Rapid spread of mannan to the immune system, skin and joints within 6 hours after local exposure.

Psoriasis (Ps), psoriatic arthritis (PsA) and rheumatoid arthritis (RA) are common diseases dependent on environmental factors that activate the immune system in unknown ways. Mannan is a group of polysaccharides common in the environment; they are potentially pathogenic, because at least some of them induce Ps-, PsA- and RA-like inflammation in mice. Here, we used positron emission tomography/computed tomography to examine in-vivo transport and spread of mannan labelled with fluorine-18 [18 F]. The results showed that mannan was transported to joints (knee) and bone marrow (tibia) of mice within 6 h after intraperitoneal injection. The time it took to transport mannan, and its presence in blood, indicated cellular transport of mannan within the circulatory system. In addition, mannan was filtered mainly through the spleen and liver. [18 F]fluoromannan was excreted via kidneys, small intestine and, to some extent, the mouth. In conclusion, mannan reaches joints rapidly after injection, which may explain why mannan-induced inflammatory disease is targeted to these tissues.

VAV1 regulates experimental autoimmune arthritis and is associated with anti-CCP negative rheumatoid arthritis.

Rheumatoid arthritis (RA) patients can be stratified into two subgroups defined by the presence or absence of antibodies against citrullinated circular peptides (anti-CCP) with most of the genetic association found in anti-CCP positive RA. Here we addressed the role of VAV1, previously associated to multiple sclerosis (MS), in the pathogenesis of RA in experimental models and in a genetic association study. Experimental arthritis triggered by pristane or collagen type II was induced in DA rats and in the DA.BN-R25 congenic line that carries a polymorphism in Vav1. Difference in arthritis severity was observed only after immunization with pristane. In a case-control study, 34 SNPs from VAV1 locus were analyzed by Immunochip genotyping in 11475 RA patients (7573 anti-CCP positive and 3902 negative) and 15,870 controls in six cohorts of European Caucasians. A combination of the previous MS-associated haplotype and two additional SNPs was associated with anti-CCP negative RA (alleles G-G-A-A of rs682626-rs2546133-rs2617822-rs12979659, OR=1.13, P=1.27 × 10-5). The same markers also contributed to activity of RA at baseline with the strongest association in the anti-CCP negative group for the rs682626-rs12979659 G-A haplotype (ß=-0.283, P=0.0048). Our study suggests a role for VAV1 and T-cell signaling in the pathology of anti-CCP-negative RA.

The SKG Mutation in ZAP-70 also Confers Arthritis Susceptibility in C57 Black Mouse Strains.

Various rodent models of arthritis are essential to dissect the full complexity of human rheumatoid arthritis (RA), a common autoimmune disease affecting joints. The SKG model of arthritis originates from a spontaneous mutation in ZAP-70 found in a BALB/c colony. This mutation affects T cell selection due to reduced TCR signalling, which allows leakage of self-reactive T cells from the thymus. To further expand the practical applicability of this unique model in arthritis research, we investigated the arthritogenicity of the SKG mutation in two common black mouse strains C57BL/6.Q and C57BL/10.Q and compared to BALB/c.Q. Mice retained the reduced TCR signalling characteristic of SKG.BALB/c mice, which leads to similar alteration in thymic selection. Importantly, mice also retained susceptibility to chronic arthritis after a single injection of mannan from Saccharomyces cerevisiae, with comparable prevalence and severity regardless of the genetic background. Further characterization of CD4(+) T cells revealed a similar bias towards IL-17 production and activated T cell phenotype in all SKG strains compared to respective wild type controls. Finally, transfer of SKG thymocytes conferred susceptibility to recipients, which confirm the intrinsic defect and pathogenicity of T cells. Overall, these results underline the strong impact that the W163C ZAP-70 mutation has on T cell-driven arthritis, and they support the use of the SKG model in black mice, which is useful for further investigations of this distinctive arthritis model to better understand autoimmunity.

NADPH oxidase elevations in pyramidal neurons drive psychosocial stress-induced neuropathology.

