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BACKGROUND: Mesenchymal stromal cells (MSC) have been proven to have potent immunosuppressive action and hence have been proposed for the treatment of severe Graft Versus Host Disease. However,in most models,MSC were added at the same time of lymphocyte stimulation,which is quite different from what occurs in vivo. AIMS: To investigate how the timing of lymphocyte activation and the exposure to activation-related cytokines (licensing) can influence the immunosuppressive action of Wharton's jelly stromal cells (WJSC). METHODS: WJSC,licensed or not with activation-related cytokines,were added lymphocytes the same time or 24 hours after their stimulation with phytohaemoagglutinin. Proliferation of lymphocytes and cytokines production was measured after three days co-culture. RESULTS: Lymphocytes stimulated in the presence of WJSC displayed a dramatic decrease in proliferation and production of cytokines,in spite of normal expression of activation markers. The suppression was weakened when targeted lymphocytes were seperated by a membrane and partially rescued by the addition of exogenous l-tryptophan,suggesting a major role for indoleamine 2,3-dioxigenase with a probable paracrine effect. Licensing of WJSC increased the immunosuppressive effect,in both contact and non-contact settings. The timing of WJSC licensing was crucial for the immunosuppressive action. Lymphocytes pre-stimulated alone for 24 h,and added afterwards to non-licensed WJSC,showed normal or even increased proliferation. On the other hand,their proliferation was strongly inhibited by licensed WJSC. CONCLUSIONS: WJSC have a potent immunosuppressive function best realized with direct contact,and increased by licensing signals before and during lymphocyte stimulation. Our results could contribute to the set up of new WJSC-based therapies for severe autoimmuno disorders. View Publication

In ischemic congestive heart failure (CHF),anemia is associated with poor prognosis. Whether anemia develops in nonischemic CHF is uncertain. The hematopoietic inhibitors TNF-alpha and nitric oxide (NO) are activated in ischemic CHF. We examined whether mice with ischemic or nonischemic CHF develop anemia and whether TNF-alpha and NO are involved. We studied mice (n = 7-9 per group) with CHF either due to myocardial infarction (MI) or to overexpression of the Ca(2+)-binding protein calsequestrin (CSQ) or to induced cardiac disruption of the sarcoplasmic reticulum Ca(2+)-ATPase 2 gene (SERCA2 KO). Hematopoiesis was analyzed by colony formation of CD34(+) bone marrow cells. Hemoglobin concentration was 14.0 +/- 0.4 g/dl (mean +/- SD) in controls,while it was decreased to 10.1 +/- 0.4,9.7 +/- 0.4,and 9.6 +/- 0.3 g/dl in MI,CSQ,and SERCA2 KO,respectively (P textless 0.05). Colony numbers per 100,000 CD34(+) cells in the three CHF groups were reduced to 33 +/- 3 (MI),34 +/- 3 (CSQ),and 39 +/- 3 (SERCA2 KO) compared with 68 +/- 4 in controls (P textless 0.05). Plasma TNF-alpha nearly doubled in MI,and addition of anti-TNF-alpha antibody normalized colony formation. Inhibition of colony formation was completely abolished with blockade of endothelial NO synthase in CSQ and SERCA2 KO,but not in MI. In conclusion,the mechanism of anemia in CHF depends on the etiology of cardiac disease; whereas TNF-alpha impairs hematopoiesis in CHF following MI,NO inhibits blood cell formation in nonischemic murine CHF. View Publication

The epithelium of the mammary gland exists in a highly dynamic state,undergoing dramatic morphogenetic changes during puberty,pregnancy,lactation,and regression. The recent identification of stem and progenitor populations in mouse and human mammary tissue has provided evidence that the mammary epithelium is organized in a hierarchical manner. Characterization of these normal epithelial subtypes is an important step toward understanding which cells are predisposed to oncogenesis. This review summarizes progress in the field toward defining constituent cells and key molecular regulators of the mammary epithelial hierarchy. Potential relationships between normal epithelial populations and breast tumor subtypes are discussed,with implications for understanding the cellular etiology underpinning breast tumor heterogeneity. View Publication

