Long-term treatment with human immunoglobulin for antisynthetase syndrome-associated interstitial lung disease.

BACKGROUND: Interstitial lung disease-associated antisynthetase syndrome (AS-ILD) carries significant morbidity and mortality. Corticosteroids and immunosuppressive drugs are the mainstay of treatment. Human immunoglobulin (IVIg), an immunomodulator without immunosuppressive properties, is effective in myositis but the evidence supporting its use in ILD is scarce. OBJECTIVE: To describe clinical outcomes of AS-ILD patients receiving IVIg. METHODS: Retrospective analysis of AS-ILD patients. Linear mixed models using restricted maximum likelihood estimation was used to estimate the change in lung function and corticosteroid dose over time. RESULTS: Data from 17 patients was analyzed. Median follow-up was 24.6 months. Fourteen patients had refractory disease. The mean percent-predicted forced vital capacity (FVC%) (p = 0.048) and percent-predicted diffusing capacity of the lung for carbon monoxide (DLCO%) (p = 0.0223) increased over time, while the mean prednisone dose (p < 0.001) decreased over time. Seven patients achieved a >10% increase in FVC%, including two who used IVIg as initial treatment. Five patients showed a >10% increase in DLCO% and TLC%. Nine (53%) patients experienced side effects. CONCLUSIONS: IVIg may be a useful complementary therapy in active progressive AS-ILD but is associated with potential side effects. Fssssurther investigation is required to determine the value of IVIg as an initial treatment in AS-ILD.

Sporadic inclusion body myositis - a myodegenerative disease or an inflammatory myopathy.

Sporadic inclusion body myositis (sIBM) is an insidious late-onset progressive myopathy that typically affects patients over the age of 50. Clinically, patients develop a characteristic pattern of weakness that affects the forearm flexors and knee extensors. Muscle biopsy, often utilized in the diagnosis, demonstrates a chronic myopathy with mixed pathologies harbouring intramyofiber protein inclusions and endomysial inflammation. The co-existence of these pathologic features (that is, inflammation and protein aggregation) has divided the field of sIBM research into two opposing (albeit slowly unifying) camps regarding disease pathogenesis. The present review explores the recent evidence supporting these distinct pathogenic mechanisms. Future therapies that are designed to target both aspects of sIBM pathologies will likely be necessary to treat sIBM.

Clinical and pathologic differences in interstitial lung disease based on antisynthetase antibody type.

BACKGROUND: Interstitial lung disease (ILD) is a common extramuscular manifestation of the idiopathic inflammatory myopathies (IIMs), dermatomyositis (DM) and polymyositis (PM). Patients with antisynthetase antibodies (ASA) demonstrate some or all of the features of the antisynthetase syndrome including IIM and ILD. It has been hypothesized that the clinical expression of antisynthetase syndrome varies between specific ASAs. OBJECTIVE: We sought to determine whether the myositis-associated ILD (MA-ILD) phenotype differs based on the presence of ASAs and by ASA subtype. METHODS: A cross-sectional and longitudinal analysis of consecutive patients enrolled at the Johns Hopkins Myositis Center with ILD in the setting of clinically diagnosed autoimmune myositis was conducted. RESULTS: Seventy-seven subjects were included; 36 were ASA negative, 28 were anti-Jo1 positive, and 13 were non-Jo1 ASA positive (5 anti-PL-12, 4 anti-PL-7, 2 anti-EJ, and 2 anti-OJ). Non-Jo1 ASA positive participants were more likely to be African-American than Caucasian as compared to both the anti-Jo1 positive (p = 0.01) and ASA negative groups (p < 0.01). ASA negative participants had better mean forced vital capacity percent predicted (FVC%) and total computed tomography scores over time compared to those with anti-Jo1 after controlling for potential confounders. CONCLUSIONS: ASA status was significantly different by race. Those with anti-Jo1 antibodies had worse lung function and CT scores over time compared to those without detectable antisynthetase antibodies. Further prospective study in a larger cohort is needed to determine whether these apparent antibody-specific differences in demographics and manifestations of disease translate into meaningful disparities in clinical outcomes.

Immunohistochemistry of affected tissue may guide cGVHD treatment decisions.

