In-vivo quantitative assessment of the therapeutic response in a mouse model of collagen-induced arthritis using 18 F-fluorodeoxyglucose positron emission tomography.

Mouse collagen-induced arthritis (CIA) is the most commonly used animal model to investigate underlying pathogenesis of autoimmune arthritis and to demonstrate the therapeutic efficacy of novel drugs in autoimmune arthritis. The conventional read-outs of CIA are clinical score and histopathology, which have several limitations, including (i) subjected to observer bias; and (ii) longitudinal therapeutic efficacy of a new drug cannot be determined. Thus, a robust, non-invasive, in-vivo drug screening tool is currently an unmet need. Here we have assessed the utility of 18 F-fluorodeoxyglucose positron emission tomography (18 F-FDG) as an in-vivo screening tool for anti-inflammatory drugs using the mouse CIA model. The radiotracer 18 F-FDG and a PET scanner were employed to monitor CIA disease activity before and after murine anti-tumour necrosis factor (TNF)-α antibody (CNTO5048) therapy in the mouse CIA model. Radiotracer concentration was derived from PET images for individual limb joints and on a per-limb basis, and Spearman's correlation coefficient (ρ) was determined with clinical score and histology of the affected limbs. CNTO5048 improved arthritis efficiently, as evidenced by clinical score and histopathology. PET showed an increased uptake of 18 F-FDG with the progression of the disease and a significant decrease in the post-treatment group. 18 F-FDG uptake patterns showed a strong correlation with clinical score (ρ = 0·71, P < 0·05) and histopathology (ρ = 0·76, P < 0·05). This study demonstrates the potential of 18 F-FDG PET as a tool for in-vivo drug screening for inflammatory arthritis and to monitor the therapeutic effects in a longitudinal setting.

Fine-mapping the MHC locus in juvenile idiopathic arthritis (JIA) reveals genetic heterogeneity corresponding to distinct adult inflammatory arthritic diseases.

OBJECTIVES: Juvenile idiopathic arthritis (JIA) is a heterogeneous group of diseases, comprising seven categories. Genetic data could potentially be used to help redefine JIA categories and improve the current classification system. The human leucocyte antigen (HLA) region is strongly associated with JIA. Fine-mapping of the region was performed to look for similarities and differences in HLA associations between the JIA categories and define correspondences with adult inflammatory arthritides. METHODS: Dense genotype data from the HLA region, from the Immunochip array for 5043 JIA cases and 14 390 controls, were used to impute single-nucleotide polymorphisms, HLA classical alleles and amino acids. Bivariate analysis was performed to investigate genetic correlation between the JIA categories. Conditional analysis was used to identify additional effects within the region. Comparison of the findings with those in adult inflammatory arthritic diseases was performed. RESULTS: We identified category-specific associations and have demonstrated for the first time that rheumatoid factor (RF)-negative polyarticular JIA and oligoarticular JIA are genetically similar in their HLA associations. We also observe that each JIA category potentially has an adult counterpart. The RF-positive polyarthritis association at HLA-DRB1 amino acid at position 13 mirrors the association in adult seropositive rheumatoid arthritis (RA). Interestingly, the combined oligoarthritis and RF-negative polyarthritis dataset shares the same association with adult seronegative RA. CONCLUSIONS: The findings suggest the value of using genetic data in helping to classify the categories of this heterogeneous disease. Mapping JIA categories to adult counterparts could enable shared knowledge of disease pathogenesis and aetiology and facilitate transition from paediatric to adult services.

IL-17 and IL-17R: an auspicious therapeutic target for psoriatic disease.