Retroviruses can establish persistent infection despite induction of a multipartite antiviral immune response. Whether collective failure of all parts of the immune response or selective deficiency in one crucial part underlies the inability of the host to clear retroviral infections is currently uncertain. We examine here the contribution of virus-specific CD4(+) T cells in resistance against Friend virus (FV) infection in the murine host. We show that the magnitude and duration of the FV-specific CD4(+) T-cell response is directly proportional to resistance against acute FV infection and subsequent disease. Notably,significant protection against FV-induced disease is afforded by FV-specific CD4(+) T cells in the absence of a virus-specific CD8(+) T-cell or B-cell response. Enhanced spread of FV infection in hosts with increased genetic susceptibility or coinfection with Lactate dehydrogenase-elevating virus (LDV) causes a proportional increase in the number of FV-specific CD4(+) T cells required to control FV-induced disease. Furthermore,ultimate failure of FV/LDV coinfected hosts to control FV-induced disease is accompanied by accelerated contraction of the FV-specific CD4(+) T-cell response. Conversely,an increased frequency or continuous supply of FV-specific CD4(+) T cells is both necessary and sufficient to effectively contain acute infection and prevent disease,even in the presence of coinfection. Thus,these results suggest that FV-specific CD4(+) T cells provide significant direct protection against acute FV infection,the extent of which critically depends on the ratio of FV-infected cells to FV-specific CD4(+) T cells. View Publication

TLR4 is a unique TLR because downstream signaling occurs via two separate pathways,as follows: MyD88 and Toll IL-1 receptor (TIR) domain-containing adaptor-inducing IFN-beta (TRIF). In this study,we compared and contrasted the interplay of these pathways between murine dendritic cells (DCs) and macrophages during LPS stimulation. During TLR4 activation,neither pathway on its own was critical for up-regulation of costimulatory molecules in DCs,whereas the up-regulation of costimulatory molecules was largely TRIF dependent in macrophages. LPS-induced secreted factors,of which type I IFNs were one of the active components,played a larger role in promoting the up-regulation of costimulatory molecules in macrophages than DCs. In both cell types,MyD88 and TRIF pathways together accounted for the inflammatory response to LPS activation. Furthermore,signaling of both adaptors allowed maximal T cell priming by LPS-matured DCs,with MyD88 playing a larger role than TRIF. In sum,in our experimental systems,TRIF signaling plays a more important role in LPS-induced macrophage activation than in DC activation. View Publication

To better understand endogenous parameters that influence pluripotent cell differentiation we used human embryonic stem cells (hESCs) as a model system. We demonstrate that differentiation trajectories in aggregate (embryoid body [EB])-induced differentiation,a common approach to mimic some of the spatial and temporal aspects of in vivo development,are affected by three factors: input hESC composition,input hESC colony size,and EB size. Using a microcontact printing approach,size-specified hESC colonies were formed by plating single-cell suspensions onto micropatterned (MP) extracellular matrix islands. Subsequently,size-controlled EBs were formed by transferring entire colonies into suspension culture enabling the independent investigation of colony and aggregate size effects on differentiation induction. Gene and protein expression analysis of MP-hESC populations revealed that the ratio of Gata6 (endoderm-associated marker) to Pax6 (neural-associated marker) expression increased with decreasing colony size. Moreover,upon forming EBs from these MP-hESCs,we observed that differentiation trajectories were affected by both colony and EB size-influenced parameters. In MP-EBs generated from endoderm-biased (high Gata6/Pax6) input hESCs,higher mesoderm and cardiac induction was observed at larger EB sizes. Conversely,neural-biased (low Gata6/Pax6) input hESCs generated MP-EBs that exhibited higher cardiac induction in smaller EBs. Our analysis demonstrates that heterogeneity in hESC colony and aggregate size,typical in most differentiation strategies,produces subsets of appropriate conditions for differentiation into specific cell types. Moreover,our findings suggest that the local microenvironment modulates endogenous parameters that can be used to influence pluripotent cell differentiation trajectories. View Publication

