A randomized study of alglucosidase alfa in late-onset Pompe's disease.

BACKGROUND: Pompe's disease is a metabolic myopathy caused by a deficiency of acid alpha glucosidase (GAA), an enzyme that degrades lysosomal glycogen. Late-onset Pompe's disease is characterized by progressive muscle weakness and loss of respiratory function, leading to early death. We conducted a randomized, placebo-controlled trial of alglucosidase alfa, a recombinant human GAA, for the treatment of late-onset Pompe's disease. METHODS: Ninety patients who were 8 years of age or older, ambulatory, and free of invasive ventilation were randomly assigned to receive biweekly intravenous alglucosidase alfa (20 mg per kilogram of body weight) or placebo for 78 weeks at eight centers in the United States and Europe. The two primary end points were distance walked during a 6-minute walk test and percentage of predicted forced vital capacity (FVC). RESULTS: At 78 weeks, the estimated mean changes from baseline in the primary end points favored alglucosidase alfa (an increase of 28.1+/-13.1 m on the 6-minute walk test and an absolute increase of 3.4+/-1.2 percentage points in FVC; P=0.03 and P=0.006, respectively). Similar proportions of patients in the two groups had adverse events, serious adverse events, and infusion-associated reactions; events that occurred only in patients who received the active study drug included anaphylactic reactions and infusion-associated reactions of urticaria, flushing, hyperhidrosis, chest discomfort, vomiting, and increased blood pressure (each of which occurred in 5 to 8% of the patients). CONCLUSIONS: In this study population, treatment with alglucosidase alfa was associated with improved walking distance and stabilization of pulmonary function over an 18-month period. (ClinicalTrials.gov number, NCT00158600.)

A phase I trial of adeno-associated virus serotype 1-γ-sarcoglycan gene therapy for limb girdle muscular dystrophy type 2C.

γ-Sarcoglycanopathy or limb girdle muscular dystrophy type 2C is an untreatable disease caused by autosomal recessively inherited mutations of the γ-sarcoglycan gene. Nine non-ambulatory patients (two males, seven females, mean age 27 years; range 16-38 years) with del525T homozygous mutation of the γ-sarcoglycan gene and no γ-sarcoglycan immunostaining on muscle biopsy were divided into three equal groups to receive three escalating doses of an adeno-associated virus serotype 1 vector expressing the human γ-sarcoglycan gene under the control of the desmin promoter, by local injection into the extensor carpi radialis muscle. The first group received a single injection of 3 × 10(9) viral genomes in 100 µl, the second group received a single injection of 1.5 × 10(10) viral genomes in 100 µl, and the third group received three simultaneous 100-µl injections at the same site, delivering a total dose of 4.5 × 10(10) viral genomes. No serious adverse effects occurred during 6 months of follow-up. All nine patients became adeno-associated virus serotype 1 seropositive and one developed a cytotoxic response to the adeno-associated virus serotype 1 capsid. Thirty days later, immunohistochemical analysis of injected-muscle biopsy specimens showed γ-sarcoglycan expression in all three patients who received the highest dose (4.7-10.5% positively stained fibres), while real-time polymerase chain reaction detected γ-sarcoglycan messenger RNA. In one patient, γ-sarcoglycan protein was detected by western blot. For two other patients who received the low and intermediate doses, discrete levels of γ-sarcoglycan expression (<1% positively stained fibres) were also detectable. Expression of γ-sarcoglycan protein can be induced in patients with limb girdle muscular dystrophy type 2C by adeno-associated virus serotype 1 gene transfer, with no serious adverse effects.

Correlation of anti-signal recognition particle autoantibody levels with creatine kinase activity in patients with necrotizing myopathy.

