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Broadly neutralizing antibodies have been associated with spontaneous clearance of the hepatitis C infection as well as viral persistence by immune escape. Further study of neutralizing antibody epitopes is needed to unravel pathways of resistance to virus neutralization, and to identify conserved regions for vaccine design. All reported broadly neutralizing antibody (BNAb) epitopes in the HCV Envelope (E2) glycoprotein were identified. The critical contact residues of these epitopes were mapped onto the linear E2 sequence. All publicly available E2 sequences were then downloaded and the contact residues within the BNAb epitopes were assessed for the level of conservation, as well as the frequency of occurrence of experimentally-proven resistance mutations. Epitopes were also compared between two sequence datasets obtained from samples collected at well-defined time points from acute ( 180days) infections, to identify any significant differences in residue usage. The contact residues for all BNAbs were contained within 3 linear regions of the E2 protein sequence. An analysis of 1749 full length E2 sequences from public databases showed that only 10 out of 29 experimentally-proven resistance mutations were present at a frequency >5%. Comparison of subtype 1a viral sequences obtained from samples collected during acute or chronic infection revealed significant differences at positions 610 and 655 with changes in residue (p

Deficiency of acid alpha-glucosidase (GAA) results in widespread cellular deposition of lysosomal glycogen manifesting as myopathy and cardiomyopathy. When GAA-/- mice were treated with rhGAA (20 mg/kg/week for up to 5 months), skeletal muscle cells took up little enzyme compared to liver and heart. Glycogen reduction was less than 50%, and some fibers showed little or no glycogen clearance. A dose of 100 mg/kg/week resulted in approximately 75% glycogen clearance in skeletal muscle. The enzyme reduced cardiac glycogen to undetectable levels at either dose. Skeletal muscle fibers with residual glycogen showed immunoreactivity for LAMP-1/LAMP-2, indicating that undigested glycogen remained in proliferating lysosomes. Glycogen clearance was more pronounced in type 1 fibers, and histochemical analysis suggested an increased mannose-6-phosphate receptor immunoreactivity in these fibers. Differential transport of enzyme into lysosomes may explain the strikingly uneven pattern of glycogen removal. Autophagic vacuoles, a feature of both the mouse model and the human disease, persisted despite glycogen clearance. In some groups a modest glycogen reduction was accompanied by improved muscle strength. These studies suggest that enzyme replacement therapy, although at much higher doses than in other lysosomal diseases, has the potential to reverse cardiac pathology and to reduce the glycogen level in skeletal muscle.

Objective The etiology and pathogenesis of human inflammatory myopathies remain unclear. Findings of several studies suggest that the degree of inflammation does not correlate consistently with the severity of clinical disease or of structural changes in the muscle fibers, indicating that nonimmune pathways may contribute to the pathogenesis of myositis. This study was undertaken to investigate these pathways in myositis patients and in a class I major histocompatibility complex (MHC)–transgenic mouse model of myositis. Methods We examined muscle tissue from human myositis patients and from class I MHC–transgenic mice for nonimmune pathways, using biochemical, immunohistochemical, and gene expression profiling assays. Results Up-regulation of class I MHC in skeletal muscle fibers was an early and consistent feature of human inflammatory myopathies. Class I MHC staining in muscle fibers of myositis patients showed both cell surface and a reticular pattern of internal reactivity. The pathways of endoplasmic reticulum (ER) stress response, the unfolded protein response (glucose-regulated protein 78 pathway), and the ER overload response (NF-κB pathway) were significantly activated in muscle tissue of human myositis patients and in the mouse model. Ectopic expression of wild-type mouse class I MHC (H-2Kb) but not degradable glycosylation mutants of H-2Kb induced ER stress response in C2C12 skeletal muscle cells. Conclusion These results indicate that the ER stress response may be a major nonimmune mechanism responsible for skeletal muscle damage and dysfunction in autoimmune myositis. Strategies to interfere with this pathway may have therapeutic value in patients with this disease.

