Methods: Upon isolation of PBMC from peripheral blood, we proceeded HLA-A2 phenotyping, intracellular staining and flow cytometry. Results

Our results shown that patients under Golimumab demonstrated high sensitivity to stimulation with the FLU-EBV-CMV peptide pool. Higher frequencies and diverse responses were observed in this group. For the first time, we reported higher frequencies of CD8+ T cells specific for HLA-A2 restricted GILGFVFTL peptide in RA patients as compared to the healthy control group.

Guillain-Barre syndrome (GBS) is an autoimmune disorder known as acute inflammatory demyelinating polyradiculoneuropathy. However, the variants known as acute motor axonal neuropathy (AMAN) and acute motor-sensory axonal neuropathy (AMSAN) also are well recognized.

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A 28-year-old male presented with symmetric weakness of bilateral upper and lower extremities. He had an episode of fever and diarrhea approximately 1 week before symptom development. Muscle strength on the Medical Research Council (MRC) scale was grade 1 for the upper and lower limb muscles bilaterally. The sensory examination was normal. Deep tendon reflexes were absent in the bilateral upper and lower limbs. In laboratory investigations, erythrocyte sedimentation rate mildly increased and serum antibody to Campylobacter jejuni was positive. On day 2, motor nerve conduction study (NCS) demonstrated no compound muscle action potentials (CMAPs) in the bilateral median, ulnar, peroneal, and tibial nerves, while sensory nerve conduction studies were completely normal. He underwent intravenous immunoglobulin treatment for 5 days was given. One week after onset, partial recovery was observed in muscle strength. From twenty days after symptom onset, serial follow-up motor NCS revealed progressively increased distal CMAP amplitude and definite partial conduction block in the forearm segment of most of nerves. Needle EMG showed significant improvements in motor unit recruitment. Two months after onset, he was discharged with mild weakness (MRC grade 4).

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Based on the clinical, laboratory, electrophysiological findings, subgroups of GBS should be defined. Viral infection may precede acute motor axonal neuropathy causing rapid progressive ascending weakness with no sensory nerve involvements.

We report a case of an twenty years old Slovenian caucasian male with chronic asymptomatic pancreatic hyperenzymemia, serum increased IgG4 level and with clinicaly and laboratory changes characteristic for autoimmune hepatitis (AIH). Jaudince, characteristic elevation of liver enzymes, anti smooth muscle antibodies and antinuclear antibodies were present. Since pancreatic hyperenzymemia is not frequently seen in patients with clinical manifestation of AH, we investigated hepatic and pancreatic histopathlogy. Abdominal ultrasound, abdominal computed tomography and magnetic resonance cholangiopancreatography showed no abnormalities in the extrahepatic bile ducts and pancreatic duct, but there were diffuse tissue changes present in both organs on all scans. An ultrasound-guided liver biopsy showed changes associated with autoimmune hepatitis: interface hepatitis, lobular hepatitis and marked plasma cell infiltration. Endoscopic ultrasound (EUS)-guided fine needle aspiration (FNA) of pancreatic tissue was performed for the evaluation of autoimmune pancreatitis (AIP); AIP was confirmed based on the presence of lymphoplasmacytic sclerosing pancreatitis and immunoglobulin IgG4 positive plasma cells in the infiltrate. For the definitive diagnosis, we used the Fukuoka 2010 International Consensus of Diagnostic Criteria (ICDC) for autoimmune pancreatitis and existing international criteria for diagnosis of AIH. The patient was put on steroid with satisfactory response on liver enzymes, jaundice and pancreatic hyperenzymemia. For maintenance therapy and preventing relaps of both diseases we succsessfuly convert steroids to azathioprin therapy in maximal dose daily. We concluded that there might exist an overlap syndrome between Type 1 AIH and AP which was in Slovenia detected for the first time.

Background and Aims: FXR is a highly expressed hepatic nuclear receptor that exerts an important role in immune regulation. We postulated that the cellular events that follow FXR activation include modulation of myeloid-derived suppressor cells (MDSCs), a heterogeneous population with a remarkable ability to regulate innate immunity. Methods: To address this issue, we induced hepatitis using both Con A and a-GalCer to monitor the natural history of liver pathology in these two murine models of hepatitis with and without FXR activation, including use of animals depleted of MDSCs and study of mechanisms of action using flow cytometry and adoptive cell transfer. We also monitored the interactions of FXR activation and expression of PIR-B both in vivo and in vitro using luciferase reporter and CHIP assays. Finally, we studied the potential of FXR activation to alter hepatic MDSCs homing. Results: We report herein that FXR activation reduces the inflammatory injury induced either by a-GalCer or Con A; such treatment expands CD11b⁺Ly6C⁺ MDSCs. The protective effect of FXR activation is dependent on expansion of MDSCs, particularly liver CD11b⁺Ly6C^high cells. Indeed, FXR activation enhances the suppressor function of MDSCs through upregulation of PIR-B by binding the PIR-B promoter. FXR activation drives the accumulation of MDSCs to liver through upregulation of S100A8. Conclusions: FXR activation facilitates homing and function of MDSCs, which function as a critical negative feedback loop in immune-mediated liver injury. These novel mechanisms emphasize the importance of defining liver lymphoid subpopulations.

