Anti-dystrophin T cell responses in Duchenne muscular dystrophy: prevalence and a glucocorticoid treatment effect.

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.

Effects of angiotensin-converting enzyme inhibitors and/or beta blockers on the cardiomyopathy in Duchenne muscular dystrophy.

Cardiomyopathy is a consequence of Duchenne muscular dystrophy (DMD). Suggested treatments include angiotensin-converting enzyme (ACE) inhibitors and/or ß blockers (BBs), but few large series have been reported. We present 42 patients with DMD and cardiomyopathy treated with an ACE inhibitor or an ACE inhibitor plus a BB. Serial echocardiograms were recorded. Adequate ejection fractions (EFs) were obtained at initiation of therapy (EF <55%). ACE inhibitor dosage adjustments were made if a continued decrease in EF was noted. BB therapy was initiated when average heart rate on Holter monitoring exceeded 100 beats/min. Data were analyzed using paired t test and linear regression. Before ACE inhibition, patients (n = 22) demonstrated decreased EF over time (r(2) = 0.23). At ACE inhibitor therapy initiation, mean age was 14.1 ± 4.6 years and mean EF was 44.2 ± 6.8%. BB therapy was used in 24 of 42 patients. Mean age for the ACE inhibitor + BB group was 15.7 ± 3.9 years. The 2 groups showed significant improvement (p <0.0001 for ACE inhibitor and ACE inhibitor plus BB) compared to the pretherapy group. No significant differences were noted between treatment groups. Patients with DMD demonstrated a gradual decrease in myocardial function. Treatment with ACE inhibitor or ACE inhibitor plus BB resulted in significant improvement compared to pretherapy. No significant difference occurred in EF improvement between treatment groups. In conclusion, treatment with ACE inhibitor or ACE inhibitor plus BB can delay progression of cardiomyopathy.

Knee extensor strength exhibits potential to predict function in sporadic inclusion-body myositis.

INTRODUCTION: In this study we address the challenging issue of potential use of muscle strength to predict function in clinical trials. This has immediate relevance to translational studies that attempt to improve quadriceps strength in sporadic inclusion-body myositis (sIBM). METHODS: Maximum voluntary isometric contraction testing as a measure of muscle strength and a battery of functional outcomes were tested in 85 ambulatory subjects with sIBM. RESULTS: Marked quadriceps weakness was noted in all patients. Strength was correlated with distance walked at 2 and 6 minutes. Additional correlations were found with time to get up from a chair, climb stairs, and step up on curbs. CONCLUSIONS: Quadriceps (knee extensor) strength correlated with performance in this large cohort of sIBM subjects, which demonstrated its potential to predict function in this disease. These data provide initial support for use of muscle strength as a surrogate for function, although validation in a clinical trial is required.

Aminoglycoside-induced mutation suppression (stop codon readthrough) as a therapeutic strategy for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is the most common, lethal, X-linked genetic disease, affecting 1 in 3500 newborn males. It is caused by mutations in the DMD gene. Owing to the large size of the gene, the mutation rate in both germline and somatic cells is very high. Nearly 13-15% of DMD cases are caused by nonsense mutations leading to premature termination codons in the reading frame that results in truncated dystrophin protein. Currently there is no cure for DMD. The only available treatment is the use of glucocorticoids that have modest beneficial effects accompanied by significant side effects. Different therapeutic strategies have been developed ranging from gene therapy to exon skipping and nonsense mutation suppression to produce the full-length protein. These strategies have shown promise in the mdx mouse model of muscular dystrophy where they have been reported to ameliorate the dystrophic phenotype and correct the physiological defects in the membrane. Each of these molecular approaches are being investigated in clinical trials. Here we review nonsense mutation suppression by aminoglycosides as a therapeutic strategy to treat DMD with special emphasis on gentamicin-induced readthrough of disease-causing premature termination codons.

Gentamicin-induced readthrough of stop codons in Duchenne muscular dystrophy.