OBJECTIVE: Anti-signal recognition particle (anti-SRP) autoantibodies are associated with severe acquired necrotizing myopathies. The role of these autoantibodies remains elusive, and the evolution of anti-SRP levels over time is unknown. In this study, we developed an addressable laser bead immunoassay (ALBIA) technique to investigate a correlation between anti-SRP levels, serum creatine kinase (CK) levels, and muscle strength in patients with necrotizing myopathy. METHODS: The diagnostic value of the ALBIA assay was determined by comparing serum levels of anti-SRP autoantibodies in 31 anti-SRP immunodot-positive patients to those in 190 healthy blood donors and 199 control patients with different inflammatory/autoimmune conditions or polyclonal hypergammaglobulinemia. Among the 31 anti-SRP-positive patients, serum samples from 8 patients were monitored over time for levels of anti-SRP autoantibodies and levels of CK (determined at least 3 times, consecutively, over a mean followup period of 783 days). The relationship between levels of anti-SRP autoantibodies and levels of CK was tested using a linear mixed model. RESULTS: The assay yielded positive results for anti-SRP in all anti-SRP immunodot-positive serum samples tested, while all control sera tested negative. The 8 anti-SRP-positive patients who were followed up longitudinally were found to have normalized CK levels and improved muscle strength. There was a striking correlation between the degree of myolysis, as measured by CK levels, in patients receiving therapy and the anti-SRP54 autoantibody levels in these same patients (P = 0.002). CONCLUSION: Anti-SRP-positive myositis appears to be one of the few autoimmune diseases in which specific autoantibody levels are correlated with surrogate disease activity markers. These results reveal the usefulness of monitoring anti-SRP autoantibody levels in patients receiving therapy, and may also suggest a possible pathogenic role for anti-SRP autoantibodies in the necrotizing myopathies.

Long-term observational study of sporadic inclusion body myositis.

We describe a long-term observational study of a large cohort of patients with sporadic inclusion body myositis and propose a sporadic inclusion body myositis weakness composite index that is easy to perform during a clinic. Data collection from two groups of patients (Paris and Oxford) was completed either during a clinic visit (52%), or by extraction from previous medical records (48%). One hundred and thirty-six patients [57% males, 61 (interquartile range 55-69) years at onset] were included. At the last visit all patients had muscle weakness (proximal British Medical Research Council scale <3/5 in 48%, distal British Medical Research Council scale <3/5 in 40%, swallowing problems in 46%). During their follow-up, 75% of patients had significant walking difficulties and 37% used a wheelchair (after a median duration from onset of 14 years). The sporadic inclusion body myositis weakness composite index, which correlated with grip strength (correlation coefficient: 0.47; P < 0.001) and Rivermead Mobility Index (correlation coefficient: 0.85; P < 0.001), decreased significantly with disease duration (correlation coefficient: -0.47; P < 0.001). The risk of death was only influenced by older age at onset of first symptoms. Seventy-one (52%) patients received immunosuppressive treatments [prednisone in 91.5%, associated (in 64.8%) with other immunomodulatory drugs (intravenous immunoglobulins, methotrexate or azathioprine) for a median duration of 40.8 months]. At the last assessment, patients who had been treated were more severely affected on disability scales (Walton P = 0.007, Rivermead Mobility Index P = 0.004) and on the sporadic inclusion body myositis weakness composite index (P = 0.04). The first stage of disease progression towards handicap for walking was more rapid among patients receiving immunosuppressive treatments (hazard ratio = 2.0, P = 0.002). This study confirms that sporadic inclusion body myositis is slowly progressive but not lethal and that immunosuppressive treatments do not ameliorate its natural course, thus confirming findings from smaller studies. Furthermore, our findings suggest that immunosuppressant drug therapy could have modestly exacerbated progression of disability. The sporadic inclusion body myositis weakness composite index might be a valuable outcome measure for future clinical trials, but requires further assessment and validation.

Role of regulatory T cells in a new mouse model of experimental autoimmune myositis.