Chronic graft-vs-host disease (cGVHD) myositis is a rare complication of hematopoietic SCT, for which the pathogenesis and optimal therapy are unclear. We performed immunohistochemistry on muscle biopsies from pediatric cGVHD myositis and typical cases of autoimmune dermatomyositis and polymyositis. The immunostaining pattern of cGVHD myositis was distinct from that of typical cases of autoimmunity. There was a high proportion of CD20+ and CD68+ cells, and the best therapeutic response was achieved with rituximab (anti-CD20). These results suggest that cGVHD myositis may be mediated by different leukocytes than similar autoimmune diseases and that treatment may be optimized by targeting the specific cellular infiltrates identified in affected tissue.

The development of excitatory synapses in cultured spinal neurons.

Immunohistochemical studies of synapses in the CNS have demonstrated that glutamate receptors (GluRs) are concentrated at postsynaptic sites in vivo and in vitro (Baude et al., 1995). The mechanisms leading to receptor clustering at excitatory synapses are far less understood than those governing acetylcholine receptor accumulation at the neuromuscular junction () or glycine receptor aggregation at central inhibitory synapses (). Using cultured rat spinal cord neurons, we demonstrate that clustering of the AMPA receptor subunit GluR1 is among the earliest events in excitatory synapse formation in vitro, coincident with the onset of miniature EPSCs and in many cases preceding presynaptic vesicle accumulation. Postsynaptic receptor clustering is induced in a highly specific and reiterative pattern, independent of receptor activation, by contact with a subset of axons capable of inducing receptor clusters. The subunit composition of AMPA receptor clusters varied significantly between neurons but was invariant within a given neuron. The presence of either GluR2 or GluR3 was common to all receptor clusters. Neither high-affinity glutamate transporters nor NMDA receptors appeared to be concentrated with AMPA receptor subunits at these excitatory synapses.

Redistribution and stabilization of cell surface glutamate receptors during synapse formation.

Although the regulation of neurotransmitter receptors during synaptogenesis has been studied extensively at the neuromuscular junction, little is known about the control of excitatory neurotransmitter receptors during synapse formation in central neurons. Using antibodies against extracellular N-terminal (N-GluR1) and intracellular C-terminal (C-GluR1) domains of the AMPA receptor subunit GluR1, combined with surface biotinylation and metabolic labeling studies, we have characterized the redistribution and metabolic stabilization of the AMPA receptor subunit GluR1 during synapse formation in culture. Before synapse formation, GluR1 is distributed widely, both on the surface and within the dendritic cytoplasm of these neurons. The diffuse cell surface pool of receptor appears to be mobile within the membrane and can be induced to cluster by the addition of N-GluR1 to live neurons. As cultures mature and synapses form, there is a redistribution of surface GluR1 into clusters at excitatory synapses where it appears to be immobilized. The change in the distribution of GluR1 is accompanied by an increase in both the half-life of the receptor and the percentage of the total pool of GluR1 that is present on the cell surface. Blockade of postsynaptic AMPA and NMDA receptors had no effect on the redistribution of GluR1. These results begin to characterize the events regulating the distribution of AMPA receptors and demonstrate similarities between synapse formation at the neuromuscular junction and at excitatory synapses in cultured neurons.

Phosphorylation of the alpha-amino-3-hydroxy-5-methylisoxazole4-propionic acid receptor GluR1 subunit by calcium/calmodulin-dependent kinase II.

Modulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic Acid (AMPA) receptors in the brain by protein phosphorylation may play a crucial role in the regulation of synaptic plasticity. Previous studies have demonstrated that calmodulin (CaM) kinase II can phosphorylate and modulate AMPA receptors. However, the sites of CaM kinase phosphorylation have not been unequivocally identified. In the current study, we have generated two phosphorylation site-specific antibodies to analyze the phosphorylation of the glutamate receptor GluR1 subunit. These antibodies recognize GluR1 only when it is phosphorylated on serine residues 831 or 845. We have used these antibodies to demonstrate that serine 831 is specifically phosphorylated by CaM kinase II in transfected cells expressing GluR1 as well as in hippocampal slice preparations. Two-dimensional phosphopeptide mapping experiments indicate that Ser-831 is the major site of CaM kinase II phosphorylation on GluR1. In addition, treatment of hippocampal slice preparations with phorbol esters and forskolin increase the phosphorylation of serine 831 and 845, respectively, indicating that protein kinase C and protein kinase A phosphorylate these residues in hippocampal slices. These results identify the site of CaM kinase phosphorylation of the GluR1 subunit and demonstrate that GluR1 is multiply phosphorylated by protein kinase A, protein kinase C, and CaM kinase II in situ.

