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1.  Allospecific Tregs Expanded After Anergization Remain Suppressive in Inflammatory Conditions but Lack Expression of Gut-homing Molecules 
Molecular Therapy  2016;24(6):1126-1134.
Cell therapy with antigen-specific regulatory T-cells (Treg) has great potential to selectively control unwanted immune responses after allogeneic stem-cell or solid organ transplantation and in autoimmune diseases. Ex vivo allostimulation with costimulatory blockade (alloanergization) of human T-cells expands populations of alloantigen-specific Treg, providing a cellular strategy to control donor T-cell alloresponses causing graft-versus-host disease after allogeneic hematopoietic stem-cell transplantation. Crucially, it is not known if Treg expanded in this way are stable in proinflammatory conditions encountered after transplantation, or if they possess capacity to migrate to key target organs. Using an in vitro model to functionally characterize human Treg expanded after alloanergization, we now show that these cells remain potently allosuppressive in the presence of relevant exogenous inflammatory signals. Expanded allospecific Treg retained expression of molecules conferring migratory capacity to several organs but small intestine-specific chemotaxis was markedly impaired, in keeping with the preponderance of gut graft-versus-host disease in previous clinical studies using this strategy. Importantly, impaired gut-specific chemotaxis could be partially corrected by pharmacological treatment. These findings will facilitate more effective application of this cellular approach to limit T-cell alloresponses after hematopoietic stem-cell transplantation and the wider application of the strategy to other clinical settings.
PMCID: PMC4923329  PMID: 27049761
2.  Neuroanatomical Alterations in Tinnitus Assessed with Magnetic Resonance Imaging 
Previous studies of anatomical changes associated with tinnitus have provided inconsistent results, with some showing significant cortical and subcortical changes, while others have found effects due to hearing loss, but not tinnitus. In this study, we examined changes in brain anatomy associated with tinnitus using anatomical scans from 128 participants with tinnitus and hearing loss, tinnitus with clinically normal hearing, and non-tinnitus controls with clinically normal hearing. The groups were matched for hearing loss, age and gender. We employed voxel- and surface-based morphometry (SBM) to investigate gray and white matter volume and thickness within regions-of-interest (ROI) that were based on the results of previous studies. The largest overall effects were found for age, gender, and hearing loss. With regard to tinnitus, analysis of ROI revealed numerous small increases and decreases in gray matter and thickness between tinnitus and non-tinnitus controls, in both cortical and subcortical structures. For whole brain analysis, the main tinnitus-related significant clusters were found outside sensory auditory structures. These include a decrease in cortical thickness for the tinnitus group compared to controls in the left superior frontal gyrus (SFG), and a decrease in cortical volume with hearing loss in left Heschl’s gyrus (HG). For masked analysis, we found a decrease in gray matter volume in the right Heschle’s gyrus for the tinnitus group compared to the controls. We found no changes in the subcallosal region as reported in some previous studies. Overall, while some of the morphological differences observed in this study are similar to previously published findings, others are entirely different or even contradict previous results. We highlight other discrepancies among previous results and the increasing need for a more precise subtyping of the condition.
PMCID: PMC5030287  PMID: 27708577
tinnitus; brain anatomy; auditory cortex; voxel-based morphometry; surface-based morphometry
3.  Combining CD19- redirection and alloanergization to generate tumor-specific human T cells for allogeneic cell therapy of B-cell malignancies 
Cancer research  2010;70(10):3915-3924.
Allogeneic hematopoietic stem-cell transplantation can cure some patients with high-risk B-cell malignancies, but disease relapse following transplantation remains a significant problem. One approach that could be used to augment the donor T cell-mediated anti-tumor effect is the infusion of allogeneic donor-derived T cells expressing a chimeric antibody receptor (CAR) specific to the B-cell antigen CD19. However, the use of such cells might result in toxicity in the form of graft-versus-host disease mediated by CD19-specific (CD19-CAR) T cells possessing alloreactive endogenous T cell receptors. We therefore investigated whether non-alloreactive tumor-specific human T cells could be generated from peripheral blood mononuclear cells of healthy donors by the combination of CD19-redirection via CAR expression and subsequent alloanergization by allostimulation and concomitant blockade of CD28-mediated costimulation. Alloanergization of CD19-CAR T cells resulted in efficient and selective reduction of alloresponses in both CD4+ and CD8+ T cells including allospecific proliferation and cytokine secretion. Importantly, T-cell effector functions including CAR-dependent proliferation and specific target cytolysis and cytokine production were retained after alloanergization. Our data supports the application of CD19-redirection and subsequent alloanergization to generate allogeneic donor T cells for clinical use possessing increased anti-tumor activity, but limited capacity to mediate graft-versus-host disease. Therapy with such cells could potentially reduce disease relapse after allogeneic transplantation without increasing toxicity, thereby improving the outcome of patients undergoing allogeneic transplantation for high-risk B-cell malignancies.
