Intercellular adhesion molecule-1 expression by skeletal muscle cells augments myogenesis.

We previously demonstrated that the expression of intercellular adhesion molecule-1 (ICAM-1) by skeletal muscle cells after muscle overload contributes to ensuing regenerative and hypertrophic processes in skeletal muscle. The objective of the present study is to reveal mechanisms through which skeletal muscle cell expression of ICAM-1 augments regenerative and hypertrophic processes of myogenesis. This was accomplished by genetically engineering C2C12 myoblasts to stably express ICAM-1, and by inhibiting the adhesive and signaling functions of ICAM-1 through the use of a neutralizing antibody or cell penetrating peptide, respectively. Expression of ICAM-1 by cultured skeletal muscle cells augmented myoblast-myoblast adhesion, myotube formation, myonuclear number, myotube alignment, myotube-myotube fusion, and myotube size without influencing the ability of myoblasts to proliferate or differentiate. ICAM-1 augmented myotube formation, myonuclear accretion, and myotube alignment through a mechanism involving adhesion-induced activation of ICAM-1 signaling, as these dependent measures were reduced via antibody and peptide inhibition of ICAM-1. The adhesive and signaling functions of ICAM-1 also facilitated myotube hypertrophy through a mechanism involving myotube-myotube fusion, protein synthesis, and Akt/p70s6k signaling. Our findings demonstrate that ICAM-1 expression by skeletal muscle cells augments myogenesis, and establish a novel mechanism through which the inflammatory response facilitates growth processes in skeletal muscle.

LFA-1 and ICAM-1 expression induced during melanoma-endothelial cell co-culture favors the transendothelial migration of melanoma cell lines in vitro.

BACKGROUND: Patients with metastatic melanoma have a poor median rate of survival. It is therefore necessary to increase our knowledge about melanoma cell dissemination which includes extravasation, where cancer cells cross the endothelial barrier. Extravasation is well understood during travelling of white blood cells, and involves integrins such as LFA-1 (composed of two chains, CD11a and CD18) expressed by T cells, while ICAM-1 is induced during inflammation by endothelial cells. Although melanoma cell lines cross endothelial cell barriers, they do not express LFA-1. We therefore hypothesized that melanoma-endothelial cell co-culture might induce the LFA-1/ICAM ligand/receptor couple during melanoma transmigration. METHODS: A transwell approach has been used as well as blocking antibodies against CD11a, CD18 and ICAM-1. Data were analyzed with an epifluorescence microscope. Fluorescence intensity was quantified with the ImageJ software. RESULTS: We show here that HUVEC-conditioned medium induce cell-surface expression of LFA-1 on melanoma cell lines. Similarly melanoma-conditioned medium activates ICAM-1 expression in endothelial cells. Accordingly blocking antibodies of ICAM-1, CD11a or CD18 strongly decrease melanoma transmigration. We therefore demonstrate that melanoma cells can cross endothelial monolayers in vitro due to the induction of ICAM-1 and LFA-1 occurring during the co-culture of melanoma and endothelial cells. Our data further suggest a role of LFA-1 and ICAM-1 in the formation of melanoma cell clumps enhancing tumor cell transmigration. CONCLUSION: Melanoma-endothelial cell co-culture induces LFA-1 and ICAM-1 expression, thereby favoring in vitro melanoma trans-migration.

Immunomodulatory action of dietary fish oil and targeted deletion of intestinal epithelial cell PPARδ in inflammation-induced colon carcinogenesis.

