Von Willebrand factor propeptide as a marker of disease activity in systemic sclerosis (scleroderma)

In 44 consecutive patients with systemic sclerosis (SSc), plasma concentrations of von Willebrand factor (vWf) were higher than those of the vWf propeptide, but the propeptide showed less variability within patient subgroups. Higher values of the propeptide were observed in patients with early pulmonary involvement. A closer correlation of the propeptide than of vWf to biochemical markers of activity was also evident. Our results suggest that the propeptide, despite a shorter circulating half-time and lower plasma concentrations than vWf, is more useful in the assessment of disease activity in SSc.

Autoantibodies to calpastatin (an endogenous inhibitor for calcium-dependent neutral protease, calpain) in systemic rheumatic diseases.

We identified an autoantibody that reacts with calpastatin [an inhibitor protein of the calcium-dependent neutral protease calpain (EC 3.4.22.17)]. In early immunoblot studies, sera from patients with rheumatoid arthritis (RA) recognized unidentified 60-, 45-, and 75-kDa proteins in HeLa cell extracts. To identify these autoantigens, we used patient sera to clone cDNAs from a lambda gt11 expression library. We isolated clones of four genes that expressed fusion proteins recognized by RA sera. The 1.2-kb cDNA insert (termed RA-6) appeared to encode a polypeptide corresponding to the 60-kDa antigen from HeLa cells, since antibodies bound to the RA-6 fusion protein also reacted with a 60-kDa HeLa protein. The deduced amino acid sequence of the RA-6 cDNA was completely identical with the C-terminal 178 amino acids of human calpastatin except for one amino acid substitution. Patient sera that reacted with the RA-6 also bound pig muscle calpastatin, and a monoclonal antibody to human calpastatin recognized the RA-6 fusion protein, confirming the identity of RA-6 with calpastatin. Moreover, the purified RA-6 fusion protein inhibited the proteolytic activity of calpain, and IgG from a serum containing anti-calpastatin antibodies blocked the calpastatin activity of the RA-6 fusion protein. Immunoblots of the RA-6 product detected autoantibodies to calpastatin in 57% of RA patients; this incidence was significantly higher than that observed in other systemic rheumatic diseases, including systemic lupus erythematosus (27%), polymyositis/dermatomyositis (24%), systemic sclerosis (38%), and overlap syndrome (29%). Thus, anti-calpastatin antibodies are present most frequently in patients with RA and may participate in pathogenic mechanisms of rheumatic diseases.

Anti-DNA antibodies are a major component of the intrathecal B cell response in multiple sclerosis

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of unknown cause that afflicts the central nervous system. MS is typified by a highly clonally restricted antigen-driven antibody response that is confined largely to the central nervous system. The major antigenic targets of this response and the role of antibody in disease pathogenesis remain unclear. To help resolve these issues, we cloned the IgG repertoire directly from active plaque and periplaque regions in MS brain and from B cells recovered from the cerebrospinal fluid of a patient with MS with subacute disease. We found that high-affinity anti-DNA antibodies are a major component of the intrathecal IgG response in the patients with MS that we studied. Furthermore, we show DNA-specific monoclonal antibodies rescued from two subjects with MS as well as a DNA-specific antibody rescued from an individual suffering from systemic lupus erythematosus bound efficiently to the surface of neuronal cells and oligodendrocytes. For two of these antibodies, cell-surface recognition was DNA dependent. Our findings indicate that anti-DNA antibodies may promote important neuropathologic mechanisms in chronic inflammatory disorders, such as MS and systemic lupus erythematosus.

Reexamination of the mechanism of hydroxyl radical adducts formed from the reaction between familial amyotrophic lateral sclerosis-associated Cu,Zn superoxide dismutase mutants and H2O2

Amyotrophic lateral sclerosis (ALS) involves the progressive degeneration of motor neurons in the spinal cord and motor cortex. Mutations to Cu,Zn superoxide dismutase (SOD) linked with familial ALS are reported to increase hydroxyl radical adduct formation from hydrogen peroxide as measured by spin trapping with 5,5′-dimethyl-1-pyrrolline N-oxide (DMPO). In the present study, we have used oxygen-17-enriched water and H2O2 to reinvestigate the mechanism of DMPO/⋅OH formation from the SOD and SOD mutants. The relative ratios of DMPO/⋅17OH and DMPO/⋅16OH formed in the Fenton reaction were 90% and 10%, respectively, reflecting the ratios of H217O2 to H216O2. The reaction of the WT SOD with H217O2 in bicarbonate/CO2 buffer yielded 63% DMPO/⋅17OH and 37% DMPO/⋅16OH. Similar results were obtained from the reaction between familial ALS SOD mutants and H217O2: DMPO/⋅17OH (64%); DMPO/⋅16OH (36%) from A4V and DMPO/⋅17OH (62%); and DMPO/⋅16OH (38%) from G93A. These results were confirmed further by using 5-diethoxyphosphoryl-5-methyl-1-pyrroline N-oxide spin trap, a phosphorylated analog of DMPO. Contrary to earlier reports, the present results indicate that a significant fraction of DMPO/⋅OH formed during the reaction of SOD and familial ALS SOD mutants with H2O2 is derived from the incorporation of oxygen from water due to oxidation of DMPO to DMPO/⋅OH presumably via DMPO radical cation. No differences were detected between WT and mutant SODs, neither in the concentration of DMPO/⋅OH or DEPMPO/⋅OH formed nor in the relative incorporation of oxygen from H2O2 or water.