Polymyositis is a rare and severe inflammatory muscle disorder. Treatments are partially efficacious but have many side effects. New therapeutic approaches must be first tested in a relevant animal model. Regulatory CD4+CD25+ T cells (Tregs) have been rediscovered as a pivotal cell population in the control of autoimmunity, but the connection between polymyositis and Tregs is currently unknown. To develop a reproducible experimental autoimmune myositis model of polymyositis, mice were immunized once a week for 3 weeks with 1 mg of partially purified myosin emulsified in complete Freund's adjuvant. All mice injected with myosin and complete Freund's adjuvant developed myositis. The infiltrates were composed of CD4(+) and CD8(+) cells, as well as macrophages, but did not contain B lymphocytes. In mice that were depleted of Tregs, the myositis was more severe, as determined by quantitative scoring of muscle inflammation (2.36 +/- 0.9 vs. 1.64 +/- 0.8, P = 0.019). In contrast, injection of in vitro expanded polyclonal Tregs at the time of immunization significantly improved the disease (quantitative score of inflammation 0.87 +/- 1.06 vs. 2.4 +/- 0.67, P = 0.047). Transfer of sensitized or CD4(+)-sorted cells from the lymph nodes of experimental autoimmune myositis mice induced myositis in naïve, irradiated, recipient mice. Thus, experimental autoimmune myositis is a reproducible, transferable disease in mice, both aggravated by Treg depletion and improved by polyclonal Treg injection.

Long-term evolution and determinants of renal function in HIV-infected patients who began receiving combination antiretroviral therapy in 1997-1999, ANRS CO8 APROCO-COPILOTE.

Among 1121 patients (90% Caucasian) infected by the human immunodeficiency virus (HIV), the glomerular filtration rate increased (+0.72 mL/min/1.73 m(2)/month) from treatment initiation to month 16 (the rate increase was lower among men and those with low body mass index, AIDS, or receipt of indinavir), then remained stable up to 7 years. Kidney function should be monitored in patients previously exposed to indinavir.

Endocarditis due to Neisseria bacilliformis in a patient with a bicuspid aortic valve.

We report a case of endocarditis due to the rod-shaped Neisseria species Neisseria bacilliformis. The phenotypic characterization of this recently characterized bacteria is difficult, and the identification requires the sequencing of the 16S rRNA gene. The resolution of the disease was complete after appropriate antibiotic therapy, and surgery was not required.

Massive expansion of regulatory T-cells following interleukin 2 treatment during a phase I-II dendritic cell-based immunotherapy of metastatic renal cancer.

Cytotoxic chemotherapy is ineffective in metastatic renal cancer. However, systemic administration of interleukin 2 (IL-2) or infusion of dendritic cells (DCs) loaded with tumor extracts can lead to some response rates with concomitant survival improvements. We report the results of a phase I-II pilot study combining DCs and IL-2 where six patients were included. DCs were derived from bone marrow CD34+ cells and loaded with autologous tumor extracts. CD34-DC vaccines were infused subcutaneously at day 45, 52, 59, 90 and 120 following surgery in combination with IL-2, that was subsequently administrated after the 3rd and 4th DC vaccinations. Preparation of tumor extracts and CD34-DCs were satisfactory in all patients but one. Due to rapid tumor progression, one patient was excluded before vaccination. In the 4 remaining patients, two received 3 vaccinations, while the 2 others received 5 vaccinations and the full IL-2 treatment. No adverse effect due to the vaccinations was observed. A specific immune response against autologous tumor cells was observed in the 2 patients who completed the treatment. Interestingly, these 2 patients had a more prolonged survival than the patients receiving 3 vaccinations. Importantly, a transient and massive increase of circulating natural regulatory T-cells (nTregs) was evidenced in 3 patients following IL-2 administration. Overall, the use of CD34-DC vaccines is feasible, safe and non-toxic. A specific anti-tumor immune response can be detected. However, our data highlights that IL-2 is a potent inducer of nTregs in vivo and as such may have a negative impact on cancer immunotherapy.

Distal inflammatory myopathy: unusual presentation of polymyositis or new entity?

New classification of idiopathic inflammatory myopathy (IIM) defined three major entities, polymyositis (PM), dermatomyositis (DM) and sporadic inclusion body myositis (s-IBM). We report the clinical, electrophysiological and pathological characteristics of three patients with a rare form of IIM not fulfilling the diagnostic criteria for any of these three major entities. The three patients presented with a subacute, distal asymmetrical weakness in upper limbs. Muscle biopsy showed an active myositis, with necrosis and regeneration, T cell infiltrates with invasion of non-necrotic fibers, without rimmed vacuoles, and diffuse major histocompatibility complex-I (MHC-I) immunostaining in muscle fibers. All patients responded to immunosuppressive agents. Seven others cases were identified in the literature. It is important to recognize this atypical presentation as it seems to respond to immunosuppressive agents.

