scattered nests of MGCs separated by small bundles of pleomorphic, fusate to polygonal cells. Between spindle cells, osteoid was very sparse and arranged in thin strands. Immunohistochemical stains for vimentin were positive, with no detectable cellular staining for cytokeratin, S-100 protein, or Class II major histocompatibility complex. Ultrastructurally, MGCs contained profiles of rough endoplasmic reticulum; no lysosomes were observed. The origin of MGCs in osteosarcoma remains obscure, and our results confirm their ambiguous identity.

Serum deprivation improves seeding and repopulation of acellular matrices with valvular interstitial cells

Cell-extracted valvular tissues (acellular scaffolds, or aScaffolds) offer unique advantages over synthetic polymers for cardiac valve engineering applications in that they retain extracellular matrix molecules to support cellular ingrowth. The extracellular matrix is important in directing many cellular pathways, such as adhesion, proliferation, migration, differentiation, and survival. However, repopulating this type of scaffold often requires high seeding densities or recurrent cell delivery. The optimization of valvular interstitial cell (VIC) seeding onto aScaffolds is reported herein. VICs (the most prevalent cell type in valve leaflets) have maximal growth in 15-20% serum concentrations on tissue-culture polystyrene. Interestingly, after VIC seeding onto aScaffolds, a reduction of serum content, from 15% serum to 5% or less, was found to increase significantly the number of adherent cells, as well as induce transfer of VICs from a tissue-culture polystyrene surface to the aScaffold. aScaffolds seeded and cultured with periods of reduced serum levels were shown to support and enhance VIC viability and attachment, as well as accelerate VIC migration into the aScaffold, leading to a uniformly repopulated valve leaflet construct after 4 weeks of static culture.

Crosslinked hyaluronan scaffolds as a biologically active carrier for valvular interstitial cells

Hyaluronic acid (HA), a major component of the cardiac jelly during heart morphogenesis, is a polysaccharide that upon modification can be photopolymerized into hydrogels. Previous work in our lab has found that photopolymerizable HA hydrogels are suitable scaffolds for the culture and proliferation of valvular interstitial cells (VICs), the most prevalent cell type in native heart valves. The physical properties of HA gels are easily modified through alteration in material crosslink density or by copolymerizing with other reactive macromolecules. Degradation products of HA gels and the starting macromers significantly increased VIC proliferation when added to cell cultures. With low molecular weight HA (<6700 Da) exhibiting greatest stimulation of VIC proliferation. Low molecular weight HA degradation products added to VIC cultures also resulted in a four-fold increase in total matrix production and a two-fold increase in elastin production over untreated controls. VIC internalization of HA, as shown by cellular uptake of fluorescently labeled HA, likely activates signaling cascades resulting in the biological responses seen here. Lastly, VICs encapsulated within HA hydrogels remained viable, and significant elastin production was observed after 6 weeks of culture. This work shows promise for the creation of a tissue-engineered heart valve utilizing the synergistic relationship between hyaluronic acid and VICs.

Entire mitogen activated protein kinase (MAPK) pathway is present in preimplantation mouse embryos.

To understand how mitogenic signals are transduced into the trophoblasts in preimplantation embryos, the expression of mitogen-activated protein kinase (MAPK) pathway molecules was tested. We used immunocytochemical means and reverse transcriptase-polymerase chain reaction to test whether MAPK pathway molecule gene products exist at the protein and phosphoprotein level in the zygote and the RNA level in the egg and zygote. In addition, all antibodies detected the correct-sized major band in Westerns of placental cell lines representing the most prevalent cell type in preimplantation embryos. A majority of mRNA transcripts of MAPK pathway genes were detected in unfertilized eggs, and all were expressed in the zygote. We found that the MAPK pathway protein set consisting of the following gene products was present: FRS2 alpha, GRB2, GAB1, SOS1, Ha-ras, Raf1/RafB, MEK1,2,5, MAPK/ERK1,2, MAPK/ERK5, and RSK1,2,3 (see abbreviations). These proteins were detected in trophoblasts in embryonic day (E) 3.5 embryos when they could mediate mitogenic fibroblast growth factor signals from the embryo or colony stimulating factor-1 signals from the uterus. The phosphorylation state and position of the phosphoproteins in the cells suggested that they might function in mediating mitogenic signals. Interestingly, a subtle transition from maternal MAPK function to zygotic function was suggested by the localization for three MAPK pathway enzymes between E2.5 and E3.5, Raf1 phospho is largely cell membrane-localized at E2.5 and E3.5, and MEK1,2 phospho accumulates in the nucleus on E2.5 and E3.5. However, MAPK phospho shifts from nuclear accumulation at E2.5 to cytoplasmic accumulation at E3.5. This finding is similar to the cytoplasmic MAPK phospho localization reported in fibroblast growth factor signaling fields in postimplantation embryos (Corson et al. [2003] Development 130:4527-4537). This spatial and temporal expression study lays a foundation to plan and analyze perturbation studies aimed at understanding the role of the major mitogenic pathway in preimplantation mouse embryos.