The neuroprotective agent SR 57746A abrogates experimental autoimmune encephalomyelitis and impairs associated blood–brain barrier disruption: Implications for multiple sclerosis treatment

Experimental autoimmune encephalomyelitis (EAE) is a T cell autoimmune disorder that is a widely used animal model for multiple sclerosis (MS) and, as in MS, clinical signs of EAE are associated with blood–brain barrier (BBB) disruption. SR 57746A, a nonpeptide drug without classical immunosuppressive properties, efficiently protected the BBB and impaired intrathecal IgG synthesis (two conventional markers of MS exacerbation) and consequently suppressed EAE clinical signs. This compound inhibited EAE-induced spinal cord mononuclear cell invasion and normalized tumor necrosis factor α and IFN-γ mRNA expression within the spinal cord. These data suggested that pharmacological intervention aimed at inhibiting proinflammatory cytokine expression within the central nervous system provided protection against BBB disruption, the first clinical sign of EAE and probably the key point of acute MS attacks. This finding could lead to the development of a new class of compounds for oral therapy of MS, as a supplement to immunosuppressive agents.

Molecular identification of a novel retrovirus repeatedly isolated from patients with multiple sclerosis

The partial molecular characterization of multiple sclerosis (MS)-associated retrovirus (MSRV), a novel retrovirus previously called LM7, is reported. MSRV has been isolated repeatedly from leptomeningeal, choroid plexus and from Epstein–Barr virus-immortalized B cells of MS patients. A strategy based on reverse transcriptase PCR with RNA-purified extracellular virions yielded an initial pol fragment from which other regions of the retroviral genome were subsequently obtained by sequence extension. MSRV-specific PCR primers amplified a pol region from RNA present at the peak of reverse transcriptase activity, coinciding with extracellular viral particles in sucrose density gradients. The same sequence was detected in noncellular RNA from MS patient plasma and in cerebrospinal fluid from untreated MS patients. MSRV is related to, but distinct from, the endogenous retroviral sequence ERV9. Whether MSRV represents an exogenous retrovirus with closely related endogenous elements or a replication-competent, virion-producing, endogenous provirus is as yet unknown. Further molecular epidemiological studies are required to determine precisely the apparent association of virions containing MSRV RNA with MS.

Elevated free nitrotyrosine levels, but not protein-bound nitrotyrosine or hydroxyl radicals, throughout amyotrophic lateral sclerosis (ALS)-like disease implicate tyrosine nitration as an aberrant in vivo property of one familial ALS-linked superoxide dismutase 1 mutant

Mutations in superoxide dismutase 1 (SOD1; EC 1.15.1.1) are responsible for a proportion of familial amyotrophic lateral sclerosis (ALS) through acquisition of an as-yet-unidentified toxic property or properties. Two proposed possibilities are that toxicity may arise from imperfectly folded mutant SOD1 catalyzing the nitration of tyrosines [Beckman, J. S., Carson, M., Smith, C. D. & Koppenol, W. H. (1993) Nature (London) 364, 584] through use of peroxynitrite or from peroxidation arising from elevated production of hydroxyl radicals through use of hydrogen peroxide as a substrate [Wiedau-Pazos, M., Goto, J. J., Rabizadeh, S., Gralla, E. D., Roe, J. A., Valentine, J. S. & Bredesen, D. E. (1996) Science 271, 515–518]. To test these possibilities, levels of nitrotyrosine and markers for hydroxyl radical formation were measured in two lines of transgenic mice that develop progressive motor neuron disease from expressing human familial ALS-linked SOD1 mutation G37R. Relative to normal mice or mice expressing high levels of wild-type human SOD1, 3-nitrotyrosine levels were elevated by 2- to 3-fold in spinal cords coincident with the earliest pathological abnormalities and remained elevated in spinal cord throughout progression of disease. However, no increases in protein-bound nitrotyrosine were found during any stage of SOD1-mutant-mediated disease in mice or at end stage of sporadic or SOD1-mediated familial human ALS. When salicylate trapping of hydroxyl radicals and measurement of levels of malondialdehyde were used, there was no evidence throughout disease progression in mice for enhanced production of hydroxyl radicals or lipid peroxidation, respectively. The presence of elevated nitrotyrosine levels beginning at the earliest stages of cellular pathology and continuing throughout progression of disease demonstrates that tyrosine nitration is one in vivo aberrant property of this ALS-linked SOD1 mutant.

