The results support efforts to achieve drug-induced mutation suppression of stop codons. The immunogenic epitope resulting from readthrough emphasizes the importance of monitoring T-cell immunity during clinical studies that suppress stop codons. Similar principles apply to other molecular strategies, including exon skipping and gene therapy.

Objectives Alpha-sarcoglycan deficiency is a severe form of muscular dystrophy (LGMD2D) without treatment. Gene replacement represents a strategy for correcting the underlying defect. Questions related to this approach were addressed in this clinical trial, particularly the need for immunotherapy, and persistence of gene expression. Methods A double-blind, randomized controlled trial using rAAV1.tMCK.hSGCA injected into the extensor digitorum brevis (EDB) muscle was conducted. Control sides received saline. A three-day course of methylprednisolone accompanied gene transfer without further immune suppression. Results No adverse events were encountered. SGCA gene expression increased 4-5 fold over control sides when examined at 6 weeks (two subjects) and 3 months (one subject). The full sarcoglycan complex was restored in all subjects and muscle fiber size was increased in the 3-month subject. AAV1 neutralizing antibodies were seen as early as 2 weeks. Neither CD4+ nor CD8+ cells were increased over contralateral sides. Scattered foci of inflammation could be found but showed features of programmed cell death. ELISpot showed no IFN-γ response to α-SG or AAV1 capsid peptide pools with the exception of a minimal capsid response in one subject. Restimulation to detect low frequency capsid specific T cells by ELISpot assays was negative. Results of the first three subjects successfully achieved study aims precluding the need for additional enrollment. Interpretation The finding of this gene replacement study in LGMD2D has important implications for muscular dystrophy. Sustained gene expression was seen, but studies over longer time periods without immunotherapy will be required for design of vascular delivery gene therapy trials

The events that contribute to the progression to AIDS during the acute phase of a primate lentiviral infection are still poorly understood. In this study, we used pathogenic and nonpathogenic simian models of simian immunodeficiency virus (SIV) infection of rhesus macaques (RMs) and African green monkeys (AGMs), respectively, to investigate the relationship between apoptosis in lymph nodes and the extent of viral replication, immune activation, and disease outcome. Here, we show that, in SIVmac251-infected RMs, a marked increased in lymphocyte apoptosis is evident during primary infection at the level of lymph nodes. Interestingly, the levels of apoptosis correlated with the extent of viral replication and the rate of disease progression to AIDS, with higher apoptosis in RMs of Indian genetic background than in those of Chinese origin. In stark contrast, no changes in the levels of lymphocyte apoptosis were observed during primary infection in the nonpathogenic model of SIVagm-sab infection of AGMs, despite similarly high rates of viral replication. A further and early divergence between SIV-infected RMs and AGMs was observed in terms of the dynamics of T-and B-cell proliferation in lymph nodes, with RMs showing significantly higher levels of cycling cells (Ki67 ؉ ) in the T-cell zones in association with relatively low levels of Ki67؉ in the B-cell zones, whereas AGMs displayed a low frequency of Ki67 ؉ in the T-cell area but a high proportion of Ki67 ؉ cells in the B-cell area. As such, this study suggests that species-specific host factors determine an early immune response to SIV that predominantly involves either cellular or humoral immunity in RMs and AGMs, respectively. Taken together, these data are consistent with the hypotheses that (i) high levels of T-cell activation and lymphocyte apoptosis are key pathogenic factors during pathogenic SIV infection of RMs and (ii) low T-cell activation and apoptosis are determinants of the AIDS resistance of SIVagm-infected AGMs, despite high levels of SIVagm replication.

Duchenne muscular dystrophy (DMD) typically occurs as a result of truncating mutations in the DMD gene that result in a lack of expression of the dystrophin protein in muscle fibers. Various therapies under development are directed toward restoring dystrophin expression at the subsarcolemmal membrane, including gene transfer. In a trial of intramuscular adeno-associated virus (AAV)-mediated delivery of a therapeutic minidystrophin construct, we identified in two of six subjects the presence of a population of T cells that had been primed to recognize dystrophin epitopes before transgene delivery. As the presence of preexisting T cell immunity may have a significant effect on the success of therapeutic approaches for restoring dystrophin, we sought to determine the prevalence of such immunity within a DMD cohort from our Muscular Dystrophy Association clinic. Dystrophin-specific T cell immunity was evaluated in subjects with DMD who were either receiving the glucocorticoid steroid prednisone (n=24) or deflazacort (n=29), or who were not receiving steroids (n=17), as well as from normal age-matched control subjects (n=21). We demonstrate that increasing age correlates with an increased risk for the presence of anti-dystrophin T cell immunity, and that treatment with either corticosteroid decreases risk compared with no treatment, suggesting that steroid therapy in part may derive some of its benefit through modulation of T cell responses. The frequency of dystrophin-specific T cells detected by enzyme-linked immunospot assay was lower in subjects treated with deflazacort versus prednisone, despite similar overall corticosteroid exposure, suggesting that the effects of the two corticosteroids may not be identical in patients with DMD. T cells targeted epitopes upstream and downstream of the dystrophin gene mutation and involved the CD4⁺ helper and/or CD8⁺ cytotoxic subsets. Our data confirm the presence of preexisting circulating T cell immunity to dystrophin in a sizable proportion of patients with DMD, and emphasize the need to consider this in the design and interpretation of clinical gene therapy trials.