The immune responses of T cells are central in cell-mediated immunity and its activation is tightly regulated using co-stimulatory surface molecules CD28, CTLA-4, PD-1, Tim-3. Programmed cell death-1 (PD-1) and T cell immunoglobulin mucin-3 (Tim-3) have been established as a negative regulatory molecule and plays a critical role in immune tolerance.

PD-1⁺ Tim-3⁺ population is more dysfunctional than the PD-1⁺Tim-3^-, PD-1^-Tim-3^- population and these phenotypes are abundant in patients with cancer and chronic diseases.



Population classified by expression of PD-1 and Tim-3

There is solid evidence on the negative properties of these two molecules, however the mechanism behind the synergistic amplification of the negative signal when these molecules expressed simultaneously, and the interconnections between the signals of these two molecules have not been elucidated yet.

In this research, the expression of PD-1 and Tim-3 show inverse relationship in vitro culture system and in vivo tumor model using wild-type, PD-1 Tg and PD-1 KO mice. In vitro culture, PD-1 expression of the PD-1 Tg, WT, PD-1 KO was highly expressed in order, on the other hand, Tim-3 was expressed lower in that order. In vivo tumor model, activated T cells persist in the tumor environment were more in PD-1 Tg mice, therefore tumor sizes after 30days were smaller in PD-1 Tg compared to WT.

In conclusion, larger PD-1 but lower Tim-3 expression in PD-1 Tg suggests inverse relationship of these two molecules and further study about these phenomenons are needed.

Very little is known about the role pre-natal environment plays in the pathogenesis of autoimmunity. Regulatory T cells (Treg) maintain peripheral tolerance by inhibiting effector T (Teff) cells. Imbalance Treg function plays a unique role in the development of autoimmune diseases. Previously, we exposed dams to a diet supplemented with methyl donors (MS) or not (control) during mating and pregnancy, and found the MS diet suppresses the development of atherosclerosis in F1 mice. Here, we studied the effects of these prenatal diets on Treg function in F1 mice. Dams were fed either MS or control diets prior to mating, and during pregnancy and lactation. At 4 weeks of age, F1 mice from both groups were either analyzed or placed on the standard NIH-31 chow and allowed to age. Prenatal MS diet exposure is associated with lower Treg numbers in F1 mice, and this decrease is sustained into late life. Both FoxP3 mRNA and protein were decreased in mice on the MS diet, compared to the control diet. The difference in Treg numbers is not due to differences in food consumption or weight gain between dams exposed to either prenatal diet. Lower Treg cell numbers are associated with hyper-methylation of the FoxP3 promoter and enhancer regions. Despite their lower numbers, Tregs from F1-MS mice produced more IL-10 and showed higher redox-dependent Teff suppression, compared to Treg from F1-control mice. Together, these results show that a methyl-donor-rich prenatal diet lowers total Treg numbers, though these cells maintain an anti-inflammatory phenotype.

In our previous study new CD19⁺CD21^lowCD38^lowIgM⁺CD27^-CD24^+/- B cell subpopulations have been observed in patients with common variable immunodeficiency and controls (CO). The presence of B cells expressing VH4-34 gene known as coding for anti-dsDNA autoantibodies has been confirmed only in patients with CVID. The aim was to search for such B cells in patients with rheumatoid arthritis (RA, N=2) and systemic lupus erythematosus (SLE, N=2), and to analyse their molecular features using single-cell Ig specific RT-PCR.

All analysed B cells overexpressed IgM transcripts having mutational frequency in both CD24⁺ and CD24^- RA/SLE B cells decreased as compared to COs (1.74% and 1.47% vs. 2.35% and 1.96%). Most interestingly, the VH repertoire was in RA/SLE B cells remarkably restricted to only 10 VH genes. In addition, the significant predominance of VH4-34 gene was found in patients« B cells of both subsets whereas in COs this gene was completely absent (0% vs. 43%; p=0.0092). The most surprising observation was extremely high degree of clonal relation of patients« B cells while in COs this was not found (76% vs. 0%; p<0.0001). Moreover, over 60% of clonally related RA/SLE B cell used VH 4-34 gene segment and their CDR3 regions were remarkably shortened (5-11 amino acids).

Observed data, namely prevalence of VH4-34 gene segment coding for autoantibodies might suggest the susceptibility of these cells to escape negative selection and to serve as a source of autoreactive B cells under autoimmune condition.

