Parallel Session: Parallel Session 10: Ankylosing Spondylitis and Immunoregulation

Spondyloarthropathies (SpA) including inflammatory arthritic, skin and bowel diseases share genetic susceptibility, interleukin (IL)-23-dependence and involvement of the microbiota. However, it is unclear how the host genetic background influences gut microbiota, and the microbiotaÕs relationship to organ inflammation in SpA. BALB/c ZAP70^W163C-mutant (SKG) mice develop SpA, including progressive peripheral and axial disease, unilateral uveitis, CrohnÕs-like ileitis and psoriasis-like skin inflammation, triggered by microbial beta-glucan (curdlan) injection. Their phenotype thus recapitulates human SpA with ileal inflammatory bowel disease (IBD).We found that microbiota content and response to beta-glucan varied in mice with impaired T cell signal strength due to the SKG ZAP70^W163C mutation. Beta-glucan triggered acute inflammation and spondyloarthritis regardless of host genetic background or microbiota. However, reconstitution of germ free SKG mice with a limited microbiota attenuated spondyloarthritis severity. In contrast, ileal IL-23 expression, ER stress, lymph node IL-17 production, goblet cell loss and ileitis development were microbiota-dependent. IBD but not peripheral arthritis was suppressed by microbiota transfer upon co-housing SKG with BALB/c mice, as well as by TLR4 deficiency. These studies show that the interaction of genetic background and host microbiota leads to an IL-23-dependent loss of mucosal function triggering ileitis in response to beta-glucan. Moreover, genetic susceptibility to SpA can be modified by alteration of microbiota prior to exposure to an infectious trigger.

Spondyloarthritis (SpA), is a chronic inflammatory disease involving spine, peripheral joints and peri-articular soft tissues. Up to 45% of patients may have extra-articular manifestations, such as uveitis, psoriasis, bowel inflammation and nephropathy. The etiology is unknown, but probably related to genetics and immune dysregulation that was triggered by environmental factors, mainly infection and trauma.

About 90% of AS patients are HLA-B27 positive. The positive rate of HLA-B27 is about 5% in normal Caucasian and Chinese population. Thus, the odds ratio for AS in HLA-B27 carriers is greater than 100. HLA-B60 is weakly associated with AS, with an odds ratio of 3.6. Recently, IL23R and ERAP1 have been reported in genome-wide association studies.

Several hypothesis including molecular mimicry theory, arthritogenic peptide theory, ER stress theory, and HLA-B27 free-heavy-chain hypothesis have been proposed to explain the immunopathogenesis of AS and SpA. Klebsiella pneumoniae (KP) was one of the most famous microorganism that involving in the pathogenesis of AS. Biomechanical stress is another possible mechanism of causing inflammation in AS. HLA-B27 molecule was found to be unfolded in the endoplasmic reticulum (ER), causing overloading ER stress and unfolded protein response (UPR). Innate immunity and mucosal inflammation are major players in the pathogenesis of spondyloarthritis. IL-23 and IL-17 in Th17 axis are of interest in recent studies in the pathogenesis of SpA. The mechanism of new bone formation in SpA is possibly due to sequelae of inflammation and fatty degeneration after inflammation, or activation of osteoblast and bone morphogenetic proteins (BMP), though DKK-1, sclerostin or wnt pathway.