Genome scan of human systemic lupus erythematosus: Evidence for linkage on chromosome 1q in African-American pedigrees

Systemic lupus erythematosus (SLE) is an autoimmune disorder characterized by production of autoantibodies against intracellular antigens including DNA, ribosomal P, Ro (SS-A), La (SS-B), and the spliceosome. Etiology is suspected to involve genetic and environmental factors. Evidence of genetic involvement includes: associations with HLA-DR3, HLA-DR2, Fcγ receptors (FcγR) IIA and IIIA, and hereditary complement component deficiencies, as well as familial aggregation, monozygotic twin concordance >20%, λs > 10, purported linkage at 1q41–42, and inbred mouse strains that consistently develop lupus. We have completed a genome scan in 94 extended multiplex pedigrees by using model-based linkage analysis. Potential [log10 of the odds for linkage (lod) > 2.0] SLE loci have been identified at chromosomes 1q41, 1q23, and 11q14–23 in African-Americans; 14q11, 4p15, 11q25, 2q32, 19q13, 6q26–27, and 12p12–11 in European-Americans; and 1q23, 13q32, 20q13, and 1q31 in all pedigrees combined. An effect for the FcγRIIA candidate polymorphism) at 1q23 (lod = 3.37 in African-Americans) is syntenic with linkage in a murine model of lupus. Sib-pair and multipoint nonparametric analyses also support linkage (P < 0.05) at nine loci detected by using two-point lod score analysis (lod > 2.0). Our results are consistent with the presumed complexity of genetic susceptibility to SLE and illustrate racial origin is likely to influence the specific nature of these genetic effects.

A genome-wide search for susceptibility genes in human systemic lupus erythematosus sib-pair families

Systemic lupus erythematosus (SLE) is an autoimmune multisystem inflammatory disease characterized by the production of pathogenic autoantibodies. Previous genetic studies have suggested associations with HLA Class II alleles, complement gene deficiencies, and Fc receptor polymorphisms; however, it is likely that other genes contribute to SLE susceptibility and pathogenesis. Here, we report the results of a genome-wide microsatellite marker screen in 105 SLE sib-pair families. By using multipoint nonparametric methods, the strongest evidence for linkage was found near the HLA locus (6p11-p21) [D6S257, logarithm of odds (lod) = 3.90, P = 0.000011] and at three additional regions: 16q13 (D16S415, lod = 3.64, P = 0.000022), 14q21–23 (D14S276, lod = 2.81, P = 0.00016), and 20p12 (D20S186, lod = 2.62, P = 0.00025). Another nine regions (1p36, 1p13, 1q42, 2p15, 2q21–33, 3cent-q11, 4q28, 11p15, and 15q26) were identified with lod scores ≥1.00. These data support the hypothesis that multiple genes, including one in the HLA region, influence susceptibility to human SLE.

Results of MDR-1 vector modification trial indicate that granulocyte/macrophage colony-forming unit cells do not contribute to posttransplant hematopoietic recovery following intensive systemic therapy

To formally test the hypothesis that the granulocyte/macrophage colony-forming unit (GM-CFU) cells can contribute to early hematopoietic reconstitution immediately after transplant, the frequency of genetically modified GM-CFU after retroviral vector transduction was measured by a quantitative in situ polymerase chain reaction (PCR), which is specific for the multidrug resistance-1 (MDR-1) vector, and by a quantitative GM-CFU methylcellulose plating assay. The results of this analysis showed no difference between the transduction frequency in the products of two different transduction protocols: “suspension transduction” and “stromal growth factor transduction.” However, when an analysis of the frequency of cells positive for the retroviral MDR-1 vector posttransplantation was carried out, 0 of 10 patients transplanted with cells transduced by the suspension method were positive for the vector MDR-1 posttransplant, whereas 5 of 8 patients transplanted with the cells transduced by the stromal growth factor method were positive for the MDR-1 vector transcription unit by in situ or in solution PCR assay (a difference that is significant at the P = 0.0065 level by the Fisher exact test). These data suggest that only very small subsets of the GM-CFU fraction of myeloid cells, if any, contribute to the repopulation of the hematopoietic tissues that occurs following intensive systemic therapy and transplantation of autologous hematopoietic cells.