PMCID: PMC2873153  PMID: 20424114
Cellular Immunotherapy; Anergy; Chimeric antigen receptor; CD19; Gene therapy; allogeneic stem cell transplantation
4.  Induction of Alloantigen-specific Anergy in Human Peripheral Blood Mononuclear Cells by Alloantigen Stimulation with Co-stimulatory Signal Blockade 
Allogeneic hematopoietic stem cell transplantation (AHSCT) offers the best chance of cure for many patients with congenital and acquired hematologic diseases. Unfortunately, transplantation of alloreactive donor T cells which recognize and damage healthy patient tissues can result in Graft-versus-Host Disease (GvHD)1. One challenge to successful AHSCT is the prevention of GvHD without associated impairment of the beneficial effects of donor T cells, particularly immune reconstitution and prevention of relapse. GvHD can be prevented by non-specific depletion of donor T cells from stem cell grafts or by administration of pharmacological immunosuppression. Unfortunately these approaches increase infection and disease relapse2-4. An alternative strategy is to selectively deplete alloreactive donor T cells after allostimulation by recipient antigen presenting cells (APC) before transplant. Early clinical trials of these allodepletion strategies improved immune reconstitution after HLA-mismatched HSCT without excess GvHD5, 6. However, some allodepletion techniques require specialized recipient APC production6, 7and some approaches may have off-target effects including depletion of donor pathogen-specific T cells8and CD4 T regulatory cells9.One alternative approach is the inactivation of alloreactive donor T cells via induction of alloantigen-specific hyporesponsiveness. This is achieved by stimulating donor cells with recipient APC while providing blockade of CD28-mediated co-stimulation signals10.This "alloanergization" approach reduces alloreactivity by 1-2 logs while preserving pathogen- and tumor-associated antigen T cell responses in vitro11. The strategy has been successfully employed in 2 completed and 1 ongoing clinical pilot studies in which alloanergized donor T cells were infused during or after HLA-mismatched HSCT resulting in rapid immune reconstitution, few infections and less severe acute and chronic GvHD than historical control recipients of unmanipulated HLA-mismatched transplantation12. Here we describe our current protocol for the generation of peripheral blood mononuclear cells (PBMC) which have been alloanergized to HLA-mismatched unrelated stimulator PBMC. Alloanergization is achieved by allostimulation in the presence of monoclonal antibodies to the ligands B7.1 and B7.1 to block CD28-mediated costimulation. This technique does not require the production of specialized stimulator APC and is simple to perform, requiring only a single and relatively brief ex vivo incubation step. As such, the approach can be easily standardized for clinical use to generate donor T cells with reduced alloreactivity but retaining pathogen-specific immunity for adoptive transfer in the setting of AHSCT to improve immune reconstitution without excessive GvHD.
PMCID: PMC3197423  PMID: 21445041
Immunology;  Issue 49;  Allogeneic stem cell transplantation;  alloreactivity;  Graft-versus-Host Disease;  T cell costimulation;  anergy;  mixed lymphocyte reaction.
5.  Selective Genomic Targeting by FRA-2/FOSL2 Transcription Factor 
The Journal of Biological Chemistry  2011;286(17):15227-15239.
FRA-2/FOSL2 is a basic region-leucine zipper motif transcription factor that is widely expressed in mammalian tissues. The functional repertoire of this factor is unclear, partly due to a lack of knowledge of genomic sequences that are targeted. Here, we identified novel, functional FRA-2 targets across the genome through expression profile analysis in a knockdown transgenic rat. In this model, a nocturnal rhythm of pineal gland FRA-2 is suppressed by a genetically encoded, dominant negative mutant protein. Bioinformatic analysis of validated sets of FRA-2-regulated and -nonregulated genes revealed that the FRA-2 regulon is limited by genomic target selection rules that, in general, transcend core cis-sequence identity. However, one variant AP-1-related (AP-1R) sequence was common to a subset of regulated genes. The functional activity and protein binding partners of a candidate AP-1R sequence were determined for a novel FRA-2-repressed gene, Rgs4. FRA-2 protein preferentially associated with a proximal Rgs4 AP-1R sequence as demonstrated by ex vivo ChIP and in vitro EMSA analysis; moreover, transcriptional repression was blocked by mutation of the AP-1R sequence, whereas mutation of an upstream consensus AP-1 family sequence did not affect Rgs4 expression. Nocturnal changes in protein complexes at the Rgs4 AP-1R sequence are associated with FRA-2-dependent dismissal of the co-activator, CBP; this provides a mechanistic basis for Rgs4 gene repression. These studies have also provided functional insight into selective genomic targeting by FRA-2, highlighting discordance between predicted and actual targets. Future studies should address FRA-2-Rgs4 interactions in other systems, including the brain, where FRA-2 function is poorly understood.
PMCID: PMC3083148  PMID: 21367864
AP-1 Transcription Factor; CBP; CREB; Gene Expression; Microarray; Pineal Gland; Rat; FRA-2; Rgs Gene
6.  The Glycinergic System in Human Startle Disease: A Genetic Screening Approach 
Human startle disease, also known as hyperekplexia (OMIM 149400), is a paroxysmal neurological disorder caused by defects in glycinergic neurotransmission. Hyperekplexia is characterised by an exaggerated startle reflex in response to tactile or acoustic stimuli which first presents as neonatal hypertonia, followed in some with episodes of life-threatening infantile apnoea. Genetic screening studies have demonstrated that hyperekplexia is genetically heterogeneous with several missense and nonsense mutations in the postsynaptic glycine receptor (GlyR) α1 subunit gene (GLRA1) as the primary cause. More recently, missense, nonsense and frameshift mutations have also been identified in the glycine transporter GlyT2 gene, SLC6A5, demonstrating a presynaptic component to this disease. Further mutations, albeit rare, have been identified in the genes encoding the GlyR β subunit (GLRB), collybistin (ARHGEF9) and gephyrin (GPHN) – all of which are postsynaptic proteins involved in orchestrating glycinergic neurotransmission. In this review, we describe the clinical ascertainment aspects, phenotypic considerations and the downstream molecular genetic tools utilised to analyse both presynaptic and postsynaptic components of this heterogeneous human neurological disorder. Moreover, we will describe how the ancient startle response is the preserve of glycinergic neurotransmission and how animal models and human hyperekplexia patients have provided synergistic evidence that implicates this inhibitory system in the control of startle reflexes.
PMCID: PMC2854534  PMID: 20407582
glycine; hyperekplexia; receptor; transporter; mutation

Results 1-6 (6)