The ligand-activated transcription factor peroxisome proliferator-activated receptor (PPAR)-δ is highly expressed in colonic epithelial cells; however, the role of PPARδ ligands, such as fatty acids, in mucosal inflammation and malignant transformation has not been clarified. Recent evidence suggests that the anti-inflammatory/chemoprotective properties of fish oil (FO)-derived n-3 polyunsaturated fatty acids (PUFAs) may be partly mediated by PPARδ. Therefore, we assessed the role of PPARδ in modulating the effects of dietary n-3 PUFAs by targeted deletion of intestinal epithelial cell PPARδ (PPARδ(ΔIEpC)). Subsequently, we documented changes in colon tumorigenesis and the inflammatory microenvironment, i.e., local [mesenteric lymph node (MLN)] and systemic (spleen) T cell activation. Animals were fed chemopromotive [corn oil (CO)] or chemoprotective (FO) diets during the induction of chronic inflammation/carcinogenesis. Tumor incidence was similar in control and PPARδ(ΔIEpC) mice. FO reduced mucosal injury, tumor incidence, colonic STAT3 activation, and inflammatory cytokine gene expression, independent of PPARδ genotype. CD8(+) T cell recruitment into MLNs was suppressed in PPARδ(ΔIEpC) mice. Similarly, FO reduced CD8(+) T cell numbers in the MLN. Dietary FO independently modulated MLN CD4(+) T cell activation status by decreasing CD44 expression. CD11a expression by MLN CD4(+) T cells was downregulated in PPARδ(ΔIEpC) mice. Lastly, splenic CD62L expression was downregulated in PPARδ(ΔIEpC) CD4(+) and CD8(+) T cells. These data demonstrate that expression of intestinal epithelial cell PPARδ does not influence azoxymethane/dextran sodium sulfate-induced colon tumor incidence. Moreover, we provide new evidence that dietary n-3 PUFAs attenuate intestinal inflammation in an intestinal epithelial cell PPARδ-independent manner.

Protein kinase R is a novel mediator of CD40 signaling and plays a critical role in modulating immunoglobulin expression during respiratory syncytial virus infection.

Effective immunoglobulin responses play a vital role in protection against most pathogens. However, the molecular mediators and mechanisms responsible for signaling and selective expression of immunoglobulin types remain to be elucidated. Previous studies in our laboratory have demonstrated that protein kinase R (PKR) plays a crucial role in IgE responses to double-stranded RNA (dsRNA) in vitro. In this study, we show that PKR plays a critical role in IgG expression both in vivo and in vitro. PKR(-/-) mice show significantly altered serum IgG levels during respiratory syncytial virus (RSV) infection. IgG2a expression is particularly sensitive to a lack of PKR and is below the detection level in mock- or RSV-infected PKR(-/-) mice. Interestingly, we show that upon activation by anti-CD40 and gamma interferon (IFN-γ), B cells from PKR(-/-) mice show diminished major histocompatibility complex class II (MHC II), CD80, and CD86 levels on the cell surface compared to wild-type (WT) mice. Our data also show that PKR is necessary for optimal expression of adhesion molecules, such as CD11a and ICAM-1, that are necessary for homotypic aggregation of B cells. Furthermore, in this report we demonstrate for the first time that upon CD40 ligation, PKR is rapidly phosphorylated and activated, indicating that PKR is an early and novel downstream mediator of CD40 signaling pathways.

Increased survival and reduced neutrophil infiltration of the liver in Rab27a- but not Munc13-4-deficient mice in lipopolysaccharide-induced systemic inflammation.

Genetic defects in the Rab27a or Munc13-4 gene lead to immunodeficiencies in humans, characterized by frequent viral and bacterial infections. However, the role of Rab27a and Munc13-4 in the regulation of systemic inflammation initiated by Gram-negative bacterium-derived pathogenic molecules is currently unknown. Using a model of lipopolysaccharide-induced systemic inflammation, we show that Rab27a-deficient (Rab27a(ash/ash)) mice are resistant to lipopolysaccharide (LPS)-induced death, while Munc13-4-deficient (Munc13-4(jinx/jinx)) mice show only moderate protection. Rab27a(ash/ash) but not Munc13-4(jinx/jinx) mice showed significantly decreased tumor necrosis factor alpha (TNF-α) plasma levels after LPS administration. Neutrophil sequestration in lungs from Rab27a(ash/ash) and Munc13-4(jinx/jinx) LPS-treated mice was similar to that observed for wild-type mice. In contrast, Rab27a- but not Munc13-4-deficient mice showed decreased neutrophil infiltration in liver and failed to undergo LPS-induced neutropenia. Decreased liver infiltration in Rab27a(ash/ash) mice was accompanied by lower CD44 but normal CD11a and CD11b expression in neutrophils. Both Rab27a- and Munc13-4-deficient mice showed decreased azurophilic granule secretion in vivo, suggesting that impaired liver infiltration and improved survival in Rab27a(ash/ash) mice is not fully explained by deficient exocytosis of this granule subset. Altogether, our data indicate that Rab27a but not Munc13-4 plays an important role in neutrophil recruitment to liver and LPS-induced death during endotoxemia, thus highlighting a previously unrecognized role for Rab27a in LPS-mediated systemic inflammation.