Systemic administration of interleukin 2 enhances the therapeutic efficacy of dendritic cell-based tumor vaccines

We have reported previously that murine bone marrow-derived dendritic cells (DC) pulsed with whole tumor lysates can mediate potent antitumor immune responses both in vitro and in vivo. Because successful therapy was dependent on host immune T cells, we have now evaluated whether the systemic administration of the T cell stimulatory/growth promoting cytokine interleukin-2 (IL-2) could enhance tumor lysate-pulsed DC-based immunizations to further promote protective immunity toward, and therapeutic rejection of, syngeneic murine tumors. In three separate approaches using a weakly immunogenic sarcoma (MCA-207), the systemic administration of nontoxic doses of recombinant IL-2 (20,000 and 40,000 IU/dose) was capable of mediating significant increases in the potency of DC-based immunizations. IL-2 could augment the efficacy of tumor lysate-pulsed DC to induce protective immunity to lethal tumor challenge as well as enhance splenic cytotoxic T lymphocyte activity and interferon-γ production in these treated mice. Moreover, treatment with the combination of tumor lysate-pulsed DC and IL-2 could also mediate regressions of established pulmonary 3-day micrometastases and 7-day macrometastases as well as established 14- and 28-day s.c. tumors, leading to either significant cure rates or prolongation in overall survival. Collectively, these findings show that nontoxic doses of recombinant IL-2 can potentiate the antitumor effects of tumor lysate-pulsed DC in vivo and provide preclinical rationale for the use of IL-2 in DC-based vaccine strategies in patients with advanced cancer.

Acute systemic inflammation up-regulates secretory sphingomyelinase in vivo: A possible link between inflammatory cytokines and atherogenesis

Inflammation plays a critical role in atherogenesis, yet the mediators linking inflammation to specific atherogenic processes remain to be elucidated. One such mediator may be secretory sphingomyelinase (S-SMase), a product of the acid sphingomyelinase gene. The secretion of S-SMase by cultured endothelial cells is induced by inflammatory cytokines, and in vivo data have implicated S-SMase in subendothelial lipoprotein aggregation, macrophage foam cell formation, and possibly other atherogenic processes. Thus, the goal of this study was to seek evidence for S-SMase regulation in vivo during a physiologically relevant inflammatory response. First, wild-type mice were injected with saline or lipopolysaccharide (LPS) as a model of acute systemic inflammation. Serum S-SMase activity 3 h postinjection was increased 2- to 2.5-fold by LPS (P < 0.01). To determine the role of IL-1 in the LPS response, we used IL-1 converting enzyme knockout mice, which exhibit deficient IL-1 bioactivity. The level of serum S-SMase activity in LPS-injected IL-1 converting enzyme knockout mice was ≈35% less than that in identically treated wild-type mice (P < 0.01). In LPS-injected IL-1-receptor antagonist knockout mice, which have an enhanced response to IL-1, serum S-SMase activity was increased 1.8-fold compared with LPS-injected wild-type mice (P < 0.01). Finally, when wild-type mice were injected directly with IL-1β, tumor necrosis factor α, or both, serum S-SMase activity increased 1.6-, 2.3-, and 2.9-fold, respectively (P < 0.01). These data show regulation of S-SMase activity in vivo and they raise the possibility that local stimulation of S-SMase may contribute to the effects of inflammatory cytokines in atherosclerosis.

A human fibrosarcoma inhibits systemic angiogenesis and the growth of experimental metastases via thrombospondin-1

Concomitant tumor resistance refers to the ability of some large primary tumors to hold smaller tumors in check, preventing their progressive growth. Here, we demonstrate this phenomenon with a human tumor growing in a nude mouse and show that it is caused by secretion by the tumor of the inhibitor of angiogenesis, thrombospondin-1. When growing subcutaneously, the human fibrosarcoma line HT1080 induced concomitant tumor resistance, preventing the growth of experimental B16/F10 melanoma metastases in the lung. Resistance was due to the production by the tumor cells themselves of high levels of thrombospondin-1, which was present at inhibitory levels in the plasma of tumor-bearing animals who become unable to mount an angiogenic response in their corneas. Animals carrying tumors formed by antisense-derived subclones of HT1080 that secreted low or no thrombospondin had weak or no ability to control the growth of lung metastases. Although purified human platelet thrombospondin-1 had no effect on the growth of melanoma cells in vitro, when injected into mice it was able to halt the growth of their experimental metastases, providing clear evidence of the efficacy of thrombospondin-1 as an anti-tumor agent.