Expression of the Epstein ± Barr virus (EBV) transforming LMP1 in B cells activates the transcription factor NF-kB and induces phenotypic changes through two distinct domains in the cytoplasmic C-terminus of the protein. The aa 187 ± 231 domain of LMP1, which is important for growth transformation, binds tumour necrosis factor (TNF) receptor associated factor (TRAF) 1 and TRAF3 and this interaction mediates subsequent signalling events. The TRAFs also associate with CD40, a member of the TNFR family, which upon ligation activates NF-kB and induces phenotypic changes similar to those mediated by LMP1. This study demonstrates that LMP1 expression in carcinoma cell lines and SV40-transformed keratinocytes results in induction of the pleiotropic cytokine interleukin 6 (IL6), an e ect which is also observed upon CD40 ligation. The mechanism by which either LMP1 expression or CD40 ligation induces IL6 production was found to be NF-kB-dependent. Mutational analysis identi®ed domains in the C-terminus of LMP1 which are important for NF-kB activation and IL6 secretion. LMP1 and CD40 share a common PxQxT core TRAF binding motif and mutations in or adjacent to this sequence impaired the ability of LMP1 or CD40 to induce NF-kB activation and IL6 secretion. The importance of TRAF interactions in mediating these e ects was con®rmed using dominant negative TRAF2 and TRAF3 mutants which also identi®ed di erences in the signalling events mediated by the two NF-kB activating domains of LMP1. A20, an anti-apoptotic protein which interacts with TRAF2 and blocks CD40-mediated NF-kB activity, also blocked NF-kB and IL6 secretion in LMP1-transfected epithelial cells. These results suggest that LMP1 regulates IL6 production in epithelial cells in a manner similar to CD40 ligation and implicate TRAFs as common mediators in the transduction of signals generated via the CD40 and LMP1 pathways. As a role for IL6 in regulating epithelial cell growth has previously been suggested, the control of IL6 secretion via the CD40 and LMP1 pathways may have implications for the growth of both normal and transformed epithelial cells.

In a neonatal and pediatric intensive care unit population, ventilation in neurally adjusted ventilatory assist mode was associated with improved patient-ventilator synchrony and lower peak airway pressure when compared with pressure-support ventilation with a pneumatic trigger. Ventilating patients in this new mode seem to be safe and well tolerated.

In this study, we find that CD45RO+ memory populations of CD4+ T lymphocytes express the vascular endothelial growth factor (VEGF) receptors KDR and Flt-1 at both the mRNA and protein levels. Furthermore, by Western blot analysis, we find that VEGF increases the phosphorylation and activation of ERK and Akt within CD4+CD45RO+ T cells. These VEGF-mediated signaling responses were inhibited by a KDR-specific small interfering RNA in a VEGF receptor-expressing Jurkat T cell line and by SU5416, a pharmacological KDR inhibitor, in CD4+CD45RO+ T cells. We also find that VEGF augments mitogen-induced production of IFN-γ in a dose-dependent manner (p < 0.001) and significantly (p < 0.05) increases directed chemotaxis of this T cell subset. Collectively, our results for the first time define a novel function for VEGF and KDR in CD45RO+ memory T cell responses that are likely of great pathophysiological importance in immunity.

The chromosome region 17p13.3 is thought to encode a tumour suppressor gene involved in sporadic breast cancer and other malignancies. Physical ordering of markers has been carried out by a series of multicolour fluorescent in situ hybridisation (FISH) experiments, using isolated yeast artificial chromosomes (YACs) and cosmids. Eight polymorphic markers ordered within this new physical map and one external marker were used to investigate the pattern of loss of heterozygosity in a panel of 40 sporadic breast tumour patients. The data revealed a region of high loss (60%) within distal 17p13.3, defined by markers D17S926, D17S695 and D17S849 which mapped close together. A contig of YACs was constructed physically linking these three markers.

In these studies, we find that the vascular endothelial growth factor (VEGF) receptor KDR is expressed on subsets of mitogenactivated CD4 ؉ and CD8 ؉ T cells in vitro. We also found that KDR colocalizes with CD3 on mitogen-activated T cells in vitro and on infiltrates within rejecting human allografts in vivo. To evaluate whether VEGF and KDR mediate lymphocyte migration across endothelial cells (ECs), we used an in vitro live-time transmigration model and observed that both anti-VEGF and anti-KDR antibodies inhibit the transmigration of both CD4 ؉ and CD8 ؉ T cells across tumor necrosis factor␣ (TNF␣)-activated, but not unactivated ECs. In addition, we found that interactions among CD4 ؉ or CD8 ؉ T cells and TNF␣-activated ECs result in the induction of KDR on each T cell subset, and that KDRexpressing lymphocytes preferentially transmigrate across TNF␣-activated ECs. Finally, using a humanized severe combined immunodeficient mouse model of lymphocyte trafficking, we found that KDRexpressing lymphocytes migrate into human skin in vivo, and that migration is reduced in mice treated with a blocking anti-VEGF antibody. IntroductionVascular endothelial growth factor (VEGF), an angiogenesis factor, is established to function in the migration, proliferation, and survival of endothelial cells (ECs). 1,2 VEGF is well known to function in wound healing, organ development, and tumor growth and it serves to promote tissue protection and repair after acute injury. 3 VEGF is also expressed in association with cell-mediated immune inflammation and acute and chronic inflammatory reactions. 4 In chronic inflammatory disease processes, VEGF fails to elicit effective tissue repair, and rather may induce a pathologic form of angiogenesis that has been proposed to augment disease activity. 4,5 Indeed, several studies have demonstrated that blockade of VEGF may attenuate the progression of chronic diseases such as arthritis, atherosclerosis, and allograft rejection. [6][7][8] Although relatively underappreciated, VEGF has potent proinflammatory properties including an ability to mediate leukocyte trafficking into sites of cell-mediated immunity. [7][8][9][10][11][12][13] The proinflammatory properties of VEGF are reported to be dependent on its ability to interact directly with monocytes resulting in chemotaxis, 10 its ability to induce the expression of endothelial adhesion molecules 9,11 and chemokine production, 8,12,14 and its ability to enhance vascular permeability. 2 Furthermore, VEGF has been reported to have direct chemoattractant effects on murine and human T cells, 13,15 and blockade of VEGF in vivo has been found to inhibit lymphocyte trafficking into skin and rejecting cardiac allografts. 8,16,17 However, the mechanism underlying the ability of VEGF to interact with T cells is not known, and the molecular basis for its ability to facilitate lymphocyte chemotaxis in vitro or in vivo is poorly understood.Several recent studies have determined that the VEGF receptors Flt-1 (VEGF receptor 1), KDR (VEGF receptor 2) and neu...