Regulatory T cells (Treg) of the FoxP3⁺CD4⁺CD25⁺ phenotype play key roles in controlling reactivity to self-antigens and the onset of autoimmunity. It has recently been demonstrated that Neurophillin-1 (Nrp-1) distinguishes natural and inducible regulatory Treg subsets in vivo (Haribhai et al., 2011). In our laboratory, we have recently been investigating the relationship between autoimmunity, anti-tumor, and Treg responses since the precise mechanisms as to how Tregs infiltrate tumor tissues remain unclear. In the present studies, B16/F10 tumors were inoculated subcutaneously into the left flank of either IL-10^+/+ or IL10^-/- C57BL/6 female mice, and tumor volume and weight were monitored and recorded over a 21 day period. By day ⁺15, there was a significant (p<0.05) decrease in tumor volume in IL10^-/- B16/F10 bearing mice compared to IL10^+/+ B16/F10 bearing mice, and these differences remained highly significant (p<0.001) through d⁺21. These tumor volume decreases correlated with significant (p<0.001) increases in CD4⁺IFNγ⁺ and FoxP3+CD25+ Treg obtained from the spleen and lymph nodes from the IL10^-/- B16/F10 versus IL10^+/+ B16/F10 mice at d⁺9 and d⁺15, respectively. Additionally, by d⁺15, intratumoral CD8⁺ T cells were significantly (p<0.001) up regulated in the IL10^-/- B16/F10 versus IL10^+/+ B16/F10 mice, and these increases correlated with significant (p<0.05) increases of spleen-derived CD4⁺IL17⁺ Th17 cells at both d⁺9 and d⁺15. Finally, we assessed the d⁺9 expression of CD4⁺Nrp-1⁺ Treg cells in the tumor-draining lymph nodes (TDLN) and spleens of the IL10^-/- B16/F10 versus IL10^+/+ B16/F10 mice. There were highly significant (p<0.001) increases in CD4⁺Nrp-1⁺ Treg obtained from the LN of IL10^-/- B16/F10 compared to IL10^+/+ B16/F10 mice. Interestingly, we did not observe these differences in CD4⁺Nrp-1⁺ Treg obtained from the spleens of IL10^-/- B16/F10. In conclusion, our data indicate that tumor growth in IL10^-/- mice induces the specific activation of intratumoral tumor-specific CD8⁺ T cells, and at the same time, it also elevates the expression of Treg in both the TDLN and spleen with Nrp-1 down-regulation in the spleen. Our findings establish that IL-10 deficiency promotes Treg and CD4⁺IFNγ⁺ Th1 and Th17 cell populations which act to suppress tumor growth in vivo.

Since pharmaceutical compounds of new technologies, such as proteins, oligonucleotides or aptamers, etc. frequently cross-react with the corresponding drug targets in non-human primates (NHP), it is apparently a more efficient approach to evaluate their potential efficacy and pharmaceutical profile in a proper monkey disease models. Collagen-induced arthritis (CIA) in monkeys has been reported, however, it is insufficient for characteristically modeling rheumatoid arthritis (RA) due to the hampering of lower incidence and inconsistent disease. To establish a reliable RA model, we characterized the disease progression following immunization of female Cynomolgus with bovine type II collagen. In our studies, the overall incidence of individual arthritis reached 80% and the average incidence of proximal interphalangeal (PIP) joint arthritis reached near 70%, significantly higher than those reported previously. The PIP joints swelled approximately by 25% from the basal level in average in correspondence to the increase of arthritic scores starting from the third week after first immunization. The pathological changes in joint bone and cartilage were well supportive for the RA disease examined by radiopathology and bone histopathology. The biomarker analysis of C-reactive protein (CRP) and ALP levels in peripheral blood showed a close correlation to the arthritic incidence and severity along with the disease progress. The CIA monkeys also presented anemic symptoms, a solid evidence for the similarity to RA with anemia of chronic disease (ACD) in the patients.

Our results demonstrated the establishment of CIA in Cynomolgus monkeys, with the characteristics of high similarity to RA in human.
P1-002 NEUROPROTECTIVE CHANGES IN DEGENERATION-RELATED GENE EXPRESSION IN THE SUBSTANTIA NIGRA FOLLOWING ACUPUNCTURE IN AN MPTP MOUSE MODEL OF PARKINSONISM
H. Jung¹, S. Yeo¹, Y. Hong¹, Y. Choi¹, S. Lim<sup>1


