TNF-α Increases Bone Marrow Mesenchymal Stem Cell Migration to Ischemic Tissues

The objective of this study was to analyze the influence of TNF-α on rat mesenchymal stem cells (MSCs) and to assess feasibility of MSC transplantation to repair ischemic injury. In this study, adhesion molecules and cell specific surface markers on MSCs were measured after exposure to different concentrations of TNF-α. MSCs stimulated with varying concentrations of TNF-α were cultured with aortic endothelial cells, and the adhesion rate was measured. MSCs were then stimulated with an optimum concentration of TNF-α as determined in vitro, and injected intravenously into rats with ischemic hind limb injury. The number of MSCs in muscle samples from the ischemic area was counted. The results showed that (1) TNF-α induced a concentration-dependent increase in VCAM-1 expression in MSCs, whereas the expression of L-selectin, ICAM-1 and VLA-4 did not change significantly. Expression of MSC-specific antigens was unchanged. (2) MSCs pretreated with 10 ng/ml TNF-α showed significantly increased adhesion to endothelial cells in vitro, and accumulated to a greater extent in the areas of ischemic damage in rat hind limbs. We were able to conclude that TNF-α has no effect on expression of MSC-specific markers, but can increase the expression of VCAM-1 on rat MSCs. Suitable concentrations of TNF-α can promote MSC adhesion to endothelial cells and migration to damaged tissue.     

Lumican – Derived Peptides Inhibit Melanoma Cell Growth and Migration

Lumican, a small leucine-rich proteoglycan of the extracellular matrix, presents potent anti-tumor properties. Previous works from our group showed that lumican inhibited melanoma cell migration and tumor growth in vitro and in vivo. Melanoma cells adhered to lumican, resulting in a remodeling of their actin cytoskeleton and preventing their migration. In addition, we identified a sequence of 17 amino acids within the lumican core protein, named lumcorin, which was able to inhibit cell chemotaxis and reproduce anti-migratory effect of lumican in vitro. The aim of the present study was to characterize the anti-tumor mechanism of action of lumcorin. Lumcorin significantly decreased the growth in monolayer and in soft agar of two melanoma cell lines – mice B16F1 and human SK-MEL-28 cells – in comparison to controls. Addition of lumcorin to serum free medium significantly inhibited spontaneous motility of these two melanoma cell lines. To characterize the mechanisms involved in the inhibition of cell migration by lumcorin, the status of the phosphorylation/dephosphorylation of proteins was examined. Inhibition of focal adhesion kinase phosphorylation was observed in presence of lumcorin. Since cancer cells have been shown to migrate and to invade by mechanisms that involve matrix metalloproteinases (MMPs), the expression and activity of MMPs were analyzed. Lumcorin induced an accumulation of an intermediate form of MMP-14 (~59kDa), and inhibited MMP-14 activity. Additionally, we identified a short, 10 amino acids peptide within lumcorin sequence, which was able to reproduce its anti-tumor effect on melanoma cells. This peptide may have potential pharmacological applications.     

The p50 Subunit of NF-κB Orchestrates Dendritic Cell Lifespan and Activation of Adaptive Immunity

Dendritic cells play a central role in keeping the balance between immunity and immune tolerance. A key factor in this equilibrium is the lifespan of DC, as its reduction restrains antigen availability leading to termination of immune responses. Here we show that lipopolysaccharide-driven DC maturation is paralleled by increased nuclear levels of p50 NF-κB, an event associated with DC apoptosis. Lack of p50 in murine DC promoted increased lifespan, enhanced level of maturation associated with increased expression of the proinflammatory cytokines IL-1, IL-18 and IFN-β, enhanced capacity of activating and expanding CD4⁺ and CD8⁺ T cells in vivo and decreased ability to induce differentiation of FoxP3⁺ regulatory T cells. In agreement, vaccination of melanoma-bearing mice with antigen-pulsed LPS-treated p50^−/− BM-DC boosted antitumor immunity and inhibition of tumor growth. We propose that nuclear accumulation of the p50 NF-κB subunit in DC, as occurring during lipopolysaccharide-driven maturation, is a homeostatic mechanism tuning the balance between uncontrolled activation of adaptive immunity and immune tolerance.     