The continuous discovery of new T cell subpopulations in human autoimmune diseases is making the immunopathological network more complex. Th17 cells are one such newly identified subset of T cells, characterized by the production of signature cytokine IL-17. In last few years, several studies have strongly established the regulatory role of Th17 cells and its signature cytokine IL-17 in autoimmune diseases including psoriasis, psoriatic arthritis, rheumatoid arthritis, inflammatory bowel disease, systemic lupus erythematosus and multiple sclerosis. Psoriasis and PsA are immune mediated hyperproliferative diseases, affecting skin and joint respectively. Before the discovery of Th17 cells, psoriasis and psoriatic diseases were thought to be chiefly Th1 mediated diseases; later on IL-17 knockout animal studies as well as human experimental data indicate the crucial role of Th17 cells and its signature cytokine IL-17 in the pathogenesis of these diseases. In vitro human studies have shown the abundance of Th17 cells in the psoriatic plaques. Subsequently our research group has extended this observation in psoriatic arthritis and found the abundance of CD4+IL-17+ T cells in the synovial fluid and majority of these T cells are of memory phenotype (CD4RO+CD45RA-CD11a+). In addition, we showed the significant presence of functional IL-17 receptor in synovial fibroblast of psoriatic arthritis patients. Considering the strong association of IL-17 and psoriatic disease, IL-17 targeted therapy have shown promises in preclinical and clinical trials. In this review article, we have discussed the pathogenic role of IL-17 in psoriatic disease and summarized the therapeutic efficacy and safety profile of different anti IL-17 therapy as an anti-psoriatic agent.

Classical HLA-DRB1 and DPB1 alleles account for HLA associations with primary biliary cirrhosis.

Susceptibility to primary biliary cirrhosis (PBC) is strongly associated with human leukocyte antigen (HLA)-region polymorphisms. To determine if associations can be explained by classical HLA determinants, we studied Italian, 676 cases and 1440 controls, genotyped with dense single-nucleotide polymorphisms (SNPs) for which classical HLA alleles and amino acids were imputed. Although previous genome-wide association studies and our results show stronger SNP associations near DQB1, we demonstrate that the HLA signals can be attributed to classical DRB1 and DPB1 genes. Strong support for the predominant role of DRB1 is provided by our conditional analyses. We also demonstrate an independent association of DPB1. Specific HLA-DRB1 genes (*08, *11 and *14) account for most of the DRB1 association signal. Consistent with previous studies, DRB1*08 (P=1.59 × 10(-11)) was the strongest predisposing allele, whereas DRB1*11 (P=1.42 × 10(-10)) was protective. Additionally, DRB1*14 and the DPB1 association (DPB1*03:01; P=9.18 × 10(-7)) were predisposing risk alleles. No signal was observed in the HLA class 1 or class 3 regions. These findings better define the association of PBC with HLA and specifically support the role of classical HLA-DRB1 and DPB1 genes and alleles in susceptibility to PBC.

Fine mapping the TAGAP risk locus in rheumatoid arthritis.

A common allele at the TAGAP gene locus demonstrates a suggestive, but not conclusive association with risk of rheumatoid arthritis (RA). To fine map the locus, we conducted comprehensive imputation of CEU HapMap single-nucleotide polymorphisms (SNPs) in a genome-wide association study (GWAS) of 5,500 RA cases and 22,621 controls (all of European ancestry). After controlling for population stratification with principal components analysis, the strongest signal of association was to an imputed SNP, rs212389 (P=3.9 × 10(-8), odds ratio=0.87). This SNP remained highly significant upon conditioning on the previous RA risk variant (rs394581, P=2.2 × 10(-5)) or on a SNP previously associated with celiac disease and type I diabetes (rs1738074, P=1.7 × 10(-4)). Our study has refined the TAGAP signal of association to a single haplotype in RA, and in doing so provides conclusive statistical evidence that the TAGAP locus is associated with RA risk. Our study also underscores the utility of comprehensive imputation in large GWAS data sets to fine map disease risk alleles.

The major risk alleles of age-related macular degeneration (AMD) in CFH do not play a major role in rheumatoid arthritis (RA).