Cellular heterogeneity presents an important challenge to the development of cell-based therapies where there is a fundamental requirement for predictable and reproducible outcomes. Transplanted Dental Pulp Stem/Progenitor Cells (DPSCs) have demonstrated early promise in experimental models of spinal cord injury and stroke, despite limited evidence of neuronal and glial-like differentiation after transplantation. Here, we report, for the first time, on the ability of single cell-derived clonal cultures of murine DPSCs to differentiate in vitro into immature neuronal-like and oligodendrocyte-like cells. Importantly, only DPSC clones with high nestin mRNA expression levels were found to successfully differentiate into Map2 and NF-positive neuronal-like cells. Neuronally differentiated DPSCs possessed a membrane capacitance comparable with primary cultured striatal neurons and small inward voltage-activated K+ but not outward Na+ currents were recorded suggesting a functionally immature phenotype. Similarly, only high nestin-expressing clones demonstrated the ability to adopt Olig1, Olig2, and MBP-positive immature oligodendrocyte-like phenotype. Together, these results demonstrate that appropriate markers may be used to provide an early indication of the suitability of a cell population for purposes where differentiation into a specific lineage may be beneficial and highlight that further understanding of heterogeneity within mixed cellular populations is required.

Background Cardiac allograft vasculopathy (CAV) is the leading cause of chronic allograft loss following pediatric heart transplantation. We hypothesize that biomarkers of endothelial injury and repair predict CAV development in pediatric heart transplant recipients. Methods Blood was collected from pediatric heart transplant recipients at the time of routine annual coronary angiography and the concentrations of 13 angiogenesis-related molecules were determined. The primary endpoint was the presence of moderate or severe CAV by angiography over a 5 y follow up period. Results Forty-eight children were enrolled. The cohort was 57% male with a median age of 15.5 y (range 2–22) and median time post-transplant of 5.8 y (range 2–15). Eight recipients developed moderate/severe CAV at a median follow-up of 4.7 y, of whom 3 died, 3 were re-transplanted, 1 had a myocardial infarction and 1 is listed for re-transplantation. Clinical characteristics associated with the development of moderate/severe CAV included prednisone use at enrollment (P=0.03) and positive recipient CMV IgG at time of transplant (P<0.01). In addition, multivariable Cox proportional hazards regression identified plasma VEGF-A concentration greater than 90 pg/mL at the time of blood draw as a significant predictor of time to moderate or severe CAV (hazard ratio of 14.3; 95% CI: 1.3–163). Receiver operating characteristic curve analysis demonstrated that VEGF-A shows moderate performance for association with the subsequent development of CAV (area under the curve of 0.77; CI: 0.61–0.92). Conclusions VEGF-A levels in pediatric heart transplant recipients are associated with clinically important CAV progression within the subsequent 5 years.

One group of sequence variants of Epstein-Barr virus is characterized by a 10-amino-acid deletion within the CTAR-2 functional domain of the latent membrane protein, LMP1. A role for this deletion in enhancing the tumorigenicity of the viral oncogene in rodent fibroblasts was recently demonstrated. We examined the effect of this deletion upon LMP1 function in four human lymphoid cell lines by using three natural variants of LMP1: the prototype B95.8 gene and the CAO and AG876 genes, both of which have codons 343 to 352 of the B95.8-LMP1 deleted. These experiments revealed that LMP1-mediated upregulation of CD40 and CD54 was markedly impaired (by 60 to 90%) with CAO-LMP1 compared with B95.8-LMP1. In contrast, the function of AG876-LMP1 was indistinguishable from that of B95.8-LMP1 in two lines and was only slightly impaired in the other two lines. Activation of NF-κB by CAO-LMP1 was not impaired in any of the lines; rather, activation of an NF-κB reporter by CAO-LMP1 was consistently about twofold greater than the activation with B95.8- or AG876-LMP1. Therefore, while the CAO-LMP1 is functionally distinct from the prototype B95.8-LMP1 in human lymphocytes, the 10-amino-acid deletion appears not to be directly responsible. This conclusion was confirmed by using a B95.8-LMP1 mutant with codons 343 to 352 deleted and chimerae of CAO- and B95.8-LMP1 in which the CTAR-2 domains of these genes were exchanged. Sequences outside the CTAR-2 domain were implicated in the distinct functional characteristics of CAO-LMP1 in human lymphoid cells.