Although many lysosomal disorders are corrected by a small amount of the missing enzyme, it has been generally accepted that 20-30% of normal acid alpha-glucosidase (GAA) activity, provided by gene or enzyme replacement therapy, would be required to reverse the myopathy and cardiomyopathy in Pompe disease. We have addressed the issue of reversibility of the disease in the Gaa(-/-) mouse model. We have made transgenic lines expressing human GAA in skeletal and cardiac muscle of Gaa(-/-) mice, and we turned the transgene on at different stages of disease progression by using a tetracycline-controllable system. We have demonstrated that levels of 20-30% of normal activity are indeed sufficient to clear glycogen in the heart of young Gaa(-/-) mice, but not in older mice with a considerably higher glycogen load. However, in skeletal muscle-a major organ affected in infantile and in milder, late-onset variants in humans-induction of GAA expression in young Gaa(-/-) mice to levels greatly exceeding wildtype values did not result in full phenotypic correction, and some muscle fibers showed little or no glycogen clearance. The results demonstrate that complete reversal of pathology in skeletal muscle or long-affected heart muscle will require much more enzyme than previously expected or a different approach.

Although acute hepatitis E virus (HEV) infection is known to induce IgM and IgG humoral host immune responses, little is known about occurrence of cellular responses in this infection. We looked for evidence of lymphocyte sensitization to HEV peptides in patients with acute HEV infection.peripheral blood lymphocytes were obtained from patients with acute hepatitis E and healthy controls. Proliferation of these lymphocytes in the presence of each of seven peptides with amino acid sequences corresponding to open reading frames 2 and 3 proteins of HEV (3 and 4 peptides, respectively) were studied; no peptide was added to control wells. Proliferative responses with stimulation indices exceeding 3.0 were taken as positive.More patients showed reactivity to two or more HEV peptides than did controls (11/21 vs 5/22, p0.05). Reactivity to one peptide corresponding to open reading frame 2 of HEV was more frequent in patients than in controls (7/21 vs 1/22, p0.05).Our results show that lymphocytes of patients with acute hepatitis E show sensitization to HEV peptides. This may have significance in understanding the pathogenetic mechanisms of liver injury in this infection.

Autoantibodies to histidyl-tRNA synthetase (HisRS) or to alanyl-, asparaginyl-, glycyl-, isoleucyl-, or threonyl-tRNA synthetase occur in approximately 25% of patients with polymyositis or dermatomyositis. We tested the ability of several aminoacyl-tRNA synthetases to induce leukocyte migration. HisRS induced CD4(+) and CD8(+) lymphocytes, interleukin (IL)-2-activated monocytes, and immature dendritic cells (iDCs) to migrate, but not neutrophils, mature DCs, or unstimulated monocytes. An NH(2)-terminal domain, 1-48 HisRS, was chemotactic for lymphocytes and activated monocytes, whereas a deletion mutant, HisRS-M, was inactive. HisRS selectively activated CC chemokine receptor (CCR)5-transfected HEK-293 cells, inducing migration by interacting with extracellular domain three. Furthermore, monoclonal anti-CCR5 blocked HisRS-induced chemotaxis and conversely, HisRS blocked anti-CCR5 binding. Asparaginyl-tRNA synthetase induced migration of lymphocytes, activated monocytes, iDCs, and CCR3-transfected HEK-293 cells. Seryl-tRNA synthetase induced migration of CCR3-transfected cells but not iDCs. Nonautoantigenic aspartyl-tRNA and lysyl-tRNA synthetases were not chemotactic. Thus, autoantigenic aminoacyl-tRNA synthetases, perhaps liberated from damaged muscle cells, may perpetuate the development of myositis by recruiting mononuclear cells that induce innate and adaptive immune responses. Therefore, the selection of a self-molecule as a target for an autoantibody response may be a consequence of the proinflammatory properties of the molecule itself.