Because activation of the alternative pathway (AP) of the complement system is an important aspect of both age-related macular degeneration (AMD) and rheumatoid arthritis (RA), we wished to address the question whether genetic risk factors of the AP inhibitor complement factor H (CFH) for AMD would also be risk factors for RA. For this purpose we genotyped single nucleotide polymorphisms (SNPs) in a Dutch set of RA patients and controls. Similarly, a meta-analysis using a Spanish cohort of RA as well as six large genome-wide association studies (GWAS) studies was performed. For these SNPs we analysed more than 6000 patients and 20,000 controls. The CFH variants, I62V, Y402H, IVS1 and IVS10, known to associate strongly with AMD, did not show a significant association with the risk of developing RA despite a strong statistical power to detect such differences. In conclusion, the major risk alleles of AMD in CFH do not have a similar effect on developing RA.

Cellular basis for neonatally induced T-suppressor activity. Primary B cell maturation is blocked by suppressor-helper interactions restricted by loci on chromosome 12.

The cellular mechanism and genetic restriction of neonatally induced HA-specific suppressor T (Ts) cells have been examined. The in vivo effect of these Ts cells on antibody production, primary B cell proliferation, B cell surface marker changes, and helper T (Th) cell priming during primary responses to HA have been determined. The results indicate that, although antigen-induced B cell proliferative responses and surface marker changes occur in the presence of Ts cells, differentiation to Ig secretion, and long-lived memory B cell production are prevented. Further, antigen-specific Th cell priming is completely ablated by Ts cells, suggesting that Ts act by preventing the delivery of Th signals required for both the later stages of primary B cell maturation, and the formation of memory B cell populations. Finally, in vivo cell mixing experiments using congenic mice indicate that this Ts-Th interaction is restricted by loci on mouse chromosome 12.

Restricted adult clonal profiles induced by neonatal immunization. Influence of suppressor T cells.

The effects of neonatal antigen exposure on the adult B cell repertoire have been examined by characterizing the influenza hemagglutinin (HA)-specific response of adult BALB/c mice given antigen soon after birth. Ligand exposure during early life exerts a profound and lasting effect upon the B cell repertoire, characterized by the expansion and preservation of particular antigen-reactive clones and the apparent loss of others. The precise subset of clonotypes selectively preserved depends upon the age at which antigen is first encountered; and is predictable given a knowledge of the emerging primary pool's dynamics and composition. The preserved (secondary) B cells differ from their unprimed precursors with respect to (a) expression of the surface marker detected by the monoclonal antibody J11d, and (b) susceptibility to T cell-mediated suppression. These studies thus demonstrate a strong relationship between the heritable dynamics of the emerging primary B cell repertoire and the effect of ligand-driven events upon repertoire phenotype. In addition, they provide a mechanistic model for certain forms of antigen-induced oligoclonal dominance, especially the phenomenon of original antigenic sin.

The regulatory role of nerve growth factor and its receptor system in fibroblast-like synovial cells.

OBJECTIVES: Investigating the role of nerve growth factor (NGF) and its receptors (NGF-R) in inflammatory diseases is an active field of research. Inflammatory diseases of the joint are the commonest cause of human morbidity but very little is known about the effect of NGF on synovial tissue biology. Here we have studied NGF/NGF-R and their functional significance on cultured fibroblast-like synovial cells (FLS) collected from the synovial tissue of five healthy subjects. METHODS: NGF/NGF-R expression was determined in the basal condition and after stimulation with tumour necrosis factor (TNF)alpha and interleukin (IL)-1beta by enzyme-linked immunosorbent assay (ELISA) and fluorescence-activated cell sorting (FACS). Proliferation studies were performed by cell count, hexosaminidase assay, and the MTT assay. The synovial fluid (SF) NGF level was studied by ELISA in 12 psoriatic arthritis (PsA), 14 rheumatoid arthritis (RA), and 10 osteoarthritis (OA) patients. RESULTS: FACS studies showed that unstimulated FLS expressed low levels of NGF and the high-affinity NGF-tyrosine kinase receptor TrkA, and TNFalpha and IL-1beta increased NGF and TrkA expression in FLS. NGF (100 ng/mL) increased FLS proliferation by 400% compared to the control (medium only). The NGF level was significantly higher in the PsA group (365.5+/-85.2 pg/mL) than in the RA (120+/-35 pg/mL) and OA groups (30+/-6 pg/mL). CONCLUSIONS: Upregulation of NGF/TrkA in proinflammatory cytokine-activated FLS, the mitogenic effect of NGF on FLS, and the increased NGF level in SF of inflammatory arthritis suggest that there is cross-talk between NGF/NGF-R and FLS. These results also suggest that dysregulated production of NGF may lead to synovial cell proliferation and thus could influence the inflammatory and proliferative cascades of inflammatory arthritis.

