Protein arginine deiminase 4 inhibition is sufficient for the amelioration of collagen-induced arthritis.

Citrullination of joint proteins by the protein arginine deiminase (PAD) family of enzymes is recognized increasingly as a key process in the pathogenesis of rheumatoid arthritis. This present study was undertaken to explore the efficacy of a novel PAD4-selective inhibitor, GSK199, in the murine collagen-induced arthritis model of rheumatoid arthritis. Mice were dosed daily from the time of collagen immunization with GSK199. Efficacy was assessed against a wide range of end-points, including clinical disease scores, joint histology and immunohistochemistry, serum and joint citrulline levels and quantification of synovial autoantibodies using a proteomic array containing joint peptides. Administration of GSK199 at 30 mg/kg led to significant effects on arthritis, assessed both by global clinical disease activity and by histological analyses of synovial inflammation, pannus formation and damage to cartilage and bone. In addition, significant decreases in complement C3 deposition in both synovium and cartilage were observed robustly with GSK199 at 10 mg/kg. Neither the total levels of citrulline measurable in joint and serum, nor levels of circulating collagen antibodies, were affected significantly by treatment with GSK199 at any dose level. In contrast, a subset of serum antibodies reactive against citrullinated and non-citrullinated joint peptides were reduced with GSK199 treatment. These data extend our previous demonstration of efficacy with the pan-PAD inhibitor Cl-amidine and demonstrate robustly that PAD4 inhibition alone is sufficient to block murine arthritis clinical and histopathological end-points.

Oral and topical boswellic acid attenuates mouse osteoarthritis.

OBJECTIVE: Boswellic acid is a plant-derived molecule with putative anti-inflammatory effects. This study was performed to determine whether oral or topical administration of boswellic acid can attenuate joint damage in a mouse model of osteoarthritis (OA). METHODS: Levels of boswellic acid were measured in the blood and synovium of mice treated with oral or topical boswellic acid. OA was generated by surgical destabilization of the medial meniscus (DMM). Therapy with oral or topical boswellic acid was initiated one day after surgery and continued for 12 weeks, when knees were harvested and scored histologically for degree of cartilage loss, osteophyte formation, and synovitis. Microdissected OA synovium was stimulated with IL-1ß or lipopolysaccharide (LPS) in the presence or absence of boswellic acid and cytokine production by quantitative polymerase chain reaction (PCR) or multiplex enzyme linked immunoabsorbant assay (ELISA). RESULTS: Topical treatment resulted in synovial concentrations of boswellic acid 2-6-fold higher than that measured in plasma. Cartilage loss was significantly reduced in mice treated with oral or topical boswellic acid compared with vehicle control (P < 0.01 for both oral and topical therapies). Likewise, treatment with either oral boswellic acid or boswellic acid ointment reduced of synovitis (P = 0.006 and 0.025, respectively) and osteophyte formation (P = 0.009 and 0.030, respectively). In vitro, boswellic acid was able to inhibit IL-1ß and TLR4 mediated induction of several inflammatory mediators from OA synovial explant tissue. CONCLUSIONS: Significant synovial concentration and therapeutic efficacy can be achieved with topical boswellic acid treatment. These findings suggest that boswellic acid has potential as a disease-modifying agent in OA.

Neuroprotective natural antibodies to assemblies of amyloidogenic peptides decrease with normal aging and advancing Alzheimer's disease.