Oxidative stress is thought to be involved in the development of behavioral and histopathological alterations in animal models of psychosis. Here we investigate the causal contribution of reactive oxygen species generation by the phagocyte NADPH oxidase NOX2 to neuropathological alterations in a rat model of chronic psychosocial stress. In rats exposed to social isolation, the earliest neuropathological alterations were signs of oxidative stress and appearance of NOX2. Alterations in behavior, increase in glutamate levels and loss of parvalbumin were detectable after 4 weeks of social isolation. The expression of the NOX2 subunit p47(phox) was markedly increased in pyramidal neurons of isolated rats, but below detection threshold in GABAergic neurons, astrocytes and microglia. Rats with a loss of function mutation in the NOX2 subunit p47(phox) were protected from behavioral and neuropathological alterations induced by social isolation. To test reversibility, we applied the antioxidant/NOX inhibitor apocynin after initiation of social isolation for a time period of 3 weeks. Apocynin reversed behavioral alterations fully when applied after 4 weeks of social isolation, but only partially after 7 weeks. Our results demonstrate that social isolation induces rapid elevations of the NOX2 complex in the brain. Expression of the enzyme complex was strongest in pyramidal neurons and a loss of function mutation prevented neuropathology induced by social isolation. Finally, at least at early stages, pharmacological targeting of NOX2 activity might reverse behavioral alterations.

Effects of oestradiol and raloxifene on the induction and effector phases of experimental postmenopausal arthritis and secondary osteoporosis.

Oestradiol and the selective oestrogen receptor modulator (SERM) raloxifene have been shown to ameliorate collagen-induced arthritis (CIA) in rats and in mice. One aim was to investigate if raloxifene exerts its anti-arthritic and anti-osteoporotic effects during the induction or effector phase of arthritis. A second aim was to analyse if raloxifene activates the oestrogen response element (ERE) to produce its immune-modulator effects. CIA or collagen-antibody-induced arthritis (CAIA) was induced in ovariectomized DBA/1-mice. CIA was used for evaluation of treatment during the induction, and CAIA for the effector phase of arthritis and osteoporosis development. Raloxifene, oestradiol or vehicle was administered 5 days/week. The clinical disease was evaluated continuously. Bone marrow density (BMD) was analysed with peripheral quantitative computer tomography, paws were collected for histological examination, and sera were analysed for markers of bone and cartilage turnover and proinflammatory cytokines. Transgenic luciferase (Luc)-ERE mice were immunized with collagen (CII), and after 10 days injected once with raloxifene, oestradiol or vehicle before termination. Spleens were analysed for luciferase activity to measure ERE activation. Treatment with oestradiol or raloxifene during the induction phase of CIA failed to affect arthritis. Raloxifene did not hamper disease activity in CAIA, whereas oestradiol delayed the onset and ameliorated the severity. Both raloxifene and oestradiol preserved BMD in CAIA. CII-immunization increased the oestradiol-induced ERE activation in spleen, and raloxifene activated the ERE at about 25% the intensity of oestradiol. Further experiments are needed to elucidate the exact mechanisms behind this finding.

Finemapping of the arthritis QTL Pia7 reveals co-localization with Oia2 and the APLEC locus.

In this study, we sought to determine the effect of the quantitative trait locus Pia7 on arthritis severity. The regulatory locus derived from the arthritis-resistant E3 rat strain was introgressed into the arthritis-susceptibility DA strain through continuous backcrossing. Congenic rats were studied for their susceptibility to experimental arthritis using pristane and adjuvant oil. In addition, cell number and function of various leukocyte populations were analyzed either under naive or stimulated conditions. We found that the minimal congenic fragment of DA.E3-Pia7 rats overlapped with the minimal fragment in DA.PVG-Oia2 congenic rats, which has been positionally cloned to the antigen-presenting lectin-like receptor complex (APLEC) genes. DA.E3-Pia7 congenic rats were protected from both PIA and OIA, but the protection was more pronounced in OIA. In adoptive transfer experiments we observed that the Pia7 locus controlled the priming of arthritogenic T cells and not the effector phase. In addition, Pia7 congenic rats had a significant higher frequency of B cells and granulocytes as well as TNFalpha production after stimulation, indicating a higher activation state of cells of the innate immune system. In conclusion, this study shows that the APLEC locus is a major locus regulating the severity of experimentally induced arthritis in rats.

C4b-binding protein (C4BP) inhibits development of experimental arthritis in mice.