OBJECTIVE: The objective of this study was to establish the feasibility of long-term gentamicin dosing to achieve stop codon readthrough and produce full-length dystrophin. Mutation suppression of stop codons, successfully achieved in the mdx mouse using gentamicin, represents an important evolving treatment strategy in Duchenne muscular dystrophy (DMD). METHODS: Two DMD cohorts received 14-day gentamicin (7.5mg/kg/day): Cohort 1 (n = 10) stop codon patients and Cohort 2 (n = 8) frameshift controls. Two additional stop codon DMD cohorts were gentamicin treated (7.5mg/kg) for 6 months: Cohort 3 (n = 12) dosed weekly and Cohort 4 (n = 4) dosed twice weekly. Pre- and post-treatment biopsies were assessed for dystrophin levels, as were clinical outcomes. RESULTS: In the 14-day study, serum creatine kinase (CK) dropped by 50%, which was not seen in frameshift DMD controls. After 6 months of gentamicin, dystrophin levels significantly increased (p = 0.027); the highest levels reached 13 to 15% of normal (1 in Cohort 3, and 2 in Cohort 4), accompanied by reduced serum CK favoring drug-induced readthrough of stop codons. This was supported by stabilization of strength and a slight increase in forced vital capacity. Pretreatment stable transcripts predicted an increase of dystrophin after gentamicin. Readthrough efficiency was not affected by the stop codon or its surrounding fourth nucleotide. In 1 subject, antigen-specific interferon-gamma enzyme-linked immunospot assay detected an immunogenic dystrophin epitope. INTERPRETATION: 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.

Limb-girdle muscular dystrophy type 2D gene therapy restores alpha-sarcoglycan and associated proteins.

OBJECTIVE: alpha-Sarcoglycan deficiency results in a severe form of muscular dystrophy (limb-girdle muscular dystrophy type 2D [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 muscle was conducted. Control sides received saline. A 3-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 (2 subjects) and 3 months (1 subject). The full sarcoglycan complex was restored in all subjects, and muscle fiber size was increased in the 3-month subject. Adeno-associated virus serotype 1 (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. Enzyme-linked immunospot (ELISpot) showed no interferon-gamma response to alpha-SG or AAV1 capsid peptide pools, with the exception of a minimal capsid response in 1 subject. Restimulation to detect low-frequency capsid-specific T cells by ELISpot assays was negative. Results of the first 3 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.

Utility of cystatin C to monitor renal function in Duchenne muscular dystrophy.

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. In all, 75 subjects were recruited: 35 DMD (mean age 10.8 +/- 5.4 years, corticosteroids n = 19, ambulatory n = 26), 29 healthy controls, 10 with renal disease, and one DMD with renal failure. Cystatin C levels in DMD were normal irrespective of age, ambulation, or corticosteroid treatment. Serum cystatin C was 0.67 +/- 0.11 mg/l compared to normal controls 0.69 +/- 0.09. mg/l. In these same individuals serum creatinine was severely reduced (0.27 +/- 0.12 mg/dl) versus normals (0.75 +/- 0.15 mg/dl, P < 0.01). In one DMD subject in renal failure, cystatin C was elevated. This study demonstrates the potential value of cystatin C as a biomarker for monitoring renal function in DMD. Its applicability extends to other neuromuscular diseases.

Re-examination of the electrocardiogram in boys with Duchenne muscular dystrophy and correlation with its dilated cardiomyopathy.

