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Preclinical evidence supports targeting the C5a receptor ( C5a R) in rheumatoid arthritis ( RA). To support ongoing clinical development of an anti- C5a R monoclonal antibody, we have investigated for the first time the mechanism of action and the pharmacodynamics of a blocking anti-murine C5a R (anti-m C5a R) surrogate antibody in mouse collagen-induced arthritis ( CIA). First, efficacy was demonstrated in a multiple-dose treatment study. Almost complete inhibition of clinical disease progression was obtained, including reduced bone and cartilage destruction in anti-m C5a R-treated mice. Then, the mechanism of action was examined by looking for early effects of anti-m C5a R treatment in single-dose treatment studies. We found that 48 h after single-dose treatment with anti-m C5a R, the neutrophil and macrophage infiltration into the paws was already reduced. In addition, several inflammatory markers, including tumour necrosis factor ( TNF)-α, interleukin ( IL)-6 and IL-17A were reduced locally in the paws, indicating reduction of local inflammation. Furthermore, dose-setting experiments supported a beneficial clinical effect of dosing above the C5a R saturation level. In conclusion, these preclinical data demonstrated rapid onset effects of antibody blockade of C5a R. The data have translational value in supporting the Novo Nordisk clinical trials of an anti- C5a R antibody in rheumatoid arthritis patients, by identifying potential biomarkers of treatment effects as well as by providing information on pharmacodynamics and novel insights into the mechanism of action of monoclonal antibody blockade of C5a R. [ABSTRACT FROM AUTHOR]

Zyx was proposed as a positional candidate for rat Iddm1. In this Zyx was mapped with a rat radiation hybrid (RH) panel using mouse-derived primers for PCR amplification and detection of a Single-Strand Conformational Polymorphism between hamster and rat. The amplified rat segment was 95% identical to mouse Zyx. Zyx was positioned to rat chromosome 4 between markers Prss1 and D4Mit5. Since Iddm1was positioned between markers D4Rat75 and Npy this excluded Zyx as a candidate gene for Iddm1. PCR amplification of rat genomic DNA gave rise to a 260-bp product.

Complement activation correlates to rheumatoid arthritis disease activity, and increased amounts of the complement split product C5a is observed in synovial fluids from rheumatoid arthritis patients. Blockade of C5a or its receptor (C5aR) is efficacious in several arthritis models. The aim of this study was to investigate the role of C5a and C5aR in human rheumatoid arthritis and psoriatic arthritis-both with respect to expression and function. Synovial fluid, blood and synovial samples were obtained from rheumatoid arthritis, psoriatic arthritis and osteoarthritis patients as a less inflammatory arthritis type, and blood from healthy subjects. Cells infiltrating synovial tissue were analysed by immunohistochemistry and flow cytometry. SF and blood were analysed for biomarkers by flow cytometry or ELISA. The effect of a blocking anti-human C5aR mAb on leukocyte migration was determined using a Boyden chamber. Appropriate statistical tests were applied for comparisons. C5aR+ cells were detected in most rheumatoid arthritis, in all psoriatic arthritis, but not in non-inflammatory control synovia. C5aR+ cells were primarily neutrophils and macrophages. C5aR+ macrophages were mainly found in lymphoid aggregates in close contact with T cells. C5a levels were increased in both rheumatoid arthritis and psoriatic arthritis synovial fluid compared to osteoarthritis, and in blood from rheumatoid arthritis compared to healthy subjects. Neutrophil and monocyte migration to rheumatoid arthritis synovial fluid was significantly inhibited by anti-C5aR. The data support that the C5a-C5aR axis may be driving the infiltration of inflammatory cells into the synovial fluid and synovium in both rheumatoid and psoriatic arthritis, and suggest that C5a or C5aR may be a promising treatment target in both diseases.