Shared blood and muscle CD8+ T-cell expansions in inclusion body myositis.

Inclusion body myositis (IBM) is the most frequent inflammatory myopathy over the age of fifty. Pathological findings suggest that two processes may contribute to IBM pathogenesis: a primary degenerative process affecting muscle fibre and/or an autoimmune process mediated by major histocompatibility complex (MHC) class-I-restricted cytotoxic CD8+ T cells. Previous studies have demonstrated that muscle-infiltrating CD8+ T cells in IBM display restricted expression of T-cell receptor (TCR)-BV families or evidenced oligoclonal T-cell expansions. This study was performed to investigate whether blood T cells similarly exhibit clonal expansions due to the recirculation of muscle-infiltrating T cells in the periphery. For this, we studied the T-cell repertoire of 17 IBM patients by complementarity-determining-region (CDR) 3 length distribution (immunoscope) analysis of TCR-B transcripts. Mean age was 68 years (range 53-88) and mean duration of the disease was 6.5 years (2-20). Oligoclonal T-cell expansions were observed in the blood of IBM patients. The quantitative average perturbation D index was significantly increased in IBM patients [D = 13.7% +/- 1.2%, mean +/- standard error of measurement (SEM)] as compared with 17 age-matched controls suffering from connective tissue diseases not associated with T-cell repertoire perturbation, that is, dermatomyositis (DM) and systemic sclerosis (9.3 +/- 0.6%, P < 0.005). Nevertheless, there was no correlation between the level of blood perturbation and muscle inflammation. Sorting experiments showed that these perturbations were due to oligoclonal expansions of CD8+ T cells. In the three IBM patients analysed, we could relate the blood expansions to T-cell clones also found in muscle. The clonally expanded blood T cells dramatically responded to interleukin-2 (IL-2) in vitro, suggesting that they had been primed in vivo, presumably in response to yet unknown muscle auto-antigens. Together, our results indicate that clonally expanded muscle-infiltrating CD8+ T cells re-circulate in the blood and support the concept of a CD8+ T-cell-mediated autoimmune component in IBM, similarly to what is observed in polymyositis (PM).

Marked efficacy of a therapeutic strategy associating prednisone and plasma exchange followed by rituximab in two patients with refractory myopathy associated with antibodies to the signal recognition particle (SRP).

We report two patients with myopathy associated with anti-signal recognition particle Ab, refractory to conventional therapy, who were treated with prednisone and plasma exchange, followed by rituximab. A marked response was observed in both patients, with partial to complete recovery of muscle strength, which was sustained.

Diaphragmatic dysfunction in patients with idiopathic inflammatory myopathies.

Polymyositis, dermatopolymyositis, and inclusion body myositis imply chronic inflammation of skeletal muscles. Pulmonary complications include aspiration pneumonia, interstitial pneumonitis, or respiratory muscle myositis. This study aims at better describing their impact on respiratory muscle. Twenty-three consecutive patients (12 PM, 5 DM, 6 IBM) were studied (static inspiratory and expiratory pressures; diaphragm function in terms of the mouth and transdiaphragmatic pressure responses to bilateral phrenic stimulation). Pulmonary parenchymatous abnormalities were mild (6 cases) or absent. The mouth pressure produced by phrenic stimulation was 6.83+/-3.01 cm H2O, with 18 patients (78%) diagnosed with diaphragm weakness (<10 cm H2O) and lower values in DM (4.35+/-1.48 cm H2O) than in IBM and in PM (P<0.05). Diaphragm weakness is frequent and probably overlooked in inflammatory myopathies. Further studies are needed to delineate the clinical relevance of these results.

Soluble transferrin receptor in hemochromatosis patients during phlebotomy therapy.