A number of distinct beta-amyloid (Abeta) variants or multimers have been implicated in Alzheimer's disease (AD), and antibodies recognizing such peptides are in clinical trials. Humans have natural Abeta-specific antibodies, but their diversity, abundance, and function in the general population remain largely unknown. Here, we demonstrate with peptide microarrays the presence of natural antibodies against known toxic Abeta and amyloidogenic non-Abeta species in plasma samples and cerebrospinal fluid of AD patients and healthy controls aged 21-89 years. Antibody reactivity was most prominent against oligomeric assemblies of Abeta and pyroglutamate or oxidized residues, and IgGs specific for oligomeric preparations of Abeta1-42 in particular declined with age and advancing AD. Most individuals showed unexpected antibody reactivities against peptides unique to autosomal dominant forms of dementia (mutant Abeta, ABri, ADan) and IgGs isolated from plasma of AD patients or healthy controls protected primary neurons from Abeta toxicity. Aged vervets showed similar patterns of plasma IgG antibodies against amyloid peptides, and after immunization with Abeta the monkeys developed high titers not only against Abeta peptides but also against ABri and ADan peptides. Our findings support the concept of conformation-specific, cross-reactive antibodies that may protect against amyloidogenic toxic peptides. If a therapeutic benefit of Abeta antibodies can be confirmed in AD patients, stimulating the production of such neuroprotective antibodies or passively administering them to the elderly population may provide a preventive measure toward AD.

Suppression of autoimmunity via microbial mimics of altered peptide ligands.

Molecular mimics of self-antigens can behave as altered peptide ligands and serve to ameliorate autoimmune disease. Analysis of experimental autoimmune encephalomyelitis with proteomic autoantibody microarrays reveals that there might exist a wide variety of microbes with features that mimic self-epitopes. Autoimmunity could therefore be modulated via microbial immunity, which may account for relapse and remission of ongoing disease.

Demyelination and inhibition of tumor necrosis factor (TNF).

The development of tumor necrosis factor alpha (TNFalpha) inhibitor therapy is arguably the most significant achievement in the treatment of rheumatic diseases to date. One serious potential side effect associated with these agents is the onset of neurologic signs and symptoms. In this paper we will examine the relationship of TNFalpha antagonism and demyelinating disease. Reviewing early laboratory and animal models, we discuss the mechanism of TNFalpha in central nervous system (CNS) injury and repair. Two negative studies of TNFa inhibitor therapy in the treatment of refractory multiple sclerosis (MS) are considered. From the manufacturers' clinical development programs and post-marketing adverse event reporting data, we report the current incidence of demyelinating symptoms associated with each of the commercially available anti-TNFa agents. Comparing these reports to the incidence of MS in society as a whole, we find the rate of new cases of neurologic disease in exposed patients is not different from the rate of expected cases. Finally we explore arguments that support and refute a potential biologic relationship between TNFa neutralization and demyelinating disease.

Autoantibodies in early arthritis: advances in diagnosis and prognostication.

Several excellent reviews have recently been published on the significance of autoantibodies in rheumatoid arthritis (RA) (1-4). Here we: (i) review selected longitudinal studies examining the predictive utility of autoantibodies in early arthritis and early RA cohorts; (ii) assess the relevance of autoantibodies as an independent parameter for prediction and prognostication of RA; and (iii) describe the potential of multiplex autoantibody assays, including miniaturized, high-throughput microarray technology, to improve diagnosis and prognostication in recent-onset synovitis/early arthritis patients.

Flexural composite oscillators for the measurement of anelastic and elastic properties of solids at frequencies of 1 to 10 kHz.

Longitudinal composite oscillators for measuring internal friction, piezoelectric modulus, and strain modulation effects are usually limited to a frequency range of 30 to 200 kHz. If the same crystals are vibrated in flexure, a longitudinal strain can be introduced with the resonance frequency below 3 kHz while at the same time keeping the inherent high Q of the composite system. This paper develops the theory for the strain amplitude and damping for the flexural composite oscillator made up of two quartz crystals plus specimen and, if appropriate, spacers. This high Q technique of vibrating in flexure has applications for strain modulation and damping experiments.

Allele-specific expression of the mouse B-cell surface protein CD72 on T cells.