OBJECTIVES: To assess the human complement inhibitor C4b-binding protein (C4BP) for treatment of arthritis. METHODS: We have used two mouse models of rheumatoid arthritis (RA) to assess the therapeutic effect of C4BP on different phases of arthritis, the collagen antibody-induced arthritis (CAIA), an acute antibody-induced disease and the collagen-induced arthritis (CIA), which carries the full complexity of arthritis. RESULTS: Purified human C4BP injected intraperitoneally alleviated CAIA significantly in a manner similar to cobra venom factor that depletes complement due to massive activation. Furthermore, C4BP was injected before and after the disease development into CIA mice. In the former case, the disease onset was delayed and in the latter, the severity of the disease was reduced in animals treated with C4BP. However, C4BP did not affect the anti-CII antibody synthesis. C4BP present in mouse sera decreased activity of the classical but not the alternative pathway of the complement system when these were assessed in a fluid phase. However, C4BP was efficiently inhibiting the alternative pathway when present on the activating surface. Taken together, the disease ameliorating effect of C4BP appears to be related to inhibition of both pathways of complement. CONCLUSIONS: Although human C4BP was cleared relatively fast from the circulation and was only moderately affecting complement activity, its effect on the disease severity was substantial, suggesting that minor alterations in complement activity can have significant therapeutic value in RA.

The novel small molecule drug Rabeximod is effective in reducing disease severity of mouse models of autoimmune disorders.

OBJECTIVES: Autoimmune diseases such as rheumatoid arthritis (RA) and multiple sclerosis (MS) affect a relatively large portion of the population, leading to severe disability if left untreated. Even though pharmaceutics targeting the immune system have revolutionised the therapy of these diseases, there is still a need for novel, more effective therapeutic substances. One such substance is the new chemical entity 9-chloro-2,3 dimethyl-6-(N,N-dimthylamino-2-oxoethyl)-6H-indolo [2,3-b] quionoxaline, Rabeximod, currently being investigated for efficiency in treatment of human RA. In this study we aimed to evaluate Rabeximod as a treatment for autoimmune diseases, using animal models. METHODS: In the present investigation we have evaluated Rabeximod as a treatment for autoimmune diseases using mouse models of RA and MS, ie, collagen-induced arthritis, collagen antibody induced arthritis and experimental autoimmune encephalomyelitis. RESULTS: Rabeximod efficiently prevented arthritis and encephalomyelitis in mice. In addition, this effect correlated to the timepoint when cells migrate into the joints. CONCLUSIONS: We conclude that Rabeximod reduces disease severity in animal models of autoimmunity and should be considered as a new therapeutic substance for MS and RA.

Multiple antibody reactivities to citrullinated antigens in sera from patients with rheumatoid arthritis: association with HLA-DRB1 alleles.

BACKGROUND: Autoantibodies to cyclic citrullinated peptides (anti-CCP) are present in most patients with rheumatoid arthritis (RA), and associate with HLA-DRB1 shared epitope (SE) alleles. OBJECTIVE: To investigate reactivities of anti-CCP to various citrullinated proteins/peptides, which represent potential autoantigens in RA, and to examine the relationship between such antibodies, and their association with genetic variants within HLA-DRB1 SE alleles. METHODS: Serum samples from 291 patients with established RA and 100 sex- and age-matched healthy subjects were included in this study. Sera were first analysed for presence of anti-CCP antibodies and further for IgG and IgA antibodies towards candidate autoantigens in both their native and citrullinated form including: fibrinogen, alpha-enolase peptide-1 and the C1-epitope of type II collagen (C1(III)). Antibody specificity was confirmed by cross-reactivity tests. HLA-DR genotyping was performed. RESULTS: 72% of patients with RA were anti-CCP positive. Among the candidate autoantigens examined, IgG antibodies to citrullinated fibrinogen were found in 66% of patients' sera and in 41% for both citrullinated alpha-enolase peptide-1 and citrullinated C1(III). These antibodies were mainly seen in the anti-CCP-positive patient group; they were specific for their respective antigen and displayed limited cross reactivity. IgA responses were also detected, but less frequently than IgG. Anti-CCP and anti-citrullinated protein antibodies were associated with HLA-DRB1*04 rather than with HLA-DRB1*01 alleles. CONCLUSIONS: Antibodies directed against several citrullinated antigens are present in CCP-positive RA, with many patients displaying multireactivity. All specific reactivities were primarily associated with the HLA-DRB1*04 alleles, suggesting common pathways of anti-citrulline immunity.