Whole-genome expression analysis: challenges beyond clustering.

Measuring the expression of most or all of the genes in a biological system raises major analytic challenges. A wealth of recent reports uses microarray expression data to examine diverse biological phenomena - from basic processes in model organisms to complex aspects of human disease. After an initial flurry of methods for clustering the data on the basis of similarity, the field has recognized some longer-term challenges. Firstly, there are efforts to understand the sources of noise and variation in microarray experiments in order to increase the biological signal. Secondly, there are efforts to combine expression data with other sources of information to improve the range and quality of conclusions that can be drawn. Finally, techniques are now emerging to reconstruct networks of genetic interactions in order to create integrated and systematic models of biological systems.

Fully mobilizing host defense: building better vaccines.

Developments in methods for identifying antigens from infectious agents and cancers has provided exciting new opportunities in prevention and treatment through vaccination. In many of these situations, however, traditional immunization techniques do not stimulate protective immunity because they fail to fully mobilize the appropriate immune responses. This limitation, together with new insights into the underlying mechanism of immune responses, has spurred development of several new approaches for vaccine delivery. We discuss some of the current efforts being developed to provide effective vaccine delivery systems.

Low-dose cyclophosphamide inhibition of transplantable fibrosarcoma growth by augmentation of the host immune response.

Preinjection of a low dose of cyclophosphamide (Cy) (500 microgram/mouse) either delayed or inhibited tumor appearance following the inoculation of transplantable 3-methylcholanthrene-induced fibrosarcomas in inbred male C3H/HeJ mice. This dose of Cy decreased the spleen weight by 13% and the total spleen cell count by 23%. However, the same dose could potentiate the footpad swelling reaction (FPSR) measured against Staphylococcus aureus antigen. Splenic lymphocytes from Cy-treated animals showed increased blastogenic response against phytohemagglutinin-M and bacterial lipopolysaccharide. Thus 500 micrograms Cy/animal may have depleted suppressor cell populations leading to: a) an increase in FPSR, b) increased blastogenic transformation of lymphocytes, and c) tumor growth inhibition.

Mechanism of regression of mammary adenocarcinomas in rats following plasma adsorption over protein A-containing Staphylococcus aureus.

The plasma from 7,12-dimethylbenz(a)anthracene-induced mammary tumor-bearing rats was adsorbed ex vivo with nonviable protein A-containing Staphylococcus aureus Cowan I and then injected into the rats, along with its original blood cells. Tumors in the treated rats showed significant (p less than 0.005) growth inhibition. There were fewer metastatic nodules; cellular cytotoxicity in the presence of plasma was augmented, and there was increased antitumor cytotoxic antibody activity in treated rats. Plasma from sham-treated rats, however, showed blocking activity. It appears that plasma perfusion over S. aureus decreases blocking activity and augments antitumor immunoreactivity of plasma. The exact mechanism by which growth of 7,12-dimethylbenz(a)anthracene-induced mammary tumors was inhibited in these treated Sprague-Dawley rats is not known. However, it is hypothesized that the observed tumor regression is at least partly attributable to the augmentation of antitumor immunoreactivity in the treated animals.

The induction of cytotoxic T cells and tumor regression by soluble antigen formulation.