The Bordetella pertussis adenylate cyclase toxin binds to T cells via LFA-1 and induces its disengagement from the immune synapse.

The Bordetella pertussis adenylate cyclase toxin (CyaA) assists infection by potently suppressing the host immune response. Although CyaA effectively targets T lymphocytes, its putative receptor on these cells is unknown. Here, we show that CyaA binds to T cells via the ß2 integrin LFA-1 in its active conformation. CyaA clusters with LFA-1 at the immune synapse (IS), from which it induces the premature disengagement of LFA-1 concomitant with the dissipation of talin, which tethers the integrin to the underlying actin cytoskeleton. The CyaA-induced redistribution of LFA-1 was cAMP- and protein kinase A (PKA)-dependent. These results not only identify LFA-1 as a CyaA receptor on T cells but unveil a novel mechanism of immunosuppression whereby the toxin parasitizes its interaction with LFA-1 to inhibit signaling at the IS through the local production of cAMP. The data also provide novel insights into the role of cAMP/PKA signaling in controlling the dynamics of the IS.

Gene expression profiles of T cells from hepatitis E virus infected patients in acute and resolving phase.

BACKGROUND AND AIMS: Approximately 50% of acute viral hepatitis in young adults and in pregnant women is due to hepatitis E virus (HEV) infection in developing countries. T cell-mediated immune injury probably plays a key role in the pathogenesis of acute hepatitis illness. However, there is a paucity of data on the global gene expression programs activated on T cells, which are subsequently responsible for T cell recruitment to the liver and triggering of immune injury. PATIENTS AND METHODS: We performed a flow cytometric analysis of T cells in individuals with acute hepatitis E (AVH-E; n=10), resolving phase of HEV (n=9), and ten healthy controls (HC). Further transcriptional profiling analysis was performed using Affymetrix GeneChip DNA microarrays to identify the genes that were differentially expressed in AVH-E and HC. RESULTS: Patients with AVH-E showed higher frequencies of CD8+ (27 ± 4%; P=0.02) and activated CD38+ CD69+ T cells (25% ± 3%; P=0.04) than in resolving phase patients (20 ± 2% and 9.1 ± 4%, respectively), who in turn exhibited higher CCR9 expression than cells from patients in active phase. The naïve T cell population (CD3+ CD45RA+) was decreased upon HEV infection (29 ± 4% in AVH-E vs. 53.1 ± 3.2% in HC; P=0.05); however, the CD11a high subpopulation within CD4+ CD45RA+ cells was increased in both AVH-E (6.1%) and resolving phase (7.7%) patients. Gene ontology analysis suggested that during AVH-E infection, there is in CD4+ T cells an activation of genes involved in pro-inflammatory responses. Additional RT-PCR analysis confirmed that in cells from AVH-E patients, there is an increased expression of CCR5, CCR9, CXCR3, CXCR4, STAT1, IRF-9, IFN-α, and TNF-α, together with a down-regulation of IL-2, SOCS3, and IL-10, with respect to cells from resolving phase patients. CONCLUSIONS: Our findings suggest the involvement of a circulating CD45RA+ CD11a high population with CCR5 expression in the pathogenesis processes of AVH-E. The obtained results help to understand the underlying inflammatory process occurring in HEV infection, which can lead to either resolution or immunopathology.

Forced swimming test decreases chemotactic responsiveness and expression of CD11a in rat monocytes.

