F. Shawn Galin

A novel peptide CXCR ligand derived from extracellular matrix degradation during airway inflammation

We describe the tripeptide neutrophil chemoattractant N-acetyl Pro-Gly-Pro (PGP), derived from the breakdown of extracellular matrix (ECM), which shares sequence and structural homology with an important domain on alpha chemokines. PGP caused chemotaxis and production of superoxide through CXC receptors, and administration of peptide caused recruitment of neutrophils (PMNs) into lungs of control, but not CXCR2-deficient mice. PGP was generated in mouse lung after exposure to lipopolysaccharide, and in vivo and in vitro blockade of PGP with monoclonal antibody suppressed PMN responses as much as chemokine-specific monoclonal antibody. Extended PGP treatment caused alveolar enlargement and right ventricular hypertrophy in mice. PGP was detectable in substantial concentrations in a majority of bronchoalveolar lavage samples from individuals with chronic obstructive pulmonary disease, but not control individuals. Thus, PGPs activity links degradation of ECM with neutrophil recruitment in airway inflammation, and PGP may be a biomarker and therapeutic target for neutrophilic inflammatory diseases.

Cloning and sequencing of imniunoglobulin variable-region genes using degenerate oligodeoxy-ribonucleotides and polymerase chain reaction

Abstract A procedure is described for using the polymerase chain reaction (PCR) to amplify and clone the cDNA from mouse immunoglobulin (Ig) variable (V) regions. This method uses a set of universal 5′-oligodeoxy-ribonucleotide primers that are degenerate and allow for the amplification of Ig V-region sequences from γ and μ heavy chains and from κ light chains. Selective first-strand cDNA synthesis is performed using Ig constant region primers and then a PCR is achieved by using the appropriate universal 5′-primer. The universal Ig heavy-chain primer was used to amplify the V-region cDNA from γ and μ isotypes and the universal light-chain primer was used to amplify three separate κ light V-region sequences. This procedure was used to obtain Ig V-region gene sequences from hybridomas secreting IgG1/κ, IgG2b/κ and IgM/κ isotypes.

The Presence of a Matrix-Derived Neutrophil Chemoattractant in Bronchiolitis Obliterans Syndrome after Lung Transplantation

Lung transplantation is a therapeutic modality frequently used in end-stage lung disease. Unfortunately, lung transplant recipients have poor clinical outcomes, often due to the development of bronchiolitis obliterans syndrome (BOS). This process is often characterized by the pathologic findings of obliterative bronchiolitis: neutrophil influx and extracellular matrix remodeling leading to luminal obstruction and airway inflammation. The molecular mechanisms underlying BOS are poorly understood and disease-specific biomarkers are lacking. We report that in addition to increased levels of IL-8, the level of the neutrophil chemoattractant proline-glycine-proline (PGP) is elevated in BOS patient bronchoalveolar lavage (BAL) fluid. The enzymes responsible for generating PGP, matrix metalloproteases 8 and -9 and prolyl endopeptidase, are also elevated in these samples. Together, IL-8 and PGP account for most of the neutrophil chemoattractant capacity seen in BOS BAL fluid. Using specific neutralizing Abs to both IL-8 and PGP, we demonstrate that PGP is a prominent neutrophil chemoattractant found in BAL fluid from individuals at the time of diagnosis of BOS. These findings highlight the influence of a matrix-derived neutrophil chemoattractant in posttransplantation BOS and provide opportunities for the development of unique diagnostics and therapeutics to potentially improve disease outcomes.

Neutrophils contain prolyl endopeptidase and generate the chemotactic peptide, PGP, from collagen.

Prolyl endopeptidase (PE), a protease that cleaves after proline residues in oligopeptides, is highly active in brain and degrades neuropeptides in vitro. We have recently demonstrated that PE, in concert with MMPs, can generate PGP (proline-glycine-proline), a novel, neutrophil chemoattractant, from collagen. In this study, we demonstrate that human peripheral blood neutrophils contain PE, which is constitutively active, and can generate PGP de novo from collagen after activation with LPS. This novel, pro-inflammatory role for PE raises the possibility of a self-sustaining pathway of neutrophilic inflammation and may provide biomarkers and therapeutic targets for diseases caused by chronic, neutrophilic inflammation.

Possible therapeutic vaccines for canine myasthenia gravis : Implications for the human disease and associated fatigue

Myasthenia gravis (MG) is caused by T cell-dependent antibodies reactive with acetylcholine receptors. These autoreactive antibodies cause muscle weakness by interfering with neuromuscular transmission via removal of acetylcholine receptors from the neuromuscular junction as well as changing the architecture of the junction itself. Consequently, muscle fatigue is a debilitating aspect of MG often leading to more general feelings of tiredness not directly due to muscle weakness. We have previously described two peptides that are mimetics of antigen receptors on certain autoreactive T and B cells that are involved in MG. When used as vaccines in the rat model of MG, these peptides prevented and ameliorated disease and muscle fatigue by blunting acetylcholine receptor antibody responses. Such disease protection resulted from vaccine-induced anergizing antibodies against acetylcholine receptor-specific T and B cell antigen receptors. The present study prospectively evaluated the efficacy of these two vaccines in spontaneous acquired MG in pet dogs. When compared to historical controls that were prospectively studied, the vaccines increased the proportion of remitted dogs from 17 to 75%. In comparison to retrospectively studied historical controls that spontaneously remitted from MG, the vaccines accelerated the rate of decline in acetylcholine receptor antibody titers which resulted in a 3-fold decrease in the mean time to remission. These results are suggestive of a new type of targeted therapy that can drive autoimmune responses into long-term remission and possibly afford a means of determining whether correction of a physical cause of muscle weakness also corrects the perception of chronic, generalized fatigue.

Possible alternate splicing or initiation of the pro-opiomelanocortin gene in lymphocytes

Des lymphocytes en culture transcrivent le gene de la proopiomelanocortine; cette transcription augmente quand les lymphocytes sont cultives en presence de ACTH-RH

Highly related immunoglobulin light chain sequences in different multiple sclerosis patients

Although immunoglobulin G and free light (L) chains of oligoclonal origin in cerebrospinal fluid (CSF) are the most common immunologic abnormalities in multiple sclerosis (MS), it is unknown whether homologous CSF L chain sequences are present in different individuals with MS. Using Southern blotting, a particular kappa (kappa) L chain variable region (V) probe was recently found to hybridize to Vkappa cDNA from CSF B cells from almost one half of the MS patients tested but only 10% of normal or other neurologic disease controls [Zhou, S.-R., Maier, C.C., Mitchell, G.W., LaGanke, C.C., Blalock, J.E., Whitaker, J.N., 1998. A cross-reactive idiotope in cerebrospinal fluid cells in multiple sclerosis: further evidence for the role of myelin basic protein. Neurology 50, 411-417.] Here, we report that this likely results from remarkable sequence similarity in certain Vkappa from CSF B cells from different individuals with MS. The high degree of sequence homology even extended to all three complementarity determining regions (CDR) which in part form an antibody combining site. In addition, marked sequence homology was observed between the light chains from the MS patients and those from certain mouse antibodies against myelin basic protein (MBP). The results establish, in principle, that the same or very similar kappa light chain variable regions can be shared between CSF B lymphocytes from different individuals with MS as well as with certain antibodies against MBP.