Clinical features and predictors for disease natural progression in adults with Pompe disease: a nationwide prospective observational study

Background

Due partly to physicians’ unawareness, many adults with Pompe disease are diagnosed with great delay. Besides, it is not well known which factors influence the rate of disease progression, and thus disease outcome. We delineated the specific clinical features of Pompe disease in adults, and mapped out the distribution and severity of muscle weakness, and the sequence of involvement of the individual muscle groups. Furthermore, we defined the natural disease course and identified prognostic factors for disease progression.

Methods

We conducted a single-center, prospective, observational study. Muscle strength (manual muscle testing, and hand-held dynamometry), muscle function (quick motor function test), and pulmonary function (forced vital capacity in sitting and supine positions) were assessed every 3–6 months and analyzed using repeated-measures ANOVA.

Results

Between October 2004 and August 2009, 94 patients aged between 25 and 75 years were included in the study. Although skeletal muscle weakness was typically distributed in a limb-girdle pattern, many patients had unfamiliar features such as ptosis (23%), bulbar weakness (28%), and scapular winging (33%). During follow-up (average 1.6 years, range 0.5-4.2 years), skeletal muscle strength deteriorated significantly (mean declines of ?1.3% point/year for manual muscle testing and of ?2.6% points/year for hand-held dynamometry; both p<0.001). Longer disease duration (>15 years) and pulmonary involvement (forced vital capacity in sitting position <80%) at study entry predicted faster decline. On average, forced vital capacity in supine position deteriorated by 1.3% points per year (p=0.02). Decline in pulmonary function was consistent across subgroups. Ten percent of patients declined unexpectedly fast.

Conclusions

Recognizing patterns of common and less familiar characteristics in adults with Pompe disease facilitates timely diagnosis. Longer disease duration and reduced pulmonary function stand out as predictors of rapid disease progression, and aid in deciding whether to initiate enzyme replacement therapy, or when.

     

Gene structure,transcripts and calciotropic effects of the PTH family of peptides in <Emphasis Type="Italic">Xenopus</Emphasis> and chicken

Background  

Parathyroid hormone (PTH) and PTH-related peptide (PTHrP) belong to a family of endocrine factors that share a highly conserved N-terminal region (amino acids 1-34) and play key roles in calcium homeostasis, bone formation and skeletal development. Recently, PTH-like peptide (PTH-L) was identified in teleost fish raising questions about the evolution of these proteins. Although PTH and PTHrP have been intensively studied in mammals their function in other vertebrates is poorly documented. Amphibians and birds occupy unique phylogenetic positions, the former at the transition of aquatic to terrestrial life and the latter at the transition to homeothermy. Moreover, both organisms have characteristics indicative of a complex system in calcium regulation. This study investigated PTH family evolution in vertebrates with special emphasis on Xenopus and chicken.     

Characterization of Blockade Antibody Responses in GII.2.1976 Snow Mountain Virus-Infected Subjects

Snow Mountain virus (GII.2.1976) is the prototype strain of GII.2 noroviruses (NoVs), which cause an estimated 8% of norovirus outbreaks, yet little is known about the immunobiology of these viruses. To define the human immune response induced by SMV infection and the antigenic relationship between different GII.2 strains that have circulated between 1976 and 2010, we developed a panel of four GII.2 variant virus-like particles (VLPs) and compared their antigenicities by enzyme immunoassay (EIA) and surrogate antibody neutralization (blockade) assays. Volunteers infected with GII.2.1976 developed a mean 167-fold increase in blockade response against the homotypic VLP by day 8 postchallenge. Blockade extended cross-genotype activity in some individuals but not cross-genogroup activity. Polyclonal sera from GII.2.1976-infected volunteers blocked GII.2.1976 significantly better than they blocked GII.2.2002, GII.2.2008, and GII.2.2010, suggesting that blockade epitopes within the GII.2 strains have evolved in the past decade. To potentially map these epitope changes, we developed mouse monoclonal antibodies (MAbs) against GII.2.1976 VLPs and compared their reactivities to a panel of norovirus VLPs. One MAb had broad cross-genogroup EIA reactivity to a nonblockade, linear, conserved epitope. Six MAbs recognized conformational epitopes exclusive to the GII.2 strains. Two MAbs recognized GII.2 blockade epitopes, and both blocked the entire panel of GII.2 variants. These data indicate that the GII.2 strains, unlike the predominant GII.4 strains, have undergone only a limited amount of evolution in blockade epitopes between 1976 and 2010 and indicate that the GII.2-protective component of a multivalent norovirus vaccine may not require frequent reformulation.     