CD72 is a 45 000 Mr mouse B-cell surface glycoprotein involved in B-cell proliferation and differentiation. Expression of mouse CD72 is thought to be restricted to the B-cell lineage. We recently demonstrated that the monoclonal antibodies K10.6 and B9.689, previously defined as recognizing the mouse lymphocyte alloantigens Ly-19.2 and Ly-32.2, respectively, recognize specific alleles of CD72. Early studies using antibody-mediated cytotoxicity assays demonstrated that K10.6 and B9.689 react with B cells, several T-cell lines, and a subset of peripheral T cells. These findings led us to consider the possibility that CD72 might also be expressed on a subset of T cells. In this report we demonstrate that CD72 is constitutively expressed on a fraction of peripheral T cells isolated from strains of mice expressing the CD72(b) allele, but not the CD72(a) or CD72(c) alleles. Three days after activating T cells with concanavalin A or plate-bound CD3-specific mAb, CD72 is expressed on a larger fraction of peripheral T cells as well as a fraction of thymocytes from mouse strains expressing the CD72(b) allele. CD72 is expressed on both the CD4(+) and CD8(+) thymocyte and peripheral T-cell subsets. No CD72 expression is detected on activated thymocytes or peripheral T cells from mouse strains expressing the CD72(a) or CD72(c) alleles. Expression of CD72(b) on peripheral T cells was confirmed by northern blot analysis demonstrating CD72 mRNA expression. These results demonstrate that CD72 expression is not restricted to B lineage cells in mouse strains expressing the CD72(b) allele; instead, a population of T lineage cells in these mice also expresses CD72.

Dispersal of American house dust mites (Acari:Pyroglyphidae) in a residence.

American house dust mites, Dermatophagoides farinae Hughes, were marked by adding a 4% solution of Sudan Red 7B, a microbiological stain, to mite rearing media. Marked mites were released onto a downstairs couch in a 2-story residence. Two children sat on the couch for approximately 3 h after which their clothes were examined for stained mites. Various parts of the house and family vehicle were vacuum sampled, and dust samples examined for presence of marked mites. Results of 2 trials showed the presence of marked mites on clothing, upstairs in the residence, as well as in the family vehicle. Clothing is shown to be a significant factor in the dispersal of American house dust mites. Even if concentrated in a small area (1 couch), mites were able to disperse throughout the house and into a family vehicle within a matter of weeks.

Identification of a mouse protein homologous to the human CD6 T cell surface protein and sequence of the corresponding cDNA.

CD6 is a 105/130 kDa monomeric T cell surface glycoprotein that has been shown to play a role in human T cell activation. Recently a partial mouse CD6 cDNA sequence was described. We have isolated full-length cDNA clones including the initiation codon and sequence encoding the full signal peptide, as well as an additional 39 amino acids within the cytoplasmic domain as compared to the previously reported clone. The predicted full-length mouse CD6 protein contains 665 amino acids and has the features of a type I integral membrane protein. The extracellular domain of mouse CD6 is composed of three repeated cysteine-rich domains similar to those in human CD6, mouse and human CD5, and other members of a family of proteins whose prototype is the type I macrophage scavenger receptor. In marked contrast to the previously published human CD6 sequence, the mouse sequence predicts a long cytoplasmic tail that is not closely related to other proteins and possesses two proline-rich motifs containing the SH3-domain binding consensus sequence, three protein kinase C phosphorylation site motifs, nine casein kinase-2 phosphorylation site motifs, and a serine-threonine-rich motif repeated three times. Northern blot analysis revealed that mouse CD6 mRNA is expressed predominantly in thymus, lymph node, and spleen. A polyclonal antiserum was raised against mouse CD6 by gene gun plasmid DNA immunization of rabbits with the mouse CD6 cDNA in an expression vector. In immunofluorescence analysis this polyclonal antiserum positively stained the surface of cells transfected with the mouse CD6 cDNA in an expression vector, as well as most normal mouse thymocytes and peripheral T cells. CD6 protein is expressed on most CD4+CD8+ double-positive and CD4+ or CD8+ single-positive thymocytes, and is expressed at highest levels on mature CD3high thymocytes. The expression of mouse CD6 in thymocytes and peripheral T cells correlates closely with the expression of the related CD5 molecule. The polyclonal rabbit anti-mouse CD6 Abs immunoprecipitated a major polypeptide of 128 kDa from resting and 130 kDa from PMA- and FCS-activated mouse thymocytes and lymph node cells; it is likely that this increase in size upon activation is due to phosphorylation of mouse CD6 as has been described for human CD6. These data demonstrate that mouse thymocytes and T cells express a 130-kDa cell surface protein homologous to human CD6.