OBJECTIVES: Previous reports have shown that the depressive status in humans and experimental animals is associated with decreased immune response. Since monocyte chemotaxis and expression of CD11a are pivotal mechanisms in immune response, impairment of these events could explain the diminished immune response in depression. METHODS: To test this, rats were submitted to the forced swimming test (FST) for 3 and 15 days. Animals were sacrificed at days 4 (3 days' FST), 16 (15 days' FST) and 30 (15 days' FST and 15 days of recovery time). At these times, a blood sample was obtained for serum and leukocyte isolation. Mononuclear leukocytes were obtained by Histopaque gradient. Chemotaxis responsiveness was determined in Boyden chambers using zymosan-activated rat serum. Cellular CD11a expression and serum CD11a were determined by immunofluorescence and ELISA, respectively. RESULTS: Decreased chemotaxis was observed in FST animals at days 4 and 16 with total recovery at day 30. Diminished expression of cellular CD11a was observed at day 16 and remained decreased at day 30. There were no significant differences in serum CD11a content. CONCLUSION: Decreased chemotactic response and expression of CD11a found in this experimental model of depression could be important mechanisms to induce impairment immune response in experimental and clinical depression.

Epigenetics and SLE: RFX1 downregulation causes CD11a and CD70 overexpression by altering epigenetic modifications in lupus CD4+ T cells.

DNA demethylation and histone hyperacetylation of CD11a and CD70 regulatory regions contribute to the development of autoreactivity and autoantibody overstimulation in CD4(+) T cells of patients with systemic lupus erythematosus (SLE). However, the mechanisms causing these changes remain largely unknown. We report that the expression and activity of the transcription factor RFX1 are decreased in SLE CD4(+) T cells. We demonstrate that RFX1 affects DNA methylation and histone acetylation in CD4(+) T cells by recruiting the co-repressors DNMT1 and HDAC1 to the CD11a and CD70 promoters, and thereby represses their expression. Reducing RFX1 in CD4(+) T cells is sufficient to cause lupus-like T and B cell hyperactivity, whereas overexpressing RFX1 suppresses T cell reactivity. These findings reveal a crucial role for RFX1 in regulating the epigenetic status of T cells, and demonstrate that autoimmune responses in SLE are due in part to RFX1 downregulation.

RFX1 regulates CD70 and CD11a expression in lupus T cells by recruiting the histone methyltransferase SUV39H1.

INTRODUCTION: Regulatory factor X-box 1 (RFX1) can interact with DNA methyltransferase 1 (DNMT1) and histone deacetylase 1 (HDAC1), and RFX1 down-regulation contributes to DNA hypomethylation and histone H3 hyperacetylation at the cluster of differentiation (CD) 11a and CD70 promoters in CD4(+) T cells of patients with systemic lupus erythematosus (SLE). This leads to CD11a and CD70 overexpression, thereby triggering autoimmune responses. In order to provide more insight into the epigenetic mechanisms leading to the deregulation of autoimmune-related genes in SLE, we asked whether RFX1 is involved in regulating histone 3 lysine 9 (H3K9) tri-methylation at the CD11a and CD70 promoters in SLE CD4(+) T cells. METHODS: CD4(+) T cell samples were isolated from 15 SLE patients and 15 healthy controls. H3K9 tri-methylation levels were measured by chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. CD4(+) T cells were transfected with plasmids using the Human T cell Nucleofector Kit. RFX1 and histone methyltransferase suppressor of variegation 3-9 (Drosophila) homolog 1 (SUV39H1) interaction was determined by co-immunoprecipation (co-IP) and Western blot and immunofluorescence staining. CD11a and CD70 mRNA levels were measured by real-time RT-PCR. RESULTS: H3K9 tri-methylation levels were significantly reduced within the CD11a and CD70 promoter regions in SLE CD4(+) T cells. RFX1 co-immunoprecipitated with SUV39H1 at the CD11a and CD70 promoters in healthy control CD4(+) T cells. Overexpressing or knocking-down RFX1 revealed that RFX1 expression correlated with H3K9 tri-methylation levels, as well as CD11a and CD70 expression levels in CD4(+) T cells. CONCLUSIONS: RFX1 recruits SUV39H1 to the promoter regions of the CD11a and CD70 genes in CD4(+) T cells, thereby regulating local H3K9 tri-methylation levels. These findings shed further light on the central role of RFX1 down-regulation in the epigenetic de-repression of auto-immune genes in SLE.