Identification of Cross-Reactive Norovirus CD4+ T Cell Epitopes

Immune responses and the components of protective immunity following norovirus infection in humans are poorly understood. Although antibody responses following norovirus infection have been partially characterized, T cell responses in humans remain largely undefined. In contrast, T cells have been shown to be essential for viral clearance of mouse norovirus (MNV) infection. In this paper, we demonstrate that CD4⁺ T cells secrete gamma interferon (IFN-γ) in response to stimulation with MNV virus-like particles (VLPs) after MNV infection, supporting earlier reports for norovirus-infected mice and humans. Utilizing this model, we immunized mice with alphavirus vectors (Venezuelan equine encephalitis [VEE] virus replicon particles [VRPs]) expressing Norwalk virus (NV) or Farmington Hills virus (FH) virus-like particles to evaluate T cell epitopes shared between human norovirus strains. Stimulation of splenocytes from norovirus VRP-immunized mice with overlapping peptides from complete libraries of the NV or FH capsid proteins revealed specific amino acid sequences containing T cell epitopes that were conserved within genoclusters and genogroups. Immunization with heterologous norovirus VRPs resulted in specific cross-reactive IFN-γ secretion profiles following stimulation with NV and FH peptides in the mouse. Identification of unique strain-specific and cross-reactive epitopes may provide insight into homologous and heterologous T cell-mediated norovirus immunity and provide a platform for the study of norovirus-induced cellular immunity in humans.Norovirus infection is characterized by the induction of both humoral and cellular immune responses. Humoral immunity in humans following norovirus infection has been described in detail for a limited number of norovirus strains (8, 10, 12, 17, 18, 29). Humans mount specific antibody responses to the infecting strain, which bear complex patterns of unique and cross-reactive, yet undefined, epitopes to other strains within or across genogroups (23, 29). Short-term immunity following homologous norovirus challenge has been documented, but long-term immunity remains controversial (16, 25). Furthermore, no studies to date have demonstrated cross-protection following heterologous norovirus challenge (30). While some susceptible individuals can become reinfected with multiple norovirus strains throughout their lifetimes, the mechanism of short-term protection and the impact of previous exposures on susceptibility to reinfection remain largely unknown.The role of T cells in controlling norovirus infection also remains largely undefined. A single comprehensive study detailing immune responses in genogroup II Snow Mountain virus-infected individuals revealed that CD4⁺ TH1 cells can be stimulated by virus-like particles (VLPs) to secrete gamma interferon (IFN-γ) and interleukin-2 (IL-2) (17). Furthermore, heterologous stimulation from VLPs derived from different norovirus strains within but not across genogroups also induced significant IFN-γ secretion compared to that for uninfected individuals (17). A follow-up study with genogroup I Norwalk virus (NV)-infected individuals confirmed high T cell cross-reactivity within a genogroup as measured by IFN-γ secretion (18). Further, vaccination of humans with VLPs also results in short-term IFN-γ production (27).Because norovirus infection studies in humans are confounded by previous exposure histories, the use of inbred mice maintained in pathogen-free environments allows for the study of norovirus immune responses in a naive background. While mice cannot be infected with human norovirus strains, VLP vaccines expressing norovirus structural proteins induce immune responses that can be measured and studied (14, 20). Mice immunized orally or intranasally with VLP vaccines in the presence of adjuvant similarly induced CD4⁺ IFN-γ responses in Peyer''s patches and spleen (22, 26). Induction of CD8⁺ T cells and secretion of the TH2 cytokine IL-4 were separately noted; however, it is unclear if these responses were influenced by VLPs or the coadministered vaccine adjuvants (22, 26). Further, coadministration of alphavirus adjuvant particles with multivalent norovirus VLP vaccine, including or excluding mouse norovirus (MNV) VLPs, resulted in significantly reduced MNV loads following MNV challenge (21). Multivalent VLP vaccines induced robust receptor-blocking antibody responses to heterologous human strains not included in the vaccine composition (20, 21). Moreover, natural infection with MNV supports a role for T cell immunity in viral clearance and protection (5).To advance our understanding of the scope of the cellular immune response within and between strains, we immunized mice with Venezuelan equine encephalitis (VEE) virus replicon particles (VRPs) expressing norovirus VLPs derived from the Norwalk virus (GI.1-1968) (1) or Farmington Hills virus (FH) (GII.4-2002) (19) strains and analyzed splenocytes for cytokine secretion, epitope identification, and heterologous stimulation. The data presented here indicate that the major capsid proteins of genogroup I and II noroviruses contain robust T cell epitopes that cross-react with related strains in the mouse yet also occur within regions of known variation, especially among the GII.4 noroviruses.     

Monoclonal antibody-based antigenic mapping of norovirus GII.4-2002

Noroviruses are the primary cause of epidemic gastroenteritis in humans, and GII.4 strains cause ～80% of the overall disease burden. Surrogate neutralization assays using sera and mouse monoclonal antibodies (MAbs) suggest that antigenic variation maintains GII.4 persistence in the face of herd immunity, as the emergence of new pandemic strains is accompanied by newly evolved neutralization epitopes. To potentially identify specific blockade epitopes that are likely neutralizing and evolving between pandemic strains, mice were hyperimmunized with GII.4-2002 virus-like particles (VLPs) and the resulting MAbs were characterized by biochemical and immunologic assays. All of the MAbs but one recognized GII.4 VLPs representing strains circulating from 1987 to 2009. One MAb weakly recognized GII.4-1987 and -1997 while strongly interacting with 2002 VLPs. This antibody was highly selective and effective at blocking only GII.4-2002-ligand binding. Using bioinformatic analyses, we predicted an evolving GII.4 surface epitope composed of amino acids 407, 412, and 413 and subsequently built mutant VLPs to test the impact of the epitope on MAb binding and blockade potential. Replacement of the 2002 epitope with the epitopes found in 1987 or 2006 strains either reduced or ablated enzyme immunoassay recognition by the GII.4-2002-specific blockade MAb. These data identify a novel, evolving blockade epitope that may be associated with protective immunity, providing further support for the hypotheses that GII.4 norovirus evolution results in antigenic variation that allows the virus to escape from protective herd immunity, resulting in new epidemic strains.