Blockade of the complement cascade at the C5a/C5a receptor (C5aR)-axis is believed to be an attractive treatment avenue in rheumatoid arthritis (RA). However, the effects of such interventions during the early phases of arthritis remain to be clarified. In this study we use the murine delayed-type hypersensitivity arthritis (DTHA) model to study the very early effects of a blocking, non-depleting anti-C5aR mAb on joint inflammation with treatment synchronised with disease onset, an approach not previously described. The DTHA model is a single-paw inflammatory arthritis model characterised by synchronised and rapid disease onset driven by T-cells, immune complexes and neutrophils. We show that a reduction in paw swelling, bone erosion, cartilage destruction, synovitis and new bone formation is apparent as little as 60 h after administration of a single dose of a blocking, non-depleting anti-mouse C5aR mAb. Importantly, infiltration of neutrophils into the joint and synovium is also reduced following a single dose, demonstrating that C5aR signalling during the early stage of arthritis regulates neutrophil infiltration and activation. Furthermore, the number of T-cells in circulation and in the draining popliteal lymph node is also reduced following a single dose of anti-C5aR, suggesting that modulation of the C5a/C5aR axis results in effects on the T cell compartment in inflammatory arthritis. In summary, these data demonstrate that blockade of C5aR leads to rapid and significant effects on arthritic disease development in a DTHA model strengthening the rationale of C5aR-blockade as a treatment strategy for RA, especially during the early stages of arthritis flare.

To understand the ability of regulatory T-cells to control diabetes development in clinically relevant situations, we established a new model of accelerated diabetes in young DP-BB rats by transferring purified T-cells from DR-BB rats made acutely diabetic. Transfer of 3, 5, 10, or 23 million pure in vitro-activated T-cells accelerated diabetes onset in >90% of the recipients, with the degree of acceleration being dosage dependent. Cotransfer of unfractionated leukocytes from healthy donors prevented diabetes. Full protection was achieved when protective cells were transferred 3-4 days before diabetogenic cells, whereas transfer 2 days before conferred only partial protection. Protection resided in the CD4(+) fraction, as purified CD4(+) T-cells prevented the accelerated diabetes. When CD25(+) cells were depleted from these cells before they were transferred, their ability to prevent diabetes was impaired. In contrast, two million CD4(+)CD25(+) cells (expressing Foxp3) prevented the accelerated diabetes when transferred both before and simultaneously with the diabetogenic T-cells. In addition, 2 million CD4(+)CD25(+) T-cells prevented spontaneous diabetes, even when given to rats age 42 days, whereas 20 million CD4(+)CD25(-) cells (with low Foxp3 expression) were far less effective. We thus demonstrated that CD4(+)CD25(+) cells exhibit powerful regulatory potential in rat diabetes.

Rat immune-associated nucleotide 4-like 1 (Ian4l1) encodes an antiapoptotic protein, which is essential for T-cell survival. A frameshift mutation at codon 85 in the biobreeding diabetes-prone (BBDP) rat is the cause of their life-long T-cell lymphopenia, which includes lack of regulatory T-cells--a prerequisite for spontaneous autoimmune destruction of their beta-cells. This study reports the identification of seven Ian4l1 mRNA variants. The genomic organization of the exons indicates three promoter regions. The promoter of two of the mRNAs was characterized. Rapid amplification of cDNA ends (RACE) and ribonuclease protection assay (RPA) demonstrated multiple transcription start sites (TSS) with two major sites. The localization of the core promoter and regulatory regions was identified by a luciferase assay of the 2.7-kb upstream of the TSS. The regulatory regions functioned similarly in two cell lines--one expressing Ian4l1 and one not expressing it. This indicates that the cell-specific expression is controlled by regions outside the 2.7-kb region, or by the chromatin structure or chromatin methylation level. The core promoter is TATA-less and initiator element-less, and contains putative binding sites for YY1, Sp1, and MED-1, the latter being an element believed to be important for transcription from TATA-less promoters.