This work was supported by the grant NT/11414-5 from IGA MZ CR.

Poster Session: Session 1: Novel therapies and new biologics

P1-059 HUMORAL AND CELLULAR IMMUNE RESPONSES TO DIFFERENT FASCIOLA GIGANTICA ANTIGENS
I. Aly¹, R. Zalat¹, M. Younis<sup>1


1</sup>Immunoparasitology, tbri, cairo, Egypt

In an attempt to develop a suitable vaccine against F. gigantica infection, two antigens were isolated and purified from excretory/secretory (E/S) products, cystein protease and fatty acid binding proteins (CP and FABP) of the parasite by immunoaffinity chromatography. Parasitological and immunological parameters were standardized. Non-infected (gp A) infected (gp B), immunized with CP (gp C), immunized with CP and infected (gp D), immunized with FABP (group E) and immunized with FABP and infected (gp F). The mean worm burdens and bile egg count after challenge were reduced significantly by 37.7% and 55.5%, respectively in rabbits vaccinated with CP. In contrast, low significant reduction in worm burdens and bile egg count were observed in rabbits immunized with FABP after challenge (23.5% and 35.7%, respectively). On the other hand, immunization of rabbits with CP or FABP induced a significant expression of humoral antibodies and cytokines with higher level in case of CP than FABP. Among Ig isotypes, IgG1 and IgG4 were most dominant in rabbits immunized with CP or FABP while, recording a low IgG2 level. On the other hand, among all cytokines IL-10 was most dominant in rabbits immunized with CP or FABP PI suggesting also Th2 response, which could be another mechanism of immune response in rabbits infected with F. gigantica. In conclusion, F. gigantica CP is a relevant candidate for vaccination against fascioliasis, while the level of protection used by FABP may not appear sufficient enough to protect these ruminants against the deleterious effects of Fasciola infection.

The exact causes of psoriasis, a relatively common, chronic, inflammatory and hyperproliferative skin disease, are not well understood, thus making it very difficult for therapy. The objective of this study was to investigate the molecular effect of each of these following Thai herbal extracts, Alpinia galanga L. (rhizome), Curcuma longa L. (rhizome) and Annona squamosa L. (leaf), containing anti-psoriatic activity on the mRNA expression of Egr-1, IL-6 and IL-8, the biomarkers believed to be upregulated in psoriatic skin, thus considering as one of essential determinants of psoriasis. Using HaCaT keratinocyte cell line as a model, the mRNA expression was evaluated by quantitative real time PCR. We found that none of the ethanolic extracts in this study could modulate the expression of all analyzed genes. Nevertheless, the extract of Annona squamosa L. at 0.25IC₅₀ could significantly suppress the expression of IL-8 mRNA (P<0.05). The extract of Alpinia galanga L. at IC₅₀ could significantly reduce the expression of IL-6 mRNA (P<0.05). Therefore, the result suggests that the ethanolic extracts of Annona squamosa L. (leaf) and Alpinia galanga L. (rhizome), contain certain constituents capable of downregulating these proinflammatory cytokines. This information is useful for further studies, which may provide insight into understanding therapeutic options in the future.

Psoriasis is a chronic, non-contagious autoimmune disease that affects the skin and joints. Currently, scientists have attempted to find a therapeutic approach with highest efficacy. In particularly, attention has been focused on the applications of medicinal herbs for treatment and prevention of psoriasis. In this study, we aimed at evaluating anti-inflammatory and anti-psoriatic activities of Houttuynai cordata Thunb. leaves extracted with 2 different methods. The fist technique was exhaustive extraction using petroleum ether, dichloromethane and ethanol. The second one was maceration with ethanol. Based on MTT assay, we found that Houttuynai cordata Thunb. leaf dichloromethane extract (100, 50, 25, 12.5 and 6.25 µg/mL) and petroleum ether extract (100, 50, 25 and 12.5 µg/mL) exhibited cytotoxic effect on cultured HaCaT cells. According to our ELISA assay, Houttuynai cordata Thunb. leaf dichloromethane extract (100 and 6.25 µg/mL) led to apoptosis of HaCaT. With regard to anti-inflammatory activity using ELISA for pro-inflammatory cytokines, interleukin-6 and interleukin-8, Houttuynai cordata Thunb. leaf petroleum ether extract (12.5 and 6.25 µg/mL) could suppress the production of interleukin-6, while other Houttuynai cordata Thunb. leaf extracts did not affect the expression of pro-inflammatory cytokines in HaCaT cells. Different extraction methods certainly resulted in extracts with distinct biological activities. It should be worth mentioning that other techniques should be employed for further studies. Nevertheless, the result of this current investigation is important with respective to new findings that may be useful for psoriatic patients in the future.