An Obligatory Role of NF-κB in Mediating Bone Marrow Derived Endothelial Progenitor Cell Recruitment and Proliferation Following Endotoxemic Multiple Organ Injury in Mice

Background

Recruitment of bone marrow derived endothelial progenitor cells (BMDEPCs) alleviates multiple organ injury (MOI) and improves outcomes. However, mechanisms mediating BMDEPC recruitment following septic MOI remain largely unknown. This study characterized the kinetics of BMDEPC recruitment and proliferation and defined the role of NF-κB in regulating BMDEPC recruitment and proliferation.

Methods and Main Findings

Chimeric mice with an intact or disrupted NF-κB p50 gene and BMDEPC-restricted expression of green fluorescent protein were created and injected with LPS (2 mg/kg, i.p.). BMDEPC recruitment and proliferation in multiple organs were quantified. BMDEPC recruitment and proliferation are highly organ-dependent. Lungs had the highest number of BMDEPC recruitment, whereas heart, liver and kidney had only a small fraction of the number of BMDEPCs in lungs. Number of proliferating BMDEPCs was several-fold higher in lungs than in other 3 organs. Kinetically, BMDEPC recruitment into different organs showed different time course profiles. NF-κB plays obligatory roles in mediating BMDEPC recruitment and proliferation. Universal deletion of NF-κB p50 gene inhibited LPS-induced BMDEPC recruitment and proliferation by 95% and 69% in heart. However, the contribution of NF-κB to these regulations varies significantly between organs. In liver, universal p50 gene deletion reduced LPS-induced BMDEPC recruitment and proliferation only by 49% and 35%. NF-κB activities in different tissue compartments play distinct roles. Selective p50 gene deletion either in stromal/parenchymal cells or in BM/blood cells inhibited BMDEPC recruitment by a similar extent. However, selective p50 gene deletion in BM/blood cells inhibited, but in stromal/parenchymal cells augmented BMDEPC proliferation.

Conclusions

BMDEPC recruitment and proliferation display different kinetics in different organs following endotoxemic MOI. NF-κB plays obligatory and organ-dependent roles in regulating BMDEPC recruitment and proliferation. NF-κB activities in different tissue compartments play distinct roles in regulating BMDEPC proliferation.     

Restoration of the GM2 Ganglioside Metabolism in Bone Marrow–Derived Stromal Cells from Tay-Sachs Disease Animal Model

The therapeutic potential of bone marrow–derived stromal cells for the therapy of Tay-Sachs disease is primarily related to the restoration of their own GM2 ganglioside storage. With this aim, we produced bone marrow–derived stromal cells from the adult Tay-Sachs animal model and transduced them with a retroviral vector encoding for the -subunit of the lysosomal enzyme -hexosaminidase A (E.C. 3.2.1.52). Our results demonstrate that transduced Tay-Sachs bone marrow–derived stromal cells have -hexosaminidase A comparable to that of bone marrow-derived stromal cells from wild-type mice. Moreover, -hexosaminidase A in transduced Tay-Sachs bone marrow-derived stromal cells was able to hydrolyze the GM2 ganglioside in a feeding experiment, thus demonstrating the correction of the altered phenotype.     

Real-time Imaging of Leukotriene B4 Mediated Cell Migration and BLT1 Interactions with β-arrestin