Identification of new loci controlling collagen-induced arthritis in mouse using a partial advanced intercross and congenic strains.

Collagen-induced arthritis (CIA) is a well studied mouse model of the human disease rheumatoid arthritis (RA). Both CIA and RA are complex diseases affected by multiple genes as well as environmental factors. Identifying the genes that determine susceptibility to arthritis would give invaluable clues to the largely unknown aetiology of RA. In this study, we dissected a known locus, Cia6, as well as a genomic region on chromosome 14 with no previously known arthritis loci, using a partial advanced intercross and a collection of congenic strains. The chromosome 14 congenic fragment, containing the T-cell receptor alpha (Tcra) locus, was included based on the hypothesis that the Cia6 locus is caused by a polymorphism in the Tcr beta (Tcrb) locus and that the two loci interact. Splitting up the congenic fragments revealed multiple loci affecting arthritis traits as well as production of collagen-specific autoantibodies. In total seven new loci were identified of which four were in the previously unlinked chromosome 14 region. Both Tcr loci were within CIA loci making them candidate susceptibility genes. The results demonstrate the importance of breaking up genetic regions in smaller fragments to identify the underlying complex set of loci.

17Beta-estradiol expands IgA-producing B cells in mice deficient for the mu chain.

Oestrogen is not only a sex hormone but also an important regulator of the immune system. Expression of the heavy chain of IgM (mu) is essential for B-cell differentiation. However, a small number of IgA-positive B cells can be found in mice lacking the mu chain (muMT-/-). The aim of this study was to investigate the effects of oestrogen on this alternative B-cell pathway in muMT-/- mice. Our results clearly demonstrate that oestrogen increases the frequency of IgA-producing B cells in muMT-/- mice in both bone marrow and spleen cells. We also show that mature IgM-producing B cells are not required for oestrogen-mediated suppression of granulocyte-mediated inflammation or thymic involution. In conclusion, we demonstrate that 17beta-estradiol benzoate increases the frequency of IgA-producing B cells in muMT-/- mice, suggesting that oestrogen can influence the alternative B-cell pathway found in muMT-/- mice.

Cell-specific and efficient expression in mouse and human B cells by a novel hybrid immunoglobulin promoter in a lentiviral vector.

The expression of genes specifically in B cells is of great interest in both experimental immunology as well as in future clinical gene therapy. We have constructed a novel enhanced B cell-specific promoter (Igk-E) consisting of an immunoglobulin kappa (Igk) minimal promoter combined with an intronic enhancer sequence and a 3' enhancer sequence from Ig genes. The Igk-E promoter was cloned into a lentiviral vector and used to control expression of enhanced green fluorescent protein (eGFP). Transduction of murine B-cell lymphoma cell lines and activated primary splenic B cells, with IgK-E-eGFP lentivirus, resulted in expression of eGFP, as analysed by flow cytometry, whereas expression in non-B cells was absent. The specificity of the promoter was further examined by transducing Lin(-) bone marrow with Igk-E-eGFP lentivirus and reconstituting lethally irradiated mice. After 16 weeks flow cytometry of lymphoid tissues revealed eGFP expression by CD19+ cells, but not by CD3+, CD11b+, CD11c+ or Gr-1+ cells. CD19+ cells were comprised of both marginal zone B cells and recirculating follicular B cells. Activated human peripheral mononuclear cells were also transduced with Igk-E-eGFP lentivirus under conditions of selective B-cell activation. The Igk-E promoter was able to drive expression of eGFP only in CD19+ cells, while eGFP was expressed by both spleen focus-forming virus and cytomegalovirus constitutive promoters in CD19+ and CD3+ lymphocytes. These data demonstrate that in these conditions the Igk-E promoter is cell specific and controls efficient expression of a reporter protein in mouse and human B cells in the context of a lentiviral vector.

Genetics of autoimmune diseases: a multistep process.