1</sup>Basic Korean Medical Science, Kyung HeeUniversity, Seoul, Korea

ParkinsonÕs disease (PD) is a neurodegenerative disorder characterized by the death of dopaminegenerating cells in the substantia nigra (SN). Acupuncture stimulation at GB34 and LR3 results in an enhanced survival of dopaminergic neurons in the SN in Parkinsonism animal models. The SN is known to be an important player in the neurological control of movement, and previous behavioral tests have found that acupuncture stimulation improves motor dysfunction by approximately 87.7%. The present study investigated changes in gene expression profiles in the SN region using a relatively chronic Parkinsonism model and a whole transcript array to identify genes that may be related to the inhibitory effects of acupuncture on changes in gene expression following 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication. In an MPTP model, acupuncture stimulation at GB34 and LR3 attenuated the decrease in tyrosine hydroxylase in the SN region; stimulation at non-acupoints did not suppress this decrease. GeneChip gene array analysis revealed that 22 (10 annotated genes:Cdh1, Itih2, Mpzl2, Rdh9, Serping1, Slc6a13, Slc6a20a, Slc6a4, Tph2, and Ucma) probes that were up-regulated in MPTP animals relative to controls were exclusively down-regulated by stimulation at acupoints but not non-acupoints. In addition, 17 (two annotated genes: 4921530L21Rik and Gm13931) probes that were down -regulated in MPTP animals compared to controls were exclusively upregulated by stimulation at acupoints but not at non-acupoints. These findings indicate that the 39 probes (12 annotated genes) affected by MPTP and acupuncture may be responsible for the inhibitory

The tests for anti-β2GPI screening and its isotypes (IgG, IgM and IgA) detection were done by enzyme linked immunosorbent assay using commercially available kits (Binding Site, Birmingham, UK).

The data was analyzed using SPSS software version 12 (Chicago, IL, USA).

Results: In 22 APS patients, IgA-anti-β2GPI was the most prevalent antibody compared to IgM and IgG isotypes (91% vs. 45.5% and 18% respectively). Eleven (50%) of these patients had isolated IgA. Systemic lupus erythmatosus was present in 12 patients with mostly IgA isotypes (10; 83%). The IgA-antiβ2GPI titers were moderate to high in 12 patients on initial testing and remained high in 10 patients on repeat testing.

Various manifestations of APS in these patients were recurrent thrombosis, livedo reticularis, digital infarction, pregnancy complications, arterio-venous-fistula-formation (AVF) failures and renal APS. Interestingly IgA isotypes were found in 100% of patients with pregnancy morbidity, AVF failures and renal APS while IgG-anti-β2GPI were absent in these patients.

APL were found positive and at higher levels than healthy controls only in Graves disease, whereas no significant difference was found among Hashimoto thyroiditis patients. IgM anti-CL-B2GPI, IgG anti-B2GPI and IgG anti-CL-B2GPI were found at higher levels among Graves' disease compared with healthy subjects. In addition, IgM anti-CL-B2GPI and IgG anti-B2GPI were more prevalent among Graves patients compared with controls.

Sub-groups of patients having Graves disease and positive for aPL might be at risk for thromboembolic events as should be further studied.

Antiphospholipid syndrome is the most common cause of thrombophilia. It's an autoimmune disease characterized by the association of multiple thrombosis in any vascular level (both arterial and venous). Our aim is to confirm the diagnosis of antiphospholipid syndrome.

We will present the case of a 17 year-old male patient, hospitalized for the presence of multiple ulcers with a chronic venous insufficiency history, with development of collateral circulation at the abdominal and chest wall level (jellyfish head) and giant veins on the legs. The patient was uncooperative, initially claiming that the lesions are due to the dog bites. He had undergone right nephrectomy two years before in the context of nephritic syndrome and the presence of renal vein thrombosis.

Postoperative, he showed signs of thrombosis at the inferior cava vein and iliac veins, with the development of collateral circulation. Suspecting a retroperitoneal process, he underwent extensive laboratory investigations. MRI confirmed the existence of a retroperitoneal lesion process that reduces the size of cava vein in its lower section, extended to the iliac veins axes. Biological samples showed positive anticardiolipin antibodies, positive lupus anticoagulant, deficiency of protein C and antitrombine III. Because of these changes we are entitled take into account antiphospholipid syndrome with severe skin manifestations and because of the patientÕs age we are confronted with hereditary thrombophilia.

The patient was transferred to the vascular surgery service.

The particularity of the case lies in the fact that such pathology is encountered in women between the ages of 30 and 40 years.