G-protein coupled receptors (GPCRs) belong to the seven transmembrane protein family and mediate the transduction of extracellular signals to intracellular responses. GPCRs control diverse biological functions such as chemotaxis, intracellular calcium release, gene regulation in a ligand dependent manner via heterotrimeric G-proteins^1-2. Ligand binding induces a series of conformational changes leading to activation of heterotrimeric G-proteins that modulate levels of second messengers such as cyclic adenosine monophosphate (cAMP), inositol triphosphate (IP3) and diacyl glycerol (DG). Concomitant with activation of the receptor ligand binding also initiates a series of events to attenuate the receptor signaling via desensitization, sequestration and/or internalization. The desensitization process of GPCRs occurs via receptor phosphorylation by G-protein receptor kinases (GRKs) and subsequent binding of β-arrestins³. β-arrestins are cytosolic proteins and translocate to membrane upon GPCR activation, binding to phosphorylated receptors (most cases) there by facilitating receptor internalization ^4-6.Leukotriene B₄ (LTB₄) is a pro-inflammatory lipid molecule derived from arachidonic acid pathway and mediates its actions via GPCRs, LTB₄ receptor 1 (BLT1; a high affinity receptor) and LTB₄ receptor 2 (BLT2; a low affinity receptor)^7-9. The LTB₄-BLT1 pathway has been shown to be critical in several inflammatory diseases including, asthma, arthritis and atherosclerosis^10-17. The current paper describes the methodologies developed to monitor LTB₄-induced leukocyte migration and the interactions of BLT1 with β-arrestin and , receptor translocation in live cells using microscopy imaging techniques^18-19.Bone marrow derived dendritic cells from C57BL/6 mice were isolated and cultured as previously described ^20-21. These cells were tested in live cell imaging methods to demonstrate LTB₄ induced cell migration. The human BLT1 was tagged with red fluorescent protein (BLT1-RFP) at C-terminus and β-arrestin1 tagged with green fluorescent protein (β-arr-GFP) and transfected the both plasmids into Rat Basophilic Leukomia (RBL-2H3) cell lines^18-19. The kinetics of interaction between these proteins and localization were monitored using live cell video microscopy. The methodologies in the current paper describe the use of microscopic techniques to investigate the functional responses of G-protein coupled receptors in live cells. The current paper also describes the use of Metamorph software to quantify the fluorescence intensities to determine the kinetics of receptor and cytosolic protein interactions.Download video file.^{(88M, mov)}     

Antitumor Effect of AZD4547 in a Fibroblast Growth Factor Receptor 2–Amplified Gastric Cancer Patient–Derived Cell Model

BACKGROUND: FGFR2 amplification is associated with aggressive gastric cancer (GC), and targeted drugs have been developed for treatment of GC. We evaluated the antitumor activity of an FGFR inhibitor in FGFR2-amplified GC patients with peritoneal carcinomatosis. METHODS: Two GC patients with FGFR2 amplification confirmed by fluorescence in situ hybridization showed peritoneal seeding and malignant ascites. We used the patient-derived xenograft model; patient-derived cells (PDCs) from malignant ascites were used to assess FGFR2 expression and its downstream pathway using immunofluorescence analysis and immunoblot assay in vitro. Apoptosis and cell cycle arrest after treatment of FGFR inhibitor were analyzed by Annexin V-FITC assay and cell cycle analysis. RESULTS: FGFR2 amplification was verified in both PDC lines. AZD4547 as an FGFR inhibitor decreased proliferation of PDCs, and the IC₅₀ value was estimated to be 250 nM in PDC#1 and 210 nM in PDC#2. FGFR inhibitor also significantly decreased levels of phosphorylated FGFR2 and downstream signaling molecules in FGFR2-amplified PDC lines. In cell cycle analysis, apoptosis was significantly increased in AZD4547-treated cells compared with nontreated cells. The proportion of cells in the sub-G1 stage was significantly higher in AZD4547-treated PDCs than in control cells. CONCLUSION: Our findings suggest that FGFR2 amplification is a relevant therapeutic target in GC with peritoneal carcinomatosis.     

Paracrine Effects of Bone Marrow–Derived Endothelial Progenitor Cells: Cyclooxygenase-2/Prostacyclin Pathway in Pulmonary Arterial Hypertension

Background

Endothelial dysfunction is the pathophysiological characteristic of pulmonary arterial hypertension (PAH). Some paracrine factors secreted by bone marrow–derived endothelial progenitor cells (BMEPCs) have the potential to strengthen endothelial integrity and function. This study investigated whether BMEPCs have the therapeutic potential to improve monocrotaline (MCT)-induced PAH via producing vasoprotective substances in a paracrine fashion.

Methods and Results

Bone marrow-derived mononuclear cells were cultured for 7 days to yield BMEPCs. 24 hours or 3 weeks after exposure to BMEPCs in vitro or in vivo, the vascular reactivity, cyclooxygenase-2 (COX-2) expression, prostacyclin (PGI₂) and cAMP release in isolated pulmonary arteries were examined respectively. Treatment with BMEPCs could improve the relaxation of pulmonary arteries in MCT-induced PAH and BMEPCs were grafted into the pulmonary bed. The COX-2/prostacyclin synthase (PGIS) and its progenies PGI₂/cAMP were found to be significantly increased in BMEPCs treated pulmonary arteries, and this action was reversed by a selective COX-2 inhibitor, NS398. Moreover, the same effect was also observed in conditioned medium obtained from BMEPCs culture.

