Common Gamma Chain Cytokines Promote Rapid In Vitro Expansion of Allo-Specific Human CD8+ Suppressor T Cells

Human CD8+ regulatory T cells, particularly the CD8+CD28− T suppressor cells, have emerged as an important modulator of alloimmunity. Understanding the conditions under which these cells are induced and/or expanded would greatly facilitate their application in future clinical trials. In the current study, we develop a novel strategy that combines common gamma chain (γc) cytokines IL-2, IL-7 and IL-15 and donor antigen presenting cells (APCs) to stimulate full HLA-mismatched allogeneic human CD8+ T cells which results in significant expansions of donor-specific CD8+CD28− T suppressor cells in vitro. The expanded CD8+CD28− T cells exhibit increased expressions of CTLA-4, FoxP3, and CD25, while down-regulate expressions of CD56, CD57, CD127, and perforin. Furthermore, these cells suppress proliferation of CD4+ T cells in a contact-dependent and cytokine-independent manner. Interestingly, the specificity of suppression is restricted by the donor HLA class I antigens but promiscuous to HLA class II antigens, providing a potential mechanism for linked suppression. Taken together, our results demonstrate a novel role for common γc cytokines in combination with donor APCs in the expansion of donor-specific CD8+CD28− T suppressor cells, and represent a robust strategy for in vitro generation of such cells for adoptive cellular immunotherapy in transplantation

IL-24 Inhibits lung cancer cell migration and invasion by disrupting the SDF-1/CXCR4 signaling axis

© 2015 Panneerselvam et al. Background The stromal cell derived factor (SDF)-1/chemokine receptor (CXCR)-4 signaling pathway plays a key role in lung cancer metastasis and is molecular target for therapy. In the present study we investigated whether interleukin (IL)-24 can inhibit the SDF-1/CXCR4 axis and suppress lung cancer cell migration and invasion in vitro. Further, the efficacy of IL-24 in combination with CXCR4 antagonists was investigated. Methods Human H1299, A549, H460 and HCC827 lung cancer cell lines were used in the present study. The H1299 lung cancer cell line was stably transfected with doxycycline-inducible plasmid expression vector carrying the human IL-24 cDNA and used in the present study to determine the inhibitory effects of IL-24 on SDF-1/CXCR4 axis. H1299 and A549 cell lines w ere used in transient transfection studies. The inhibitory effects of IL-24 on SDF1/CXCR4 and its downstream targets were analyzed by quantitative RT-PCR, western blot, luciferase reporter assay, flow cytometry and immunocytochemistry. Functional studies included cell migration and invasion assays. Principal Findings Endogenous CXCR4 protein expression levels varied among the four human lung cancer cell lines. Doxycycline-induced IL-24 expression in the H1299-IL24 cell line resulted in reduced CXCR4 mRNA and protein expression. IL-24 post-transcriptionally regulated CXCR4 mRNA expression by decreasing the half-life of CXCR4 mRNA ( > 40%). Functional studies showed IL-24 inhibited tumor cell migration and invasion concomitant with reduction in CXCR4 and its downstream targets (pAKTS 473 , pmTORS 2448 , pPRAS40 T246 and HIF-1α). Additionally, IL-24 inhibited tumor cell migration both in the presence and absence of the CXCR4 agonist, SDF-1. Finally, IL-24 when combined with CXCR4 inhibitors (AMD3100, SJA5) or with CXCR4 siRNA demonstrated enhanced inhibitory activity on tumor cell migration. Conclusions IL-24 disrupts the SDF-1/CXCR4 signaling pathway and inhibits lung tumor cell migration and invasion. Additionally, IL-24, when combined with CXCR4 inhibitors exhibited enhanced anti-metastatic activity and is an attractive therapeutic strategy for lung metastasi

Development of a diagnostic protocol for dizziness in elderly patients in general practice: a Delphi procedure