Duchenne muscular dystrophy (DMD) results in dilated cardiomyopathy (DC). Characteristic electrocardiographic (ECG) changes include short PR interval, right ventricular hypertrophy (RVH), prolonged QTc interval, and prominent Q waves in leads I, aVL, V5, and V6 or in leads II, III, aVF, V5, and V6. We re-examined the prevalence and correlation of ECG changes with DC in DMD. Electrograms of 115 patients with DMD were evaluated. DC was defined as an echocardiographic ejection fraction<55%. PR interval and RVH were based on age-based normal values. Abnormal Q waves were >or=4 mm. Abnormal QTc interval was >or=450 ms. ST-segment depression was defined as >0.5 mm. Fisher's exact test evaluated significant differences between groups and logistic regression determined whether number of ECG changes predicted DC. Forty had DC. No significant differences existed between the number of ECG changes in DC and non-DC groups (p=0.279). Distribution of findings included short PR interval (43%), RVH (37%), prominent Q waves in leads V5 (34%) and V6 (33%), prominent Q waves in leads I, aVL, V5, and V6 (3, 1 with DC), prominent Q waves in leads II, III, aVF, V5, and V6 (9, 4 with DC), long QTc interval (0), ST depression (2, 1 with DC), and flat/biphasic ST segments (38, 15 with DC). In conclusion, ECG changes are similar in patients with DMD regardless of presence of DC. Previously reported characteristic ECG changes are seen in a minority of DMD cases. The most common findings are short PR interval and RVH. Prominent Q waves in leads II, III, aVF, V5, and V6 are more likely.

Death of CD4+ T cells from lymph nodes during primary SIVmac251 infection predicts the rate of AIDS progression.

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.

CD8+ T cell dynamics during primary simian immunodeficiency virus infection in macaques: relationship of effector cell differentiation with the extent of viral replication.

Immunological and virological events that occur during the earliest stages of HIV-1 infection are now considered to have a major impact on subsequent disease progression. We observed changes in the frequencies of CD8(bright) T cells expressing different chemokine receptors in the peripheral blood and lymph nodes of rhesus macaques during the acute phase of the pathogenic SIVmac251 infection; the frequency of CD8(bright) T cells expressing CXCR4 decreased, while the frequency of those expressing CCR5 increased. These reciprocal changes in chemokine receptor expression were associated with changes in the proportion of cycling (Ki67(+)) CD8(bright) T cells, and with the pattern of CD8(bright) T cell differentiation as defined by expression of CCR7 and CD45RA. In contrast, during the primary phase of the attenuated SIVmac251Deltanef infection, no major change was observed. Whereas during the acute phase of the infection with pathogenic SIV (2 wk postinfection) no correlate of disease protection was identified, once the viral load set points were established (2 mo postinfection), we found that the levels of cycling and of CCR5- and CXCR4-positive CD8(bright) T cells were correlated with the extent of viral replication and therefore with SIV-infection outcome. Our data reveal that, during primary SIV infection, despite intense CD8 T cell activation and an increase in CCR5 expression, which are considered as essential for optimal effector function of CD8(+) T cells, these changes are associated with a poor prognosis for disease progression to AIDS.

HIV-1 triggers mitochondrion death.

Apoptosis, a phenotype of programmed cell death involved in development and tissue homeostasis of multicellular organisms, brings into two major pathways and implies a central sensor: the mitochondria. Abnormalities in the cell death control can lead to a variety of diseases and many pathogenic agents target the mitochondria, especially affecting its permeability in order to induce cell death. HIV infection is linked to progressive CD4 T cell depletion. Among the different hypothesis that may explain T cell depletion, apoptosis is one of the main described mechanisms. This review provides current knowledge in HIV-mediated mitochondrial damage due to (i) HIV-specific proteins, (ii) death-by-neglect and (iii) side effects of the HIV drugs.

Intrinsic and extrinsic pathways signaling during HIV-1 mediated cell death.

Infection with human immunodeficiency virus (HIV) is characterized by the gradual depletion of CD4+ T lymphocytes. The incorporation of the concept of apoptosis as a rationale to explain progressive T cell depletion has led to growing research in this field during the last 10 years. In parallel, the biochemical pathways implicated in programmed cell death have been extensively studied. Thus, the influence of mitochondrial control in the two major apoptotic pathways-the extrinsic and intrinsic pathways-is now well admitted. In this review, we summarized our current knowledge of the different pathways involved in the death of T cells in the course of HIV infection.