Tracking the total CD8 T cell response to infection reveals substantial discordance in magnitude and kinetics between inbred and outbred hosts.

Determining the magnitude and kinetics, together with the phenotypic and functional characteristics of responding CD8 T cells, is critical for understanding the regulation of adaptive immunity as well as in evaluating vaccine candidates. Recent technical advances have allowed tracking of some CD8 T cells responding to infection, and a body of information now exists describing phenotypic changes that occur in CD8 T cells of known Ag-specificity during their activation, expansion, and memory generation in inbred mice. In this study, we demonstrate that Ag but not inflammation-driven changes in expression of CD11a and CD8alpha can be used to distinguish naive from Ag-experienced (effector and memory) CD8 T cells after infection or vaccination. Interestingly and in contrast to inbred mice, tracking polyclonal CD8 T cell responses with this approach after bacterial and viral infections revealed substantial discordance in the magnitude and kinetics of CD8 T cell responses in outbred hosts. These data reveal limitations to the use of inbred mouse strains as preclinical models at vaccine development and suggest the same dose of infection or vaccination can lead to substantial differences in the magnitude and timing of Ag-specific CD8 expansion as well in differences in protective memory CD8 T cell numbers in outbred individuals. This concept has direct relevance to development of vaccines in outbred humans.

Identification of a NCR+/NKG2D+/LFA-1(low)/CD94(-) immature human NK cell subset.

CD56(bright) natural killer (NK) cells, generated in vitro from CD34+ hematopoietic progenitor cells, were characterized after a 30-day culture with flt3 ligand plus IL-15. Virtually, all CD56(bright) cells expressed CD117, CD25, natural cytotoxicity receptors (NCRs), NKG2D, CD161, and CD244, while only a subset expressed CD18-CD11a (LFA-1), and CD94 molecule, defining an immature CD56(bright)/NCRs+/NKG2D+/LFA-1(-)/CD94(-) subset. Another small subset of cells expressing CD94 but not LFA-1 integrin was also identified, suggesting that during NK differentiation LFA-1 might be upregulated later than CD94. To verify this hypothesis in vivo, we evaluated the NK cell expression of LFA-1 in both peripheral and umbilical cord blood samples. Interestingly, in these blood fluids, we have identified a lineage negative CD34(-)/LFA-1(low)/NKp46(dim)/NKG2D(dim)/CD94(-) subset that resembled an immature stage of NK cells present in lymph nodes. Altogether, the results indicate that CD18-CD11a integrin, as well as CD11b in mice, may be a useful marker to identify immature stages of NK cell differentiation.

LFA-1 and CD2 synergize for the Erk1/2 activation in the Natural Killer (NK) cell immunological synapse.

Natural killer (NK) cell recognition and formation of a conjugate with target cells, followed by intracellular signal pathway activation and degradation of cytolytic granules, are essential for NK cell cytotoxicity. In this study, NK92 cells were used to investigate synapse formation and subsequent signaling after binding to the target cell. The binding rate of the NK92-target cell was associated with NK92 cell cytotoxicity. Confocal results showed that adhesion molecules, LFA-1 (CD11a) and CD2, accumulated at the interface of the NK92-K562 contact. Ligation with K562 cells activated the Erk1/2 signal pathway of NK92 cells. The blocking of the NK-target conjugate by EDTA or anti-CD11a or/and anti-CD2 antibody decreased the phosphorylation of Erk1/2 and NK cell cytotoxicity. Inhibition of Erk1/2 phosphorylation by the chemical inhibitor U0126 suppressed the cytolytic activity of NK92 cells, but had no effect on NK-target conjugate formation. Thus, conjugate formation of the NK92-target cell was prerequisite to NK cell activation, and subsequent signal transduction was also required for NK cell cytotoxicity.

Characterization and osteogenic effects of mesenchymal stem cells on microbeads composed of hydroxyapatite nanoparticles/reconstituted collagen.