Synaptic targeting of glutamate receptors.

The proper targeting and clustering of neurotransmitter receptors at appropriate postsynaptic sites are principal requirements for the formation of functional synapses. Recently, new studies have begun to elucidate the mechanisms underlying the targeting and clustering of glutamate receptors at excitatory synapses in the brain. Members of the SAP90/PSD-95 family of proteins have emerged as potential regulators of glutamate-receptor membrane distribution. Further, targeting motifs within glutamate receptor subunits have been identified. These findings provide important clues in the effort to understand the molecular features of synaptic organization.

Characterization of multiple phosphorylation sites on the AMPA receptor GluR1 subunit.

We have characterized the phosphorylation of the glutamate receptor subunit GluR1, using biochemical and electrophysiological techniques. GluR1 is phosphorylated on multiple sites that are all located on the C-terminus of the protein. Cyclic AMP-dependent protein kinase specifically phosphorylates SER-845 of GluR1 in transfected HEK cells and in neurons in culture. Phosphorylation of this residue results in a 40% potentiation of the peak current through GluR1 homomeric channels. In addition, protein kinase C specifically phosphorylates Ser-831 of GluR1 in HEK-293 cells and in cultured neurons. These results are consistent with the recently proposed transmembrane topology models of glutamate receptors, in which the C-terminus is intracellular. In addition, the modulation of GluR1 by PKA phosphorylation of Ser-845 suggests that phosphorylation of this residue may underlie the PKA-induced potentiation of AMPA receptors in neurons.

The mouse APLP2 gene. Chromosomal localization and promoter characterization.

Senile plaques are primarily comprised of deposits of the beta-amyloid precursor-like proteins APLP1 and APLP2. proteins (APPs). APP is a member of a gene family, including amyloid precursor-like proteins APLP1 and APLP2. Using interspecific mouse backcross mapping, we localized the mouse APLP2 gene to the promixmal region of mouse chromosome 9, syntenic with a region of human 11q. We cloned an approximately 1.2-kilobase mouse genomic fragment containing the APLP2 gene promoter. The APLP2 promoter lacks a typical TATA box, is GC-rich, and contains several sequences for transcription factor binding. S1 nuclease protection analysis revealed the presence of multiple transcription start sites. The lack of a TATA box, the presence of a high GC content, and multiple transcription start sites place the APLP2 promoter in the class of promoters of "housekeeping genes." Regulatory regions within the promoter were assayed by transfection of mouse N2a and Ltk- cells with constructs containing progressive 5'-deletions of the APLP2 promoter fused to the bacterial chloramphenicol acetyl transferase (CAT) reporter gene. A minimal region that includes sequences 99 bp upstream of the predominant transcription start site of the APLP2 promoter was sufficient to direct high levels of CAT expression.

Detection of soluble forms of the beta-amyloid precursor protein in human plasma.

A approximately 40-residue fragment of the beta-amyloid precursor protein (APP) is progressively deposited in the extracellular spaces of brain and blood vessels in Alzheimer's disease (AD), Down's syndrome and aged normal subjects. Soluble, truncated forms of APP lacking the carboxyl terminus are normally secreted from cultured cells expressing this protein and are found in cerebrospinal fluid. Here, we report the detection of a similar soluble APP isoform in human plasma. This approximately 125 kDa protein, which was isolated from plasma by Affi-Gel Blue chromatography or dialysis-induced precipitation, comigrates with the larger of the two major soluble APP forms present in spinal fluid and contains the Kunitz protease inhibitor insert. It thus derives from the APP751 and APP770 precursors; a soluble form of APP695 has not yet been detected in plasma. The approximately 125 kDa plasma form lacks the C-terminal region and is unlikely to serve as a precursor for the beta-protein that forms the amyloid in AD.