BACKGROUND: The monitoring of phlebotomies in hemochromatosis patients depends on iron status measured by ferritin and transferrin saturation (TS). However, in the presence of inflammation or liver injury, soluble transferrin receptor (sTfR) determination was proposed to replace ferritin for diagnosing iron deficiency (ID). The present study evaluated performances of sTfR for the prediction of iron deficiency in a large number of hemochromatosis patients under phlebotomy therapy. METHODS: We studied 52 patients undergoing therapeutic phlebotomies and obtained 2 samples from 37 patients. Biological parameters were determined before each phlebotomy began. Performances of sTfR and TS in the diagnosis of iron deficiency were compared, according to ferritin levels under 12 microg/l. RESULTS: Ferritin and TS were correlated with removed iron (r=0.473, p<0.005 and r=0.345, p<0.05, respectively) and sTfR was correlated with the decrease in hemoglobin levels induced by phlebotomies (r=-0.678, p<0.0001). Areas under Receiver Operating Characteristics (ROC) curves for sTfR and TS were not statistically different for prediction of iron deficiency and sensitivity/specificity of sTfR at 1.64 mg/l were 67/86%. CONCLUSIONS: sTfR determination could be used to predict iron depletion induced by phlebotomies when ferritin is of limited interest, to avoid the appearance of anemia.

Efficacy of Rituximab in Refractory Inflammatory Myopathies Associated with Anti- Synthetase Auto-Antibodies: An Open-Label, Phase II Trial.

OBJECTIVE: Anti-synthetase syndrome (anti-SS) is frequently associated with myositis and interstitial lung disease (ILD). We evaluated prospectively, in a multicenter, open-label, phase II study, the efficacy of rituximab on muscle and lung outcomes. METHODS: Patients were enrolled if they were refractory to conventional treatments (prednisone and at least 2 immunosuppressants). They received 1 g of rituximab at D0, D15, and M6. The primary endpoint was muscular improvement based on manual muscular testing (MMT10, Kendall score in 10 muscles) at M12. Secondary endpoints were normalization of creatine kinase (CK) level, ILD improvement based on forced vital capacity and/or diffuse capacity for carbon monoxide, and number and/or doses of associated immunosuppressants. RESULTS: Twelve patients were enrolled, and 10 completed the study. Only 2 patients presented an improvement of at least 4 points on at least two muscle groups (primary end-point). Overall, seven patients had an increase of at least 4 points on MMT10. CK level decreased from 399 IU/L (range, 48-11,718) to 74.5 IU/L (range, 40-47,857). Corticosteroid doses decreased from 52.5 mg/d (range, 10-70) to 9 mg/d (range, 7-65) and six patients had a decrease in the burden of their associated immunosuppressants. At baseline, all 10 patients presented with ILD. At M12, improvement of ILD was observed in 5 out of the 10 patients, stabilization in 4, and worsening in 1. CONCLUSIONS: This pilot study of rituximab treatment in patients with refractory anti-SS provided data on evolution of muscular and pulmonary parameters. Rituximab should now be evaluated in a larger, controlled study for this homogenous group of patients. TRIAL REGISTRATION: Clinicaltrials.gov NCT00774462.

Highly active antiretroviral therapy corrects hematopoiesis in HIV-1 infected patients: interest for peripheral blood stem cell-based gene therapy.

OBJECTIVES: To study, in asymptomatic HIV-1-infected (HIV+) patients, whether peripheral blood hematopoietic progenitor/stem cells (PBPC) mobilized by granulocyte colony stimulating factor (G-CSF), can be used as a source of cells for retroviral gene therapy. DESIGN: PBPC from two groups of HIV+ patients (treated or untreated by highly active antiretroviral therapy) and from seronegative donors were mobilized with G-CSF. METHODS: PBPC collected by leukapheresis were enriched for CD34 cells, immunophenotypically and functionally characterized, cultured and infected with retroviral vectors. HIV proviral integration was studied on fresh and cultured cells. RESULTS: G-CSF moderately and transiently increased the viral load in untreated patients only, and induced in both groups of HIV+ patients mobilization of percentages and numbers of CD34 cells comparable to those of seronegative volunteers. The most immature CD34 cell subset, the clonogenic progenitor and long-term culture initiating cells were significantly decreased in leukapheresis products and CD34-enriched fractions from untreated HIV+ patients but not in those from treated HIV+ patients. Cell cycle activation and growth factor responses of CD34 cells from both groups of HIV+ patients were not different from those of the control group. Culture and retroviral infection of CD34 cells from HIV+ patients did not enhance HIV replication, and yielded transduction levels similar to those obtained using CD34 cells from seronegative donors. CONCLUSIONS: G-CSF-mobilized PBPC can be safely used for HIV retroviral gene therapy in asymptomatic treated patients while highly active antiretroviral therapy would control the G-CSF-induced increase in viral load and correct the defective hematopoiesis observed in untreated patients, without inhibiting the retroviral transduction of PBPC.