Natural bone is comprised of nanosized blade-like crystals of hydroxyapatite grown in close contact with collagen (Col) fibers. Characteristics of artificial bone tissue differ considerably with those of natural ones, mainly from the unusual self-organizing interaction between the apatite crystals and the proteic components. Nanoparticle spheres of hydroxyapatite (n-HA), dispersed in reconstituted fibrous Col, were prepared in three weight ratios of 75:25, 65:35, and 50:50 (n-HA:Col). Bone marrow mesenchymal stem cells (MSCs) from rabbits were seeded and cultured on the n-HA/Col microbeads and characterized. n-HA were evenly distributed throughout the Col matrix and aggregated to microbeads as determined by scanning electron microscopy. Electron and confocal microscopy showed that the MSCs spread and attached to microbeads via focal adhesions, while staining for F-actin and DNA revealed the presence of stress fibers. The phenotype of the MSCs in the flow cytometry was identified as CD11a-, CD44+, and CD90.1+. The optimal weight ratio is 65:35 for the normalized alkaline phosphatase activities. The transduced MSCs, engineered by replication-defective adenovirus to express the BMP-2 gene, demonstrated synergic osteogenic effects in the microbeads. MSCs are capable of proliferating and differentiating in appropriate combinations of n-HA/Col. Thus it is a promising composite for future clinical applications.

Reperfusion injury and inflammatory responses following acute lower limb revascularization surgery.

After revascularization of an acute arterial occlusion the development of a serious ischaemic-reperfusion injury is a menacing challenge and a hard task in peripheral vascular surgery. A whale of evidences point to oxidative stress, as an important trigger, in the complex chain of events leading to reperfusion injury. In the present study authors aimed to examine oxidative stress parameters, antioxidant-prooxidant state and leukocyte adhesion molecules (CD11a and CD18) expression following acute revascularization surgery of lower limb.10 patients were examined in the prospective randomized study. Peripheral blood sample was collected in ischaemic period, and after reperfusion in the 2nd and 24th hours, and on 7th day. Superoxide-dismutase activity, reduced glutathion concentration and leukocytes free radical production were measured. The degree of lipidperoxidation was marked with the quantity of malondialdehyde. The expressions of adhesion molecules were measured with flowcytometry.The speed and rate of free radical production significantly increased in the early reperfusion (p<0.05). The level of antioxidant enzymes decreased after revascularization. The CD11a and CD18 expression of the granulocytes significantly (p<0.05) decreased right after the revascularization, but with a gradual elevation until the 7th day they exceed the ischaemic value. Our results showed a time specific turnover of the sensitive antioxidant-prooxidant balance after revascularization operation.

Transfer of maternal colostral leukocytes promotes development of the neonatal immune system I. Effects on monocyte lineage cells.

Although it has been established that maternal leukocytes traffic from colostrum into the neonatal circulation, the effects of these cells on neonatal immunity are only beginning to be understood. This study examined the effects of maternal colostral leukocytes on development and maturation of neonatal antigen presenting cells. At birth, groups of neonatal calves received whole or cell-free colostrum (CFC) from their respective dams. Peripheral blood samples were obtained over the first 4 weeks of life, and expression of surface markers associated with cellular activation and physiological stress were monitored on monocyte lineage cells. Calves receiving cell-free colostrum at birth expressed elevated levels of CD11a, CD11c, and CD14, compared to calves receiving whole colostrum (C). Calves receiving cell-free colostrum had an elevated number of monocytes in the peripheral blood during the first 2 weeks of life, however, these cells expressed lower levels of expression of CD25 and MHC class I compared to calves receiving whole colostrum. The most significant differences in marker expression occurred within the first 7 days of life.

Integrin expression and H2O2 production in circulating and splenic leukocytes of obese rats.

OBJECTIVE: Obesity is associated with oxidative stress and inflammation. We hypothesized that the pro-inflammatory state in obesity may result in spontaneous activation and, hence, increased generation of reactive oxygen species (ROS) and integrin expression in the circulating leukocytes. METHODS: Flow cytometry was used to determine integrin expression (immunostaining) as well as superoxide and hydrogen peroxide productions (fluorescent probes) in the peripheral blood and splenic leukocyte of 24-week-old male obese normotensive and not-as-yet diabetic Zucker rats (n = 6) and their lean counterparts (n = 6). RESULTS: Obese rats had hyperlipidemia and normal arterial pressure, plasma glucose, and creatinine concentrations. Nevertheless, obese rats exhibited increased hydrogen peroxide production by circulating and splenic CD4+ and CD8+ T lymphocytes and by splenic macrophages. This was accompanied by up-regulations of CD11a expression in the peripheral blood and splenic CD4+ T cells, CD11b in circulating macrophages, and CD11a and CD18 in circulating granulocytes. CONCLUSION: The study revealed direct evidence of spontaneous leukocyte activation and increased ROS generation by T lymphocytes and monocytes in the peripheral blood of obese Zucker rats before the development of diabetes or hypertension. These findings illustrate the link between obesity, oxidative stress, and inflammation.