Migration of epidermal keratinocytes: mechanisms, regulation, and biological significance.

Keratinocytes are the prevalent cell type of the epidermis, a multilayered cornified epithelium which provides the cellular basis of the outermost barrier between the organism and its environment. By this barrier function the epidermis protects the organism against a variety of environmental hazards such as dehydration and mechanical stress. Under normal conditions, keratinocytes of all layers are interconnected by desmosomes and anchored by hemidesmosomes to a specialised type of extracellular matrix, the basement membrane. When the epidermis is injured, a vitally important response is initiated with the aim to restore the protective function of the epithelium. A fast but provisional sealing is achieved by the deposition of the fibrin clot before within 24 h after wounding keratinocytes from the wound margins begin to migrate into the wound bed, where they start to proliferate and to form the new epithelium. The development of new high-resolution assays for the study of cell migration and motility has potentiated major progress in our understanding of keratinocyte migration in vitro and in situ. The data reviewed here point to a sophisticated cooperation between soluble motogenic growth factors, cell-matrix interactions, and cell-to-cell communications as major parts of the machinery regulating keratinocyte migration.

Chronic ethanol inhibits CXC chemokine ligand 10 production in human A172 astroglia and astroglial-mediated leukocyte chemotaxis

Astroglia are the most prevalent cell type in the human central nervous system (CNS) and perform important roles in normal tissue homeostasis, during pathological events and following trauma. Astroglial-derived chemokines have important neurotrophic effects and are important to CNS immunocompetence and response to injury, in part, due to their direct role in leukocyte and microglial cell recruitment. However, while ethanol is known to induce CNS pathologies and to be peripherally immunosuppressive, ethanol effects on chemokine expression in human astroglia are essentially unknown. We have demonstrated that chemotaxis of human U937 leukocytic cells, across a 0.5 μm pore polycarbonate transmembrane insert, is induced in response to culture media collected from 10 μg/ml lipopolysaccharide (LPS)+10 ng/ml interleukin (IL)-1β-stimulated A172 human astroglia cells. The involvement of the chemokine CXCL10 (also known as interferon-γ inducible protein or IP-10) in astroglial-induced chemotaxis of U937 cells has been indicated, as chemotaxis can be reduced by an anti-CXCL10 neutralizing antibody. Interestingly, chemotaxis of U937 cells, in response to astroglial-exposed media, is reduced when astroglia are chronically (9 days) exposed to 50 mM ethanol before stimulation with LPS+IL-1β. Furthermore, we observed that LPS+IL-1β-stimulated CXCL10 production is inhibited in human A172 astroglia exposed to chronic 50 mM ethanol. Thus, alterations in astroglial CXCL10 expression may disrupt CNS immunocompetence and play an important role in ethanol-induced CNS pathologies.