CTLs specific for tumor antigens play a major role in the immunity against cancer. We have shown that class I-restricted CTLs can be induced by injecting soluble antigens mixed in an antigen formulation (AF) that consists of squalane, Tween 80, and Pluronic L121 (S. Raychaudhuri et al., Proc. Natl. Acad. Sci. USA, 89: 8308-8312, 1992). In this study, using ovalbumin and the ovalbumin-expressing transfectoma (EG7) as a tumor model system, we examined the in vivo antitumor effect of antigen-AF mixture. Vaccination of mice with ovalbumin in AF 2 or 3 days after EG7 tumor challenge showed significant inhibition of tumor growth compared to mice vaccinated with ovalbumin in alum or in saline. Depletion of CD8+ cells at the time of immunization completely abrogated the AF-induced tumor protection, indicating that CD8+ T cells are the major effectors in tumor protection in vivo. Depletion of CD4+ cells led to a marginal loss of tumor protection, which may be the result of inhibition of ovalbumin-specific CTL response due to the lack of T-helper activity. Our results demonstrate that AF can be used in subunit vaccines to stimulate CTLs and tumor regression in vivo.

Monoclonal anti-idiotypic antibodies to human melanoma-associated proteoglycan antigen: generation and characterization of anti-idiotype antibodies.

We have characterized a number of monoclonal anti-idiotype antibodies (mAb2s) made against a monoclonal antitumor antibody, MEM136. The monoclonal antibody 1 (mAb1) MEM136 recognizes an epitope on human melanoma-associated proteoglycan and blocks melanoma cell interaction with basement membrane components in vitro. The anti-idiotype antibodies (Ab2s) made against MEM136 each cross-inhibited, to varying degrees, their binding to MEM136. Thus, the mAb2s recognized overlapping idiotopes on MEM136. In an attempt to identify potential internal image candidates we set up a cell migration inhibition assay. In this assay, migration of melanoma-associated proteoglycan-positive Colo38 cells was determined through a membrane barrier impregnated with Matrigel, which is composed of extracellular matrix components, i.e., collagen type IV, heparan sulfate, and laminin. Interestingly, only Ab2s IM06 and IM32 inhibited melanoma cell migration. Additional studies indicate that of eight mAb2s tested, only IM32 and IM06 induced anti-MPG responses in rabbits. The possibility that IM32 and IM06 bear images of melanoma cell surface-associated proteoglycan epitopes is discussed.

Human high molecular weight-melanoma associated antigen mimicry by an anti-idiotypic antibody: characterization of the immunogenicity and the immune response to the mouse monoclonal antibody IMel-1.

The mouse anti-idiotype (anti-id) monoclonal antibody (mAb) IMel-1 recognizes an idiotope in the antigen combining site of the immunizing anti-human high molecular weight melanoma-associated antigen (HMW-MAA) mAb 225.28. The mAb IMel-1 is able to induce an immune response against self cross-reacting HMW-MAA in rabbits that express HMW-MAA in normal tissues. Most of the rabbit anti-anti-id antibodies recognize a spatially distant determinant(s) from that defined by anti-HMW-MAA mAb 225.28. The immunogenicity of mAb IMel-1 is enhanced by its administration with the muramyl dipeptide-derived adjuvant. Anti-HMW-MAA antibodies were not detected in sera from rabbits immunized with HMW-MAA bearing cultured human melanoma cells. The differential immunogenicity of mAb IMel-1 and cell membrane bound HMW-MAA may account for the ability of anti-id mAb to induce anti-HMW-MAA immunity in patients who have not mounted such a response to HMW-MAA present in their lesions. Rabbit anti-HMW-MAA antibodies induced by anti-id mAb IMel-1 inhibited interactions of melanoma cells with elements of extracellular matrix. This may represent an additional mechanism by which anti-HMW-MAA immunity may affect the biology of melanoma cells in patients with melanoma immunized with anti-id mAb IMel-1.

Ability of nonviable Staphylococcus aureus Cowan I to bind ionized calcium in vitro and ex vivo in an extracorporeal perfusion system.