Human CD6 possesses a large, alternatively spliced cytoplasmic domain.

Human CD6 is a monomeric 105/130-kDa T cell surface glycoprotein that is involved in T cell activation. The apparent discrepancy between the size of the cytoplasmic domain in human (44 amino acids) and mouse (243 amino acids) CD6, led us to use reverse transcriptase-polymerase chain reaction of human peripheral blood lymphocyte mRNA to isolate cDNA clones that include the carboxyl-terminal coding region of human CD6. The nucleotide sequence of the longest human cDNA clone, CD6-PB1, predicts a protein of 668 amino acids with a 244-amino acid cytoplasmic domain similar in size to and possessing 71.5% amino acid sequence identity with the cytoplasmic domain of mouse CD6. This previously unrecognized 244-amino acid cytoplasmic domain does not have significant homology to any other known protein (except mouse CD6), but does possess two proline-rich motifs containing the SH3 domain-binding consensus sequence, a serine-threonine-rich motif repeated three times, three protein kinase C phosphorylation-site motifs, and 10 casein kinase-2 phosphorylation-site motifs. These sequences are likely to play a role in the ability of CD6-specific monoclonal antibodies to stimulate T cell proliferation. Full-length CD6 cDNA containing this cytoplasmic domain sequence encodes a monomeric 105/130-kDa protein that can be immunoprecipitated from the surface of transfected cells and comigrates upon SDS-PAGE with wild-type CD6 immunoprecipitated from PBL. We also isolated two alternatively spliced forms of human CD6 cDNA lacking sequences encoding membrane-proximal regions of the cytoplasmic domain which maintain the same reading frame as CD6-PB1. The short cytoplasmic domain of the previously reported human CD6-15 cDNA clone results from a deletion of a 20-bp segment through use of an alternative 3' splice site, resulting in a frame shift and premature termination of translation relative to the clones we have isolated. These data demonstrate that human CD6 possesses a large cytoplasmic domain containing sequence motifs that are likely to be involved in signal transduction upon stimulation of T cells through CD6 ligation.

Structure of the mouse CD72 (Lyb-2) gene and its alternatively spliced transcripts.

The complete sequence of the CD72 gene from the C57L mouse, including the 5' and 3' flanking sequences, is reported. The gene spans 6830 base pairs and includes nine exons surrounding eight introns. It does not have an obvious TATAA box, so it belongs to a group of genes with TATA-less promoters that are regulated during mammalian immunodifferentiation. cDNA sequence comparisons among CD72a, CD72b, and CD72c alleles have demonstrated two distinct seven amino acid insertion/deletions among these allelic variants. Based on our genomic sequence studies as well as PCR analyses, we found that different strains of mice can alternatively or exclusively use either of two AG sites surrounding the 21-bp insertion/deletions as 3' splice sites in an allele-specific manner. Other alternative splicing events, such as exon skipping, also contribute to CD72 polymorphism. In mouse splenic B cells there are allele-specific distributions of CD72 mRNAs that contain sequences from both exon 3 and exon 4, from either exon 3 or exon 4, or from neither exon 3 nor 4. It is unclear what the in vivo function might be of the proteins encoded by the mRNA forms lacking these exon sequences.

Biochemical identity of the mouse Ly-19.2 and Ly-32.2 alloantigens with the B cell differentiation antigen Lyb-2/CD72.