Phase I study of dystrophin plasmid-based gene therapy in Duchenne/Becker muscular dystrophy.

Nine patients with Duchenne or Becker muscular dystrophy were injected via the radialis muscle with a full-length human dystrophin plasmid, either once with 200 or 600 microg of DNA or twice, 2 weeks apart, with 600 microg of DNA. In the biopsies taken 3 weeks after the initial injection, the vector was detected at the injection site in all patients. Immunohistochemistry and nested reverse transcription-polymerase chain reaction indicated dystrophin expression in six of nine patients. The level of expression was low (up to 6% weak, but complete sarcolemmal dystrophin staining, and up to 26% partial sarcolemmal labeling). No side effects were observed, nor any cellular or humoral anti-dystrophin responses. These results suggest that exogenous dystrophin expression can be obtained in Duchenne/Becker patients after intramuscular transfer of plasmid, without adverse effects, hence paving the way for future developments in gene therapy of hereditary muscular diseases.

Current protocol of a research phase I clinical trial of full-length dystrophin plasmid DNA in Duchenne/Becker muscular dystrophies. Part II: clinical protocol.

A phase I open clinical study on gene therapy in Duchenne and Becker muscular dystrophy, without direct individual benefit for the patient, is being performed at the Pitié-Salpêtrière Hospital, Paris. The aims of this project are: (a) to determine the tolerance and the safety of the intramuscular administration of dystrophin cDNA and (b) to study the quality of the gene transfer in vivo in human patients affected by Duchenne and Becker muscular dystrophy. This clinical trial is conducted sequentially and includes three cohorts of three patients each. Patients must be at least 15 years of age. Diagnosis of Duchenne and Becker muscular dystrophy was confirmed by molecular analysis of the dystrophin gene and for each patient the abnormal expression of dystrophin was confirmed, in skeletal muscle, with antibodies directed against the deleted part of the dystrophin. This phase I study is scheduled to be completed by the end of 2002.

[Pathogenesis of primary inflammatory myopathies]. / Physiopathologie des myopathies inflammatoires primitives.

COMMON ELEMENTS: Primary inflammatory myopathies consist of dermatomyositis (DM), polymyositis (PM) and inclusion body myositis (IBM). They have certain characteristics in common: progressive muscle weakness and mononuclear inflammatory infiltrates in the muscle. DIFFERENT MECHANISMS: They may be distinguished by their histological features which also reflect their different underlying pathogeneses. The mechanism of DM would be complement-mediated microangiopathy, the inflammatory infiltrate of which would be secondary to ischemic phenomena, whereas in PM the muscle fibres are damaged by cytotoxic CD8 T lymphocytes. The factors triggering-off these two forms of myositis remain unknown. IBM may be a degenerative disease with accumulation of a variety of proteins within the fibres. The inflammatory infiltrate, which is similar to that seen in PM, may be a reaction to accumulated proteins.

Four-year longitudinal study of clinical and functional endpoints in sporadic inclusion body myositis: implications for therapeutic trials.

Natural history studies in sporadic inclusion body myositis are of fundamental interest for future therapeutic trials. Previous works have demonstrated the particular relevance of knee extension strength in the follow-up of this disease. This work aimed to extend a preceding natural history over 9 months to a four year period. Thirteen patients were assessed using clinical and functional scales and dynamometry. Except wrist extension torque and manual muscle testing composite score, all the measurements presented a significant decline. The most important changes were observed for knee extension and ankle flexion and extension. The relative change in knee extension strength correlated with the level of strength at baseline. A non-linear correlation was found between 6-minute walk distance and knee extension strength. This study confirms that knee extension strength is particularly relevant to follow patients with sporadic inclusion body myositis. It also shows that a strength loss does not have linear consequences on motor ability. Finally strength and motor ability are complementing each other in the understanding of disease progression.