Multiple myeloma is characterized by the clonal expansion of neoplastic plasma cells within the bone marrow,elevated serum immunoglobulin,and osteolytic bone disease. The disease is highly responsive to a wide variety of anticancer treatments including conventional cytotoxic chemotherapy,corticosteroids,radiation therapy,and a growing number of agents with novel mechanisms of action. However,few if any patients are cured with these modalities and relapse remains a critical issue. A better understanding of clonogenic multiple myeloma cells is essential to ultimately improving long-term outcomes,but the nature of the cells responsible for myeloma regrowth and disease relapse is unclear. We review evidence that functional heterogeneity exists in multiple myeloma and discuss potential strategies and clinical implications of the stem-cell model of cancer in this disease. View Publication

The repertoire of receptors that is expressed by NK cells is critical for their ability to kill virally infected or transformed cells. However,the molecular mechanisms that determine whether and when NK receptor genes are transcribed during hemopoiesis remain unclear. In this study,we show that hypomethylation of a CpG-rich region in the mouse NKG2A gene is associated with transcription of NKG2A in ex vivo NK cells and NK cell lines. This observation was extended to various developmental stages of NK cells sorted from bone marrow,in which we demonstrate that the CpGs are methylated in the NKG2A-negative stages (hemopoietic stem cells,NK progenitors,and NKG2A-negative NK cells),and hypomethylated specifically in the NKG2A-positive NK cells. Furthermore,we provide evidence that DNA methylation is important in maintaining the allele-specific expression of NKG2A. Finally,we show that acetylated histones are associated with the CpG-rich region in NKG2A positive,but not negative,cell lines,and that treatment with the histone deacetylase inhibitor trichostatin A alone is sufficient to induce NKG2A expression. Treatment with the methyltransferase inhibitor 5-azacytidine only is insufficient to induce transcription,but cotreatment with both drugs resulted in a significantly greater induction,suggesting a cooperative role for DNA methylation and histone acetylation status in regulating gene expression. These results enhance our understanding of the formation and maintenance of NK receptor repertoires in developing and mature NK cells. View Publication

In a murine model of autoimmunity targeted against the epidermal cell Ags,Skn,adoptive transfer of Skn-immune T cells to immunosuppressed recipients elicits skin lesions in areas of mild epidermal trauma. In this study,we examined peripheral regulation of Skn-induced autoreactivity disrupted by rendering the mice immunoincompetent. We found that regulation of Skn-directed autoimmunity was restored by cotransfer of normal syngeneic spleen cells at twice the concentration of Skn-immune cells and was evidenced by significantly reduced lesion severity by days 5-7 post-cotransfer compared with animals given injections of Skn-immune cells alone. Enrichment and depletion of normal CD4(+) or CD8(+) spleen cells and RT-PCR analysis of selected cytokines identified CD4(+) cells as the regulatory cells in the cotransfer inoculum; however,significant reduction in lesion severity was observed only when there was a concomitant increase in levels of IL-7. The role of IL-7 was further supported in that mice cotransferred with Skn-immune cells plus normal spleen cells,but also treated with anti-IL-7 Ab,no longer exhibited reduced lesion severity. To determine whether IL-7 expression without normal spleen cell cotransfer could modulate lesion development,an IL-7-encoding plasmid (pCMV-Tag1-IL-7) was topically delivered to sites flanking the stressed skin site in Skn-induced autoimmune mice. Daily application of 15 mug of pCMV-Tag1-IL-7 significantly suppressed lesion severity. Our results support a mechanism for CD4(+) T cells and IL-7 in contributing to the control of autoreactivity. View Publication