Type 1 diabetes is an autoimmune disease with a complex polygenic inheritance. Until recently, only three susceptibility genes had been reproducibly identified, namely HLA, INS-VNTR, and CTLA4. During the past 7 years, a number of new putative susceptibility genes have been isolated from both human and animal models of the disease. We present eight genes implicated in type 1 diabetes etiology and discuss them in relation to the pathogenesis of the disease: VDR, IL6, IL12B, AIRE, FOXP3, B2m, Cblb, and Lyp/Ian4l1.

Iddm4 is one of several susceptibility genes that have been identified in the BB rat model of type 1 diabetes. The BB rat allele of this gene confers dominant predisposition to diabetes induction by immune perturbation in both the diabetes-prone and the diabetes-resistant substrains, whereas the Wistar Furth (WF) allele confers resistance. We have positioned the gene in a 2.8-cM region on rat Chromosome (Chr) 4, proximal to Lyp/Ian4l1. We have produced a radiation hybrid map of the Iddm4-region that includes a number of rat genes with their mouse and human orthologs. We present a comparative map of the rat Iddm4 region in rat, human, and mouse, assigning the gene to a 6.3-Mb segment between PTN and ZYX at 7q32 in the human genome, and to a 5.7-Mb segment between Ptn and Zyx in the mouse genome.

Apoptosis is a regulated cell death program controlled by extrinsic and intrinsic signaling pathways. The intrinsic pathway involves stress signals that activate pro-apoptotic members of the Bcl-2 family, inducing permeabilization of mitochondria and release of apoptogenic factors. These proteins localize to the outer mitochondrial membrane. Ian4, a mitochondrial outer membrane protein with GTP-binding activity, is normally present in thymocytes, T cells, and B cells. We and others have recently discovered that a mutation in the rat Ian4 gene results in severe T cell lymphopenia that is associated with the expression of autoimmune diabetes. The mechanism by which Ian4 controls T cell homeostasis is unknown. Here we show that the absence of Ian4 in T cells causes mitochondrial dysfunction, increased mitochondrial levels of stress-inducible chaperonins and a leucine-rich protein, and T cell-specific spontaneous apoptosis. T cell activation and caspase 8 inhibition both prevented apoptosis, whereas transfection of T cells with Ian4-specific small interfering RNA recapitulated the apoptotic phenotype. The findings establish Ian4 as a tissue-specific regulator of mitochondrial integrity.

Diabetes-prone (DP) BB rats spontaneously develop insulin-dependent diabetes resembling human type 1 diabetes. They also exhibit lifelong T-cell lymphopenia. Functional and genetic data support the hypothesis that the gene responsible for the lymphopenia, Lyp, is also a diabetes susceptibility gene, named Iddm1. We constructed a 550-kb P1-derived artificial chromosome contig of the region. Here, we present a corrected genetic map reducing the genetic interval to 0.2 cM and the physical interval to 150-290 kb. A total of 13 genes and six GenomeScan models are assigned to the homologous human DNA segment on HSA7q36.1, 8 of which belong to the family of immune-associated nucleotides (Ian genes). Two of these are orthologous to mouse Ian1 and -4, both excellent candidates for Iddm1. In normal rats, they are expressed in the thymus and T-cell regions of the spleen. In the thymus of lymphopenic rats, Ian1 exhibits wild-type expression patterns, whereas Ian4 expression is reduced. Mutational screening of their coding sequences revealed a frameshift mutation in Ian4 among lymphopenic rats. The mutation results in a truncated protein in which the COOH-terminal 215 amino acids-including the anchor localizing the protein to the outer mitochondrial membrane-are replaced by 19 other amino acids. We propose that Ian4 is identical to Iddm1.