Lyb-2/CD72 is a 45-kDa mouse B cell surface protein that binds CD5 (Ly-1) and has been shown to induce B cell proliferation upon mAb binding. The serologically defined Ly-19.2 and Ly-32.2 lymphocyte alloantigens have mouse strain distribution patterns similar to that of the Lyb-2/CD72 alleles and map to the same region on chromosome 4 as Lyb-2/CD72. Our recent isolation of the Lyb-2a, -2b, and -2c cDNA has enabled us in this report to examine the relationship between Ly-19, Ly-32, and Lyb-2/CD72. A rat T cell line transfected with a mouse Lyb-2a cDNA is recognized by Ly-19.2-specific mAb, whereas transfectants expressing the Lyb-2b cDNA are recognized by both Ly-19.2 and Ly-32.2-specific mAb. Cell surface iodination immunoprecipitation analysis from Lyb-2a cDNA transfectants using Lyb-2a- and Ly-19.2-specific mAb as well as from Lyb-2b cDNA transfectants using Lyb-2b-, Ly-19.2-, and Ly-32.2-specific Ab, produced immunoprecipitates containing comigrating 45-kDa polypeptides. Preclearing studies with these transfectants indicate that the immunoprecipitated proteins represent the same polypeptide chain. These results demonstrate that the mouse Ly-19.2 and Ly-32.2 alloantigens are in fact the B cell differentiation Ag Lyb-2/CD72.

Extensive polymorphism in the extracellular domain of the mouse B cell differentiation antigen Lyb-2/CD72.

Lyb-2/CD72 is a 45-kDa mouse B cell surface protein that binds CD5 and has been shown to play a role in B cell proliferation and differentiation. Using the polymerase chain reaction we have isolated and sequenced cDNA clones encoding the serologically defined mouse Lyb-2a, Lyb-2b, and Lyb-2c alleles. We confirmed that our full length cDNA clones encode the Lyb-2a, -2b, and -2c alleles, respectively, by transfecting the isolated Lyb-2/CD72 cDNA clones into L cells and demonstrating that the transfectants bind only the appropriate allele specific anti-Lyb-2/CD72 antibodies. Sequence comparisons demonstrate that the Lyb-2/CD72 allels are highly conserved in their cytoplasmic and transmembrane domains but exhibit a high degree of polymorphism in their extracellular domains. This polymorphism in the extracellular region involves amino acid substitutions at a minimum of 20 residues and is concentrated primarily in the membrane distal region. cDNA sequence comparisons also demonstrate two distinct seven amino acid insertion/deletions among these allelic variants. A form of Lyb-2b cDNA lacking the sequence encoding the transmembrane region was isolated from a C57B1/6 mouse and a CH12.LX subline. The Lyb-2/CD72 PCR products from mRNA of mice expressing Lyb-2a and Lyb-2c contain a DNA fragment that corresponds in size to the transmembraneless form, suggesting that these mouse strains also express this mRNA.

Measuring cypermethrin resistance in the German cockroach (Orthoptera: Blattellidae).

Cypermethrin resistance in the German cockroach, Blattella germanica (L.), was assessed by tests of surface contact and topical application. Topical application provided the most sensitive measure of resistance in a field strain. The resistance ratio (RR) measured by topical application was 122.6 for cypermethrin at LD50. As measured by surface contact, the resistance ratio at KT50 was 2.9. Differences between the walking movement of cockroaches of susceptible (VPI) and field (RHA) strains on deposits of cypermethrin influenced KT50 values. A bioassay with unrestricted movement resulted in RHA strain cockroaches accumulating a larger dose on the tarsal pads and subsequent reduction of the resistance ratio at KT50. Less walking by the VPI strain resulted in less insecticide accumulation on their tarsal pads. On a bioassay in which movement was restricted, the amount of insecticide accumulating on the tarsi was equalized, resulting in an increased resistance ratio at KT50. Differences in susceptibility were more accurately measured when the two strains were topically treated (either on the thorax or the tarsal pads) with known doses of insecticide.