Conclusions

Implantation of BMEPCs effectively ameliorates MCT-induced PAH. Factors secreted in a paracrine fashion from BMEPCs promote vasoprotection by increasing the release of PGI₂ and level of cAMP.     

Gap Junction–mediated Cell–Cell Communication Modulates Mouse Neural Crest Migration

Previous studies showed that conotruncal heart malformations can arise with the increase or decrease in α₁ connexin function in neural crest cells. To elucidate the possible basis for the quantitative requirement for α₁ connexin gap junctions in cardiac development, a neural crest outgrowth culture system was used to examine migration of neural crest cells derived from CMV43 transgenic embryos overexpressing α₁ connexins, and from α₁ connexin knockout (KO) mice and FC transgenic mice expressing a dominant-negative α₁ connexin fusion protein. These studies showed that the migration rate of cardiac neural crest was increased in the CMV43 embryos, but decreased in the FC transgenic and α₁ connexin KO embryos. Migration changes occurred in step with connexin gene or transgene dosage in the homozygous vs. hemizygous α₁ connexin KO and CMV43 embryos, respectively. Dye coupling analysis in neural crest cells in the outgrowth cultures and also in the living embryos showed an elevation of gap junction communication in the CMV43 transgenic mice, while a reduction was observed in the FC transgenic and α₁ connexin KO mice. Further analysis using oleamide to downregulate gap junction communication in nontransgenic outgrowth cultures showed that this independent method of reducing gap junction communication in cardiac crest cells also resulted in a reduction in the rate of crest migration. To determine the possible relevance of these findings to neural crest migration in vivo, a lacZ transgene was used to visualize the distribution of cardiac neural crest cells in the outflow tract. These studies showed more lacZ-positive cells in the outflow septum in the CMV43 transgenic mice, while a reduction was observed in the α₁ connexin KO mice. Surprisingly, this was accompanied by cell proliferation changes, not in the cardiac neural crest cells, but in the myocardium— an elevation in the CMV43 mice vs. a reduction in the α₁ connexin KO mice. The latter observation suggests that cardiac neural crest cells may have a role in modulating growth and development of non–neural crest– derived tissues. Overall, these findings suggest that gap junction communication mediated by α₁ connexins plays an important role in cardiac neural crest migration. Furthermore, they indicate that cardiac neural crest perturbation is the likely underlying cause for heart defects in mice with the gain or loss of α₁ connexin function.     

NK Cells Regulate CD8+ T Cell Priming and Dendritic Cell Migration during Influenza A Infection by IFN-γ and Perforin-Dependent Mechanisms

An effective immune response against influenza A infection depends on the generation of virus-specific T cells. NK cells are one of the first-line defenses against influenza A infection. We set out to delineate the role of NK cells in T cell immunity using a murine model of influenza A infection with A/PR/8/34. We show that early T cell recruitment mainly occurs in the posterior mediastinal lymph node (pMLN). Depletion of NK cells significantly impaired both dendritic cell (DC) and T cell recruitment into the pMLN. A similar reduction of T cell recruitment was observed when migration was blocked by pertussis toxin, suggesting that migration of pulmonary NK cells and DCs regulates cell recruitment to the pMLN. T cell recruitment was dependent on IFN-γ, and transfer of IFN-γ-competent naive NK cells into IFN-γ(-/-) mice restored T cell recruitment, whereas IFN-γ-deficient NK cells failed to do so. In addition, NK cell depletion reduced the uptake and transport of influenza A virus by DCs, and significantly impaired the virus-specific T cell response. Both IFN-γ(-/-) and perforin(-/-) mice showed reduced viral Ag transport by DCs, suggesting that the ability of NK cells to influence virus transport depends on IFN-γ and perforin. In summary, our data suggest that NK cells play a critical role in the initiation and shaping of the T cell response after influenza A infection.     