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Immunological and virological events that occur during the earliest stages of SIV infection are now considered to have a major impact on subsequent disease progression. In the present study, we demonstrate a clear correlation between progression to AIDS and the rate of in vitro CD4+ (but not CD8+) T cell death in lymph nodes. The dying CD4+ T cells were effector memory T cells, which are critical for the immune response to pathogens. However, there was no correlation between the rate of the viral replication within lymph nodes and the extent of Fas ligand-mediated death, despite the increased sensitivity of CD4+ T cells to death in response to recombinant human Fas ligand. CD4+ T cell death was caspase and apoptosis-inducing factor independent but was clearly associated with mitochondrion damage. Interestingly, higher expression levels of the active form of Bak, a proapoptotic molecule involved in mitochondrial membrane permeabilization, were observed in SIV-infected macaques progressing more rapidly to AIDS. Finally, we demonstrated that the strain of SIV we used requires CCR5 and BOB/GRP15 molecules as coreceptors and caused death of unstimulated noncycling primary CD4+ T cells. Altogether, these results demonstrate that CD4+ T cell death occurring early after SIV infection is a crucial determinant of progression to AIDS and that it is mediated by the intrinsic death pathway.

SIV-infected macaques exhibit distinct rates of progression to AIDS and despite significant increases in CD8 þ T cells, immune cells fail to control and eradicate SIV in vivo. Here, we investigated the interplay between viral reservoir sites, CD8 þ T-cell activation/death and outcome. Our data provide strong evidence that mesenteric (Mes) lymph nodes represent major reservoirs not only for SIV-infected macaques progressing more rapidly toward AIDS but also in controllers. We demonstrate that macaques progressing faster display greater expression of TGF-b and Indoleamine 2,3 dioxygenase in particular in intestinal tissues associated with a phosphorylation of the p53 protein on serine 15 in CD8 þ T cells from Mes lymph nodes. These factors may act as a negative regulator of CD8 þ T-cell function by inducing a Bax/Bak/Puma-dependent death pathway of effector/ memory CD8 þ T cells. Greater T-cell death and viral dissemination was associated with a low level of TIA-1 þ expressing cells. Finally, we provide evidence that abrogation of TGF-b in vitro enhances T-cell proliferation and reduces CD8 þ T-cell death. Our data identify a mechanism of T-cell exhaustion in intestinal lymphoid organs and define a potentially effective immunological strategy for the modulation of progression to AIDS. Cell Death and Differentiation (2007) 14, 1747-1758; doi:10.1038/sj.cdd.4402192; published online 6 July 2007Virus production in Human Immunodeficiency Virus 1 (HIV-1)-infected individuals is largely the result of a dynamic process involving continuous rounds of de novo infection and replication in CD4 þ cells with rapid turnover of both free virus and virus producing cells. Moreover, an increasing body of evidence suggests that reservoirs, cell types or anatomical sites ('sanctuaries'), represent a major barrier to virus eradication. 1 HIV-specific CD8 þ T lymphocytes (CTL) are considered crucial in the control of viral replication. 2 Excessive induction of apoptosis has been proposed as one major mechanism of abnormal T-cell response during HIV and Simian Immunodeficiency Virus (SIV) infections. 3 However, most studies, which investigated the potential relationship between HIV and SIV infections and apoptosis have focused on cells from peripheral blood, rather than on cells from lymphoid organs.Peripheral lymphoid tissues such as the axillary and inguinal (Ing) lymph nodes (LNs), which drain the arms and legs, respectively, and the spleen, are considered major sites of HIV/SIV replication. 4 Accumulating data also indicate that gut-associated lymphoid tissue (GALT), which contains inductive sites, Peyer's patches and mesenteric (Mes) LNs, and effectors sites located within lamina propria and epithelium of the intestinal wall, is also an early and predominant tissue site of viral replication and may be a reservoir for HIV/SIV. 5,6 Because T cells from the GALT are required to maintain a state of immunological tolerance toward a myriad of dietary and resident bacterial antigens, the chronic exposure of HIV/ SIV-specific antigens coul...

Introduction: Creatinine as a marker of renal function has limited value in Duchenne muscular dystrophy (DMD) because of reduced muscle mass. Alternative methods of assessing renal function are sorely needed. Cystatin C, a nonglycosylated protein unaffected by muscle mass, is potentially an ideal biomarker of nephrotoxicity for this population but requires validation.