It has so far been difficult to identify genes behind polygenic autoimmune diseases such as rheumatoid arthritis (RA), multiple sclerosis (MS), and type I diabetes (T1D). With proper animal models, some of the complexity behind these diseases can be reduced. The use of linkage analysis and positional cloning of genes in animal models for RA resulted in the identification of one of the genes regulating severity of arthritis in rats and mice, the Ncf1 gene. The Ncf1 gene encodes for the Ncf1 protein that is involved in production of free oxygen radicals through the NADPH oxidase complex, which opens up a new pathway for therapeutic treatment of inflammatory diseases. In most cases, however, a quantitative trait locus (QTL) is the sum effect of several genes within and outside the QTL, which make positional cloning difficult. Here we will discuss the possibilities and difficulties of gene identification in animal models of autoimmune disorders.

Nature's choice of genes controlling chronic inflammation.

Inflammation is a physiological response that may go uncontrolled and thereby develop in a chronic way. This seems to happen in many common diseases of autoimmune, degenerative, or allergic character. Rheumatoid arthritis (RA) is by definition a chronic disease with an autoimmune inflammatory attack on diarthrodial cartilaginous joints. The development of new treatment neutralizing cytokines involved in the inflammatory attack has given relief and gives the promise of more effective treatment of already established disease. It is now time to set our eyes on a new vision to develop preventive and curative treatment based on knowledge of the unique and causative pathogenic mechanisms. To do this we believe it is important to identify the natural-selected polymorphisms that are associated with disease. These have proven to be extremely difficult to identify in complex diseases such as RA, but using animal models, this work is closer to reality. Animal models have recently been developed mimicking various aspects of the human disease. We will present an example in which a genetic polymorphism associated with the development of arthritis has been identified. On the basis of this finding, a new pathway involving control of immune tolerance by reactive oxidative species has been identified and a new class of antiinflammatory agents activating the induced oxidative burst protein complex is suggested.

Gene expression profiling of arthritis using a QTL chip reveals a complex gene regulation of the Cia5 region in mice.

One of the major quantitative trait loci (QTLs) associated with arthritis in crosses between B10.RIII and RIIIS/J mice is the Cia5 on chromosome 3. Early in the congenic mapping process it was clear that the locus was complex, consisting of several subloci with small effects. Therefore, we developed two novel strategies to dissect a QTL: the partial advanced inter-cross (PAI) strategy, with which we recently found the Cia5 region to consist of three loci, Cia5, Cia21 and Cia22, and now we introduce the QTL-chip strategy, where we have combined congenic mapping with a QTL-restricted expression profiling using a novel microarray design. The expression of QTL genes was compared between parental and congenic mice in lymph node, spleen and paw samples in five biological replicates and in dye-swapped experiments at three time points during the induction phase of arthritis. The QTL chip approach revealed 4 genes located in Cia21, differently expressed in lymph nodes, and 14 genes in Cia22, located within two clusters. One cluster contains six genes, differently expressed in spleen, and the second cluster contains eight genes, differently expressed in paws. We conclude the QTL-chip strategy to be valuable in the selection of candidate genes to be prioritized for further investigation.

Influence on spontaneous tissue inflammation by the major histocompatibility complex region in the nonobese diabetic mouse.

We investigated the role of the major histocompatibility complex (MHC) region in the specificity of autoimmunity by analysing specifically the development of sialadenitis, but also insulitis, nephritis and autoantibody production in autoimmune-prone nonobese diabetic (NOD) mice where the MHC H2g7 haplotype had been exchanged for the H2q (NOD.Q) or H2p (NOD.P) haplotype. The exchange of H2 haplotype did not affect the frequency of sialadenitis because the H2q and H2p congenic NOD strains developed sialadenitis with the same incidence as NOD. However, the severity of sialadenitis varied among the strains, as NOD.Q >NOD >NOD.P. At 11-13 weeks of age, the NOD.Q (H2q) female mice developed more severe sialadenitis compared to NOD.P (H2p) (P=0.038). At 20 weeks, the NOD (H2g7) female mice showed more severe sialadenitis than NOD.P (P=0.049). This is in contrast to the development of insulitis in the present strains, because the incidence of insulitis was almost completely inhibited by the replacement of the H2g7 haplotype of NOD. The incidence of insulitis in NOD.Q was 11-22%, compared to 75% in NOD, which correlated well with lower titres of anti-glutamic acid decarboxylase (anti-GAD) antibodies in NOD.Q compared to NOD (P=0.009). However, the introduction of the H2q haplotype into the NOD strain instead directed the autoimmune response towards the production of lupus types of autoantibodies, because the incidence of antinuclear antibodies (ANA) in NOD.Q was 89% compared with 11% in NOD.P and 12% in NOD mice, which in turn correlated with a high incidence of nephritis in NOD.Q compared to NOD. Consequently, we show that different haplotypes of MHC are instrumental in directing the specificity of the spontaneous autoimmune inflammation.