Cutaneous and Bone Marrow Histoplasmosis After 18 Years of Renal Allograft Transplant

The frequency of histoplasmosis among solid organ transplant (SOT) recipients appears to be low where there are only a few case series, mostly among renal and liver transplant recipients. Herein we report a case of a 44-year-old woman who underwent a living-related renal transplant 18 years prior to evaluation, developed a nodule after followed by ulceration upon her posterior right leg and a second one upon her left leg 3 months and 2 months before her hospitalisation, respectively. The biopsy of lesion revealed the presence of Histoplasma spp. Bone marrow aspiration was performed and also revealed the same organism. She had initially received itraconazole without improvement of lesions, while a new lesion appeared on her left arm. Healing of all lesions could be observed after 40 days of liposomal amphotericin B when she was submitted to skin grafts on the legs and a surgical treatment on the arms, and the myelosuppression improved simultaneously. Histoplasmosis seems to be very uncommon among patients who underwent to organ solid transplantation. Most cases occur within 12–18 months after transplantation, although unusual cases have been presented many years post-transplant. There are cases reported in the literature, occurring from 84 days to 18 years after organ transplantation, but without cutaneous involvement. Our patient developed lesions on limbs and myelosuppression after 18 years of chronic immunosuppression medication. This case suggests that besides cutaneous histoplasmosis is an uncommon infection following iatrogenic immunosuppression and even rarer over a long period after the transplantation. Clinicians who care SOT recipient patients must bear in mind histoplasmosis infection as differential diagnosis in any case of cutaneous injury with prolonged fever and try to use as many tools as possible to make the diagnosis, once this disease presents a good prognosis if it is diagnosed and treated promptly.     

Variation of Neisseria gonorrhoeae Lipooligosaccharide Directs Dendritic Cell–Induced T Helper Responses

Gonorrhea is one of the most prevalent sexually transmitted diseases in the world. A naturally occurring variation of the terminal carbohydrates on the lipooligosaccharide (LOS) molecule correlates with altered disease states. Here, we investigated the interaction of different stable gonoccocal LOS phenotypes with human dendritic cells and demonstrate that each variant targets a different set of receptors on the dendritic cell, including the C-type lectins MGL and DC-SIGN. Neisseria gonorrhoeae LOS phenotype C constitutes the first bacterial ligand to be described for the human C-type lectin receptor MGL. Both MGL and DC-SIGN are locally expressed at the male and female genital area, the primary site of N. gonorrhoeae infection. We show that targeting of different C-type lectins with the N. gonorrhoeae LOS variants results in alterations in dendritic cell cytokine secretion profiles and the induction of distinct adaptive CD4⁺ T helper responses. Whereas N. gonorrhoeae variant A with a terminal N-acetylglucosamine on its LOS was recognized by DC-SIGN and induced significantly more IL-10 production, phenotype C, carrying a terminal N-acetylgalactosamine, primarily interacted with MGL and skewed immunity towards the T helper 2 lineage. Together, our results indicate that N. gonorrhoeae LOS variation allows for selective manipulation of dendritic cell function, thereby shifting subsequent immune responses in favor of bacterial survival.     