The Lyp locus controls diabetes development in rats. The diabetogenic allele in diabetes-prone BB rats is responsible for T cell lymphopenia characterized by the absence of regulatory T cells. We present refined genetic and radiation hybrid maps of the Lyp region on rat chromosome 4, a single 800-kb rat yeast artificial chromosome and a rat P1-derived artificial chromosome (PAC) contig corresponding to approximately 550 kb, both encompassing the entire candidate region. The contig, consisting of 48 PACs, gives 3- to 12-fold coverage. Genetic, radiation hybrid, and physical data were all in agreement and supported the same marker order. Nine genes and ESTs were identified in the contig in addition to a rat EST from the University of Iowa rat EST database-all possible candidate genes for Lyp. Alignment of our rat PAC contig with sequenced human PAC/BAC contigs confirms the position within the region of 3 of the 10 candidates and identifies an additional 8 genes/ESTs as candidates. These data will facilitate identification of Lyp., (Copyright 2000 Academic Press.)

Diazoxide and the diazoxide-analogue, NNC 55-0118, are potassium channel openers that interfere with insulin secretion from beta-cells. In vitro, we show that these two drugs inhibit insulin release from diabetes-resistant BB rat islets cultured at either low or high glucose concentration and cause an intracellular accumulation of insulin with high glucose. Preservation of beta-cells was investigated in newly diabetic BB rats treated with insulin implants from day 0-8 under oral diazoxide, NNC 55-0118 or solvent gavage once a day from day 0-7. Three of eight rats (37.5%) treated with diazoxide and three of ten (30%) treated with NNC 55-0118 retained near normal C-peptide responses when challenged with glucose/arginine on day 9, whereas none of eight (0%) solvent-treated rats showed a C-peptide response. Immunohistochemical staining for insulin and glucagon showed that all the C-peptide responding rats had insulin-positive cells in their islets. In contrast, islets from non-responding rats displayed marked inflammation or end-stage lesions. Furthermore, rats with C-peptide response and treated with NNC 55-0118 exhibited only minimal signs of islet inflammation, whereas C-peptide responding diazoxide-treated rats had low level islet inflammation. These results imply that it is conceivable to preserve residual beta-cells at diabetes onset by induction of target cell rest with potassium channel openers and continuous insulin treatment.

Caspase-2 is a member of the caspase family of cystein proteases involved in programmed cell death or apoptosis. Functional and genetic data suggest it as a candidate gene for lymphopenia (Lyp)-a susceptibility gene for rat diabetes-which is responsible for the T-cell lymphopenia in the diabetes-prone BB rat. Firstly, there is a higher frequency of apoptosis among recent thymic emigrants in the diabetes-prone BB rat than in the non-lymphopenic diabetes-resistant BB rat. Secondly, caspase-2 maps close to Tcrb on mouse Chromosome (Chr) 6, while Lyp is closely linked to Tcrb on the homologous rat Chr 4. In this paper, we report genetic fine-positioning and radiation hybrid mapping of caspase-2 in the rat. Both methods positioned caspase-2 to rat Chr 4 between markers Prss1 and D4Mit5. Since Lyp maps distally to D4Mit5, between markers D4Rat75 and Npy, we exclude caspase-2 as a candidate gene for Lyp.

Diabetes-prone DP-BB rats spontaneously develop insulin-dependent diabetes mellitus resembling type 1 diabetes mellitus in man. Expression of T cell lymphopenia and presence of at least one class II major histocompatibility complex (MHC) RT1u haplotype are required for development of diabetes. Diabetes segregation was studied in lymphopenic backcross (BC) offspring from a cross between male DP-BB/HRI and female BN/Mol rats. Diabetes occurred in 75% of BC rats with genotype RT1u/u and in 18% of those being RT1n/u in genotype. The latter developed diabetes significantly later than MHC homozygotes and parental DP-BBs. Our data further point to the existence of additional genes of minor importance for development of IDDM. One of these seemed to be positioned on the X chromosome. The recently published linkage to chromosome 18 could not be confirmed however. Finally, the BN-derived non-albino allele of the C gene was associated with higher diabetes incidence. This points to the existence of minor susceptibility genes in other strains of rats.