<p>Abstract</p> <p>Background</p> <p>Dizziness in general practice is very common, especially in elderly patients. The empirical evidence for diagnostic tests in the evaluation of dizziness is scarce. Aim of our study was to determine which set of diagnostic tests should be part of a diagnostic protocol for evaluating dizziness in elderly patients in general practice.</p> <p>Methods</p> <p>We conducted a Delphi procedure with a panel of 16 national and international experts of all relevant medical specialities in the field of dizziness. A selection of 36 diagnostic tests, based on a systematic review and practice guidelines, was presented to the panel. Each test was described extensively, and data on test characteristics and methodological quality (assessed with the Quality Assessment of Diagnostic Accuracy Studies, QUADAS) were presented. The threshold for in- or exclusion of a diagnostic test was set at an agreement of 70%.</p> <p>Results</p> <p>During three rounds 21 diagnostic tests were selected, concerning patient history (4 items), physical examination (11 items), and additional tests (6 items). Five tests were excluded, although they are recommended by existing practice guidelines on dizziness. Two tests were included, although several practice guidelines question their diagnostic value. Two more tests were included that have never been recommended by practice guidelines on dizziness.</p> <p>Conclusion</p> <p>In this study we successfully combined empirical evidence with expert opinion for the development of a set of diagnostic tests for evaluating dizziness in elderly patients. This comprehensive set of tests will be evaluated in a cross-sectional diagnostic study.</p

A scoping review and thematic analysis of social and behavioural research among HIV-serodiscordant couples in high-income settings.

CAPRISA, 2015.Abstract available in pdf

Aberrant expression of costimulatory molecules in splenocytes of the mevalonate kinase‐deficient mouse model of human hyper‐IgD syndrome (HIDS)

Objective We sought to determine the activation status and proliferative capacities of splenic lymphocyte populations from a mevalonate kinase‐deficient mouse model of hyper‐IgD syndrome (HIDS). We previously reported that murine mevalonate kinase gene ablation was embryonic lethal for homozygous mutants while heterozygotes (Mvk+/−) demonstrated several phenotypic features of human HIDS including increased serum levels of IgD, IgA, and TNFα, temperature dysregulation, hematological abnormalities, and splenomegaly. Methods and results Flow cytometric analysis of cell surface activation markers on T and B lymphocytes, and macrophage populations, demonstrated aberrant expression of B7 glycoproteins in all splenic cell types studied. Differences in expression levels between Mvk+/− and Mvk+/+ littermate controls were observed in both the basal state (unstimulated) and after Concanavalin A (Con‐A) stimulation in vitro of whole splenocyte cultures. In Mvk+/− CD4 and CD8 T cells, alterations in expression of CD25, CD80, CD152, and CD28 were observed. Mvk+/− splenic macrophages expressed altered levels of CD80, CD86, CD40, and CD11c while Mvk+/− B lymphocytes had differential expression of CD40, CD80, and CD86. Mvk+/− splenocyte subpopulations also exhibited altered proliferative capacities in response to in vitro stimulation. Conclusion We postulate that imbalances in the expression of cell surface proteins necessary for activation, proliferation, and regulation of the intensity and duration of an immune response may result in defective T cell activation, proliferation, and effector functions in our model and potentially in human HIDS

Factor VII Deficiency Impairs Cutaneous Wound Healing in Mice

Skin keratinocytes express tissue factor (TF) and are highly associated with skin wound healing. Although it has been demonstrated that perivascular TF expression in granulation tissue formed after dermal injury is downregulated during healing, studies of the mechanism of factor (F) VII, a TF ligand, in skin wound healing are lacking. We reported the use of a dermal punch model to demonstrate that low-expressing FVII mice (~1% of wild type [WT]) exhibited impaired skin wound healing compared with WT controls. These low-FVII mice showed defective reepithelialization and reduced inflammatory cell infiltration at wound sites. This attenuated reepithelialization was associated with diminished expression of the transcription factor early growth response 1 (Egr-1). In vitro, Egr-1 was shown to be essential for the FVIIa-induced regulation of keratinocyte migration and inflammation. Both Egr-1 upregulation and downstream inflammatory cytokine appearance in keratinocytes depended on FVIIa/TF/protease-activated receptor 2 (PAR-2)-induced signaling and did not require subsequent generation of FXa and thrombin. The participation of Egr-1 in FVIIa-mediated regulation of keratinocyte function was confirmed by use of Egr-1–deficient mice, wherein a significant delay in skin wound healing after injury was observed, relative to WT mice. The results from these studies demonstrate an in vivo mechanistic relationship between FVIIa, Egr-1 and the inflammatory response in keratinocyte function during the wound healing process