During immunoadsorption of plasma immunoglobulin G (IgG) and/or its complexes, using nonviable Staphylococcus aureus Cowan I (SA) as an immunoadsorbent, we observed a consistent drop in plasma calcium during every immunoadsorption procedure. The percentage of decrease in plasma calcium was directly dependent on the amount of SA adsorbent present. Although SA can bind both IgG and calcium, the degree of binding of IgG was greater than that of calcium. Calcium could not be detected in the IgG fraction of the plasma SA, however, could bind an appreciable amount of ionized calcium. Extracorporeal perfusion of plasma over SA greatly decreased plasma calcium in a hypercalcemic patient, indicating the feasibility of this method in such patients. Questions concerning the significance of lowering calcium levels as a concomitant of other effects observed in cancer patients undergoing immunoadsorption therapy using SA evolve from the above and become a new focus for attention.

Association of valine and leucine at HLA-DRB1 position 11 with radiographic progression in rheumatoid arthritis, independent of the shared epitope alleles but not independent of anti-citrullinated protein antibodies.

OBJECTIVE: For decades it has been known that the HLA-DRB1 shared epitope (SE) alleles are associated with an increased risk of development and progression of rheumatoid arthritis (RA). Recently, the following variations in the peptide-binding grooves of HLA molecules that predispose to RA development have been identified: Val and Leu at HLA-DRB1 position 11, Asp at HLA-B position 9, and Phe at HLA-DPB1 position 9. This study was undertaken to investigate whether these variants are also associated with radiographic progression in RA, independent of SE and anti-citrullinated protein antibody (ACPA) status. METHODS: A total of 4,911 radiograph sets from 1,878 RA patients included in the Leiden Early Arthritis Clinic (The Netherlands), Umeå (Sweden), Hospital Clinico San Carlos-Rheumatoid Arthritis (Spain), and National Data Bank for Rheumatic Diseases (US) cohorts were studied. HLA was imputed using single-nucleotide polymorphism data from an Immunochip, and the amino acids listed above were tested in relation to radiographic progression per cohort using an additive model. Results from the 4 cohorts were combined in inverse-variance weighted meta-analyses using a fixed-effects model. Analyses were conditioned on SE and ACPA status. RESULTS: Val and Leu at HLA-DRB1 position 11 were associated with more radiographic progression (meta-analysis P = 5.11 × 10(-7)); this effect was independent of SE status (meta-analysis P = 0.022) but not independent of ACPA status. Phe at HLA-DPB1 position 9 was associated with more severe radiographic progression (meta-analysis P = 0.024), though not independent of SE status. Asp at HLA-B position 9 was not associated with radiographic progression. CONCLUSION: Val and Leu at HLA-DRB1 position 11 conferred a risk of a higher rate of radiographic progression independent of SE status but not independent of ACPA status. These findings support the relevance of these amino acids at position 11.

Basic microarray analysis: grouping and feature reduction.

DNA microarray technologies are useful for addressing a broad range of biological problems - including the measurement of mRNA expression levels in target cells. These studies typically produce large data sets that contain measurements on thousands of genes under hundreds of conditions. There is a critical need to summarize this data and to pick out the important details. The most common activities, therefore, are to group together microarray data and to reduce the number of features. Both of these activities can be done using only the raw microarray data (unsupervised methods) or using external information that provides labels for the microarray data (supervised methods). We briefly review supervised and unsupervised methods for grouping and reducing data in the context of a publicly available suite of tools called CLEAVER, and illustrate their application on a representative data set collected to study lymphoma.

Insights into the local residual entropy of proteins provided by NMR relaxation.

A simple model is used to illustrate the relationship between the dynamics measured by NMR relaxation methods and the local residual entropy of proteins. The expected local dynamic behavior of well-packed extended amino acid side chains are described by employing a one-dimensional vibrator that encapsulates both the spatial and temporal character of the motion. This model is then related to entropy and to the generalized order parameter of the popular "model-free" treatment often used in the analysis of NMR relaxation data. Simulations indicate that order parameters observed for the methyl symmetry axes in, for example, human ubiquitin correspond to significant local entropies. These observations have obvious significance for the issue of the physical basis of protein structure, dynamics, and stability.