Murine hematopoietic stem and progenitor cells (HSPCs) home to bone marrow in part by rolling on P-selectin and E-selectin expressed on endothelial cells. Human adult CD34(+) cells,which are enriched in HSPCs,roll on endothelial selectins in bone marrow vessels of nonobese diabetic/severe combined immune deficiency (NOD/SCID) mice. Many human umbilical cord blood (CB) CD34(+) cells do not roll in these vessels,in part because of an uncharacterized defect in binding to P-selectin. Selectin ligands must be alpha1-3 fucosylated to form glycan determinants such as sialyl Lewis x (sLe(x)). We found that inadequate alpha1-3 fucosylation of CB CD34(+) cells,particularly CD34(+)CD38(-/low) cells that are highly enriched in HSPCs,caused them to bind poorly to E-selectin as well as to P-selectin. Treatment of CB CD34(+) cells with guanosine diphosphate (GDP) fucose and exogenous alpha1-3 fucosyltransferase VI increased cell-surface sLe(x) determinants,augmented binding to fluid-phase P- and E-selectin,and improved cell rolling on P- and E-selectin under flow. Similar treatment of CB mononuclear cells enhanced engraftment of human hematopoietic cells in bone marrows of irradiated NOD/SCID mice. These observations suggest that alpha1-3 fucosylation of CB cells might be a simple and effective method to improve hematopoietic cell homing to and engraftment in bone marrows of patients receiving CB transplants. View Publication

Cell enrichment methods that deal with larger volumes of peripheral blood and BM are needed for increased sensitivity of detection,characterization and quantification of isolated tumor cells (ITC). This study was designed to evaluate a new procedure,the RosetteSep-Applied Imaging Rare Event (RARE) detection method,which depletes the majority of the erythrocytes and leucocytes in a peripheral blood (PB) sample,thereby negatively enriching tumor cells if present. This enrichment procedure allows for increased sensitivity,by analyzing a 5-10 fold larger volume of blood,compared with a direct immunocytochemical (ICC) technique,with minimal impact on laboratory workload. Model experiments showed comparable tumor cell recoveries between the two tested methods,both in PB and BM. Clinical samples were evaluated using paired PB and BM samples from 95 carcinoma patients. Analysis of PB results showed that 25.3% had textgreater or = 1 tumor cell detected by the RARE procedure,compared with 5.2% after direct ICC analysis,analyzing a 10-fold larger volume by the RARE procedure. The direct ICC analysis of BM from the same patients revealed 16.8% positive. The ITC detection differed both quantitatively and qualitatively between BM and PB,as samples with high numbers of ITC in BM were still negative in PB. The clinical significance of ITC in blood still needs to be established. However,the easy access of peripheral blood,and the increased sensitivity obtained by increasing the sample volume with the RARE procedure,suggests that the value of peripheral blood analysis should be tested in parallel in studies where ITC detection in BM is performed. View Publication

High-titer,HIV-1-based lentiviral vector particles were found to transduce cytokine-mobilized rhesus macaque CD34(+) cells and clonogenic progenitors very poorly (textless 1%),reflecting the postentry restriction in rhesus cells to HIV infection. To overcome this barrier,we developed a simian immunodeficiency virus (SIV)-based vector system. A single exposure to a low concentration of amphotropic pseudotyped SIV vector particles encoding the green fluorescent protein (GFP) resulted in gene transfer into 68% +/- 1% of rhesus bulk CD34(+) cells and 75% +/- 1% of clonogenic progenitors. Polymerase chain reaction (PCR) analysis of DNA from individual hematopoietic colonies confirmed these relative transduction efficiencies. To evaluate SIV vector-mediated stem cell gene transfer in vivo,3 rhesus macaques underwent transplantation with transduced,autologous cytokine-mobilized peripheral blood CD34(+) cells following myeloablative conditioning. Hematopoietic reconstitution was rapid,and an average of 18% +/- 8% and 15% +/- 7% GFP-positive granulocytes and monocytes,respectively,were observed 4 to 6 months after transplantation,consistent with the average vector copy number of 0.19 +/- 0.05 in peripheral blood leukocytes as determined by real-time PCR. Vector insertion site analysis demonstrated polyclonal reconstitution with vector-containing cells. SIV vectors appear promising for evaluating gene therapy approaches in nonhuman primate models. View Publication