Overexpression of a hematopoietic transcriptional regulator EDAG induces myelopoiesis and suppresses lymphopoiesis in transgenic mice.

Erythroid differentiation-associated gene (EDAG) is a hematopoietic tissue-specific gene that is highly expressed in the earliest CD34+ lin- bone marrow (BM) cells and involved in the proliferation and differentiation of hematopoietic cells. To investigate the role of EDAG in hematopoiesis, we established an EDAG transgenic mouse model driven by human CD11a promoter. The transgenic mice showed increased mortality with severe organ infiltration by neutrophils, and the homeostasis of hematopoiesis was broken. The myelopoiesis was enhanced with expansion of myeloid cells in BM, increased peripheral granulocytes and extramedullary myelopoiesis in spleen. In contrast to myeloid cells, the lymphoid commitment was severely impaired with the B lymphopoiesis blocked at the transition from pro/pre-B I to pre-B II stage in BM and T thymocytes development blocked at the most immature stage (DN I). Moreover, we showed that EDAG was a transcriptional regulator which had transactivation activity and regulated the expression of several key transcription factors such as PU.1 and Pax5 in transgenic hematopoietic stem cells. These data suggested that EDAG was a key transcriptional regulator in maintaining the homeostasis of hematopoietic lineage commitment.

Paradoxical drop in circulating neutrophil count following granulocyte-colony stimulating factor and stem cell factor administration in rhesus macaques.

OBJECTIVE: Granulocyte colony-stimulating factor (G-CSF) is frequently used therapeutically to treat chronic or transient neutropenia and to mobilize hematopoietic stem cells. Shortly following G-CSF administration, we observed a dramatic transient drop in circulating neutrophil number. This article characterizes this effect in a rhesus macaque animal model. METHODS: Hematologic changes were monitored following subcutaneous (SQ) administration of G-CSF. G-CSF was administered as a single SQ dose at 10 microg/kg or 50 microg/kg. It was also administered (10 microg/kg) in combination with stem cell factor (SCF; 200 microg/kg) over 5 days. Flow cytometry was performed on serial blood samples to detect changes in cell surface adhesion protein expression. RESULTS: Neutrophil count dramatically declined 30 minutes after G-CSF administration. This decline was observed whether 10 microg/kg G-CSF was administered in combination with SCF over 5 days, or given as a single 10 microg/kg dose. At a single 50 microg/kg dose, the decline accelerated to 15 minutes. Neutrophil count returned to baseline after 120 minutes and rapidly increased thereafter. An increase in CD11a and CD49d expression coincided with the drop in neutrophil count. CONCLUSION: A transient paradoxical decline in neutrophil count was observed following administration of G-CSF either alone or in combination with SCF. This decline accelerated with the administration of a higher dose of G-CSF and was associated with an increase in CD11a and CD49d expression. It remains to be determined whether this decline in circulating neutrophils is associated with an increase in endothelial margination and/or entrance into extravascular compartments.

The role of selenite on microglial migration.

Oxidative brain damage, such as excitotoxicity and stroke, leads to primary neuronal destruction. The primary damage is further potentiated by macrophages and microglial cells, which are attracted and invade into the zone of damage resulting in secondary neuronal death. Since the essential trace element selenium has anti-inflammatory properties, we analyzed the effects of selenium on these inflammatory cells. Here, we show that the essential trace element selenium abrogates the stress-induced migration of microglial cells. Thus, the antimigratory effects of selenium may attenuate the secondary cell death cascade by preventing microglial invasion.