Chronic ethanol inhibits CXC chemokine ligand 10 production in human A172 astroglia and astroglial-mediated leukocyte chemotaxis

Astroglia are the most prevalent cell type in the human central nervous system (CNS) and perform important roles in normal tissue homeostasis, during pathological events and following trauma. Astroglial-derived chemokines have important neurotrophic effects and are important to CNS immunocompetence and response to injury, in part, due to their direct role in leukocyte and microglial cell recruitment. However, while ethanol is known to induce CNS pathologies and to be peripherally immunosuppressive, ethanol effects on chemokine expression in human astroglia are essentially unknown. We have demonstrated that chemotaxis of human U937 leukocytic cells, across a 0.5 μm pore polycarbonate transmembrane insert, is induced in response to culture media collected from 10 μg/ml lipopolysaccharide (LPS)+10 ng/ml interleukin (IL)-1β-stimulated A172 human astroglia cells. The involvement of the chemokine CXCL10 (also known as interferon-γ inducible protein or IP-10) in astroglial-induced chemotaxis of U937 cells has been indicated, as chemotaxis can be reduced by an anti-CXCL10 neutralizing antibody. Interestingly, chemotaxis of U937 cells, in response to astroglial-exposed media, is reduced when astroglia are chronically (9 days) exposed to 50 mM ethanol before stimulation with LPS+IL-1β. Furthermore, we observed that LPS+IL-1β-stimulated CXCL10 production is inhibited in human A172 astroglia exposed to chronic 50 mM ethanol. Thus, alterations in astroglial CXCL10 expression may disrupt CNS immunocompetence and play an important role in ethanol-induced CNS pathologies.

Hepatitis C virus (HCV) in lymphocyte subsets and in B lymphocytes expressing rheumatoid factor cross-reacting idiotype in type II mixed cryoglobulinaemia.

The IgMk rheumatoid factors (RF) of type II mixed cryoglobulinaemia (MC) react, in 95% of cases, with MoAbs against the cross-reactive idiotypes (CRI) Cc1 or Lc1 (corresponding to the products of the VH1 and VH4 genes). MC is closely associated with HCV infection, a virus which infects lymphocytes and may replicate in B cells. It has been suggested that HCV may induce clonal selection of B cells producing monoclonal IgMk RF in type II MC. To verify whether HCV is enriched in B cells, and in the subsets expressing Cc1 and Lc1 CRI, we studied peripheral blood lymphocytes from eight patients with MC and HCV RNA-positive sera. Seven patients had RF reacting with anti-Cc1, the other with anti-Lc1 CRI. Total lymphocytes, T cells, B cells, and Cc1+ or Lc1+, Cc1- or Lc1- B cells were purified using MoAb-coated magnetic beads. Lymphocyte subsets were then diluted to give a range of 1 x 106-1 x 103 cells and tested for HCV RNA by reverse transcriptase-polymerase chain reaction. HCV was found exclusively in B cells in seven out of eight patients. In three patients HCV was enriched in the Cc1+ cells. In one of these patients, HCV was found exclusively in Cc1+ cells, with Cc1- cells being HCV-. The data indicate that B cells from type II MC patients are almost constantly infected by HCV. In selected cases, B cell subsets expressing IgMk RF CRI are the prevalent cell type infected by HCV. Our data suggest HCV involvement in B cell dysregulation leading to cryoprecipitable IgMk RF production.

La esquizofrenia en atención primaria. Identificación y tratamiento

In human skin, melanocytes and their neighboring keratinocytes have a close functional interrelationship. Keratinocytes, which represent the prevalent cell type of human skin, regulate melanocytes through various mechanisms. Here, we use a keratinocyte and melanoma co-culture system to show for the first time that keratinocytes regulate the cell surface expression of N-cadherin through cell-cell contact. Compared to mono-cultured human melanoma A375 cells, which expressed high levels of N-cadherin, those co-cultured with the HaCaT human keratinocyte cell line showed reduced levels of N-cadherin. This reduction was most evident in areas of A375 cells that underwent cell-cell contact with the HaCaT cells, whereas HaCaT cell-derived extracellular matrix and conditioned medium both failed to reduce N-cadherin levels. The intracellular level of calcium in co-cultured A375 cells was lower than that in mono-cultured A375 cells, and treatment with a cell-permeant calcium chelator (BAPTA) reduced the N-cadherin level of mono-cultured A375 cells. Furthermore, co-culture with HaCaT cells reduced the expression levels of transient receptor potential cation channel (TRPC) 1, −3 and −6 in A375 cells, and siRNA-mediated multi-depletion of TRPC1, -3 and -6 reduced the N-cadherin level in these cells. Taken together, these data suggest that keratinocytes negatively regulate the N-cadherin levels of melanoma cells via cell-to-cell contact-mediated calcium regulation.