Molecular Bases of Cell–Cell Junctions Stability and Dynamics

Epithelial cell–cell junctions are formed by apical adherens junctions (AJs), which are composed of cadherin adhesion molecules interacting in a dynamic way with the cortical actin cytoskeleton. Regulation of cell–cell junction stability and dynamics is crucial to maintain tissue integrity and allow tissue remodeling throughout development. Actin filament turnover and organization are tightly controlled together with myosin-II activity to produce mechanical forces that drive the assembly, maintenance, and remodeling of AJs. In this review, we will discuss these three distinct stages in the lifespan of cell–cell junctions, using several developmental contexts, which illustrate how mechanical forces are generated and transmitted at junctions, and how they impact on the integrity and the remodeling of cell–cell junctions.Cell–cell junction formation and remodeling occur repeatedly throughout development. Epithelial cells are linked by apical adherens junctions (AJs) that rely on the cadherin-catenin-actin module. Cadherins, of which epithelial E-cadherin (E-cad) is the most studied, are Ca²⁺-dependent transmembrane adhesion proteins forming homophilic and heterophilic bonds in trans between adjacent cells. Cadherins and the actin cytoskeleton are mutually interdependent (Jaffe et al. 1990; Matsuzaki et al. 1990; Hirano et al. 1992; Oyama et al. 1994; Angres et al. 1996; Orsulic and Peifer 1996; Adams et al. 1998; Zhang et al. 2005; Pilot et al. 2006). This has long been attributed to direct physical interaction of E-cad with β-catenin (β-cat) and of α-catenin (α-cat) with actin filaments (for reviews, see Gumbiner 2005; Leckband and Prakasam 2006; Pokutta and Weis 2007). Recently, biochemical and protein dynamics analyses have shown that such a link may not exist and that instead, a constant shuttling of α-cat between cadherin/β-cat complexes and actin may be key to explain the dynamic aspect of cell–cell adhesion (Drees et al. 2005; Yamada et al. 2005). Regardless of the exact nature of this link, several studies show that AJs are indeed physically attached to actin and that cadherins transmit cortical forces exerted by junctional acto-myosin networks (Costa et al. 1998; Sako et al. 1998; Pettitt et al. 2003; Dawes-Hoang et al. 2005; Cavey et al. 2008; Martin et al. 2008; Rauzi et al. 2008). In addition, physical association depends in part on α-cat (Cavey et al. 2008) and additional intermediates have been proposed to represent alternative missing links (Abe and Takeichi 2008) (reviewed in Gates and Peifer 2005; Weis and Nelson 2006). Although further work is needed to address the molecular nature of cadherin/actin dynamic interactions, association with actin is crucial all throughout the lifespan of AJs. In this article, we will review our current understanding of the molecular mechanisms at work during three different developmental stages of AJs biology: assembly, stabilization, and remodeling, with special emphasis on the mechanical forces controlling AJs integrity and development.     

The Additive Effect of Mesenchymal Stem Cells and Bone Morphogenetic Protein 2 on γ-Irradiated Bone Marrow in Mice

Irradiation from γ-rays can cause severe damage to bone marrow and hematopoietic tissues. Presently, the most effective method available to treat severe hematopoietic injury is a bone marrow transplant (BMT). Allogeneic BMT is a difficult technique to perform due to the differences in human leukocyte antigen proteins between the donor and recipient, with acute graft-versus-host disease being a major complication of the technique. This limits the widespread applicability of allogeneic BMT. To develop a novel treatment for acute hematopoietic damage, we transplanted bone marrow derived mesenchymal stem cells (MSCs) into recipient mice and treated them with recombinant human bone morphogenetic protein 2 (rhBMP2) to investigate whether MSCs and rhBMP2 could additively promote the restoration of hematopoietic function. MSCs are vital components of the hematopoietic microenvironment that supports hematopoiesis, and bone morphogenic protein is a key factor in hematopoiesis. The 30-day survival rate as well as the numbers of nucleated cells, bone marrow colony-forming unit-granulocyte macrophages, spleen colony-forming units and peripheral blood cells were enumerated. The results showed that, after γ-irradiation and transplantation, MSCs and rhBMP2 additively promoted and improved hematopoietic restoration and function in vivo and in vitro. This additive effect of MSCs and rhBMP2 may one day provide a novel means of treating acute hematopoietic damage.     

Bulgarian Bone Marrow Donors Registry—past and future directions

Recently Bulgarian Bone Marrow Donors Registry (BBMDR) has been established and since August 2005 it has been a member of Bone Marrow Donors Worldwide. Currently the number of healthy donors included in the BBMDR is relatively low. All donors included in the BBMDR are typed for HLA-A, -B, -DRB loci. Phylogenetic analysis based on HLA allele frequencies shows that Bulgarians were characterized with closest genetic similarity to Macedonians, Greeks, Romanians, Cretans and Sardinians in comparison to the other European and Mediterranean populations. On the contrary the second largest ethnic minority–the Roma were the closest to the other Roma populations and North Indians. These differences were due to the predominance of alleles and haplotypes that are specific for the Asian and the other Roma populations. These specific genetic profiles in the Bulgarian ethnic minorities justify the need of an adequate representation of minorities in BBMDR. Future directions for BBMDR development are discussed, including an increase of the total number of donors and these for ethnic minorities, as well the enhancement of the level of resolution of the HLA typing for the donors in the registry.