Identification of epistasis through a partial advanced intercross reveals three arthritis loci within the Cia5 QTL in mice.

Identification of genes controlling complex diseases has proven to be difficult; however, animal models may pave the way to determine how low penetrant genes interact to promote disease development. We have dissected the Cia5/Eae3 susceptibility locus on mouse chromosome 3 previously identified to control disease in experimental models of multiple sclerosis and rheumatoid arthritis. Congenic strains showed significant but small effects on severity of both diseases. To improve the penetrance, we have now used a new strategy that defines the genetic interactions. The QTL interacted with another locus on chromosome 15 and a partial advanced intercross breeding of the two congenic strains for eight generations accumulated enough statistical power to identify interactions with several loci on chromosome 15. Thereby, three separate loci within the original QTL could be identified; Cia5 affected the onset of arthritis by an additive interaction with Cia31 on chromosome 15, whereas the Cia21 and Cia22 affected severity during the chronic phase of the disease through an epistatic interaction with Cia32 on chromosome 15. The definition of genetic interactions was a prerequisite to dissect the Cia5 QTL and we suggest the partial advanced intercross strategy to be helpful also for dissecting other QTL controlling complex phenotypes.

Methotrexate ameliorates T cell dependent autoimmune arthritis and encephalomyelitis but not antibody induced or fibroblast induced arthritis.

OBJECTIVE: To investigate the mode of action of methotrexate (MTX) in different types of models for rheumatoid arthritis (RA) and multiple sclerosis (MS). METHODS: Models for RA and MS were selected known to have different pathogenesis--that is, fibroblast induced arthritis in SCID mice, collagen induced arthritis (CIA), anticollagen II antibody induced arthritis (CAIA), and experimental autoimmune encephalomyelitis (EAE) in (Balb/c x B10.Q)F1 and B10.Q mice, and Pristane induced arthritis in DA rats (PIA). The MTX treatment was started 1 day after the onset of disease and continued for 14 days to compare effects on the different models. RESULTS: All models known to be critically dependent on T cell activation (CIA, PIA, and EAE) were effectively down regulated by titrated doses of MTX. In contrast, no effects were seen on fibroblast induced arthritis or CAIA. No effects were seen on the levels of anticollagen II antibodies in the CIA experiment. CONCLUSION: The data show that MTX has strong ameliorative effect on both classical models of RA, like CIA and PIA, but also on a model for MS, EAE. It also suggests that MTX operates only in diseases which are preceded by, and dependent on, T cell activation. A comparison of CAIA and CIA suggested that MTX operates independently of arthritogenic antibodies. These results demonstrate that different animal models reflect the complexity of the corresponding human diseases and suggest that several models should be used for effective screening of new therapeutic agents.

Oligoclonal IgG bands synthesized in the central nervous system are present in rats with experimental autoimmune encephalomyelitis.

OBJECTIVE: Oligoclonal bands (OBs) in electrophoresis of cerebrospinal fluid (CSF) are present in multiple sclerosis and here is investigated whether these also occur in experimental autoimmune encephalomyelitis (EAE). MATERIAL AND METHODS: Experimental autoimmune encephalomyelitis was induced in 42 DA rats after immunization with rat spinal chord homogenate and the occurrence of OBs were detected by electrophoresis of both sera and CSF. The relationship between disease symptoms, antibody response against myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG) and appearance of OBs was studied. RESULTS: Development of CSF-specific OB was found to occur, 6 weeks after immunization, in seven of 42 rats. OB was detected in rats with an antibody response against MBP, whereas as a role no such bands were present in rats with an antibody response against MOG. Initially severe disease symptoms were correlated to a concomitant intense oligoclonal antibody response. CONCLUSION: Cerebrospinal fluid-specific OB occurs in EAE. It is present in rats with an anti-MBP, but not in rats with an anti-MOG antibody response. A severe disease results in an intense oligoclonal antibody response, which might have an anti-inflammatory effect.