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Materials and methods: Patient samples were collected with informedconsent, and disease activity quantified by the SELENA-SLEDAI index.
Multispectral imaging flow cytometry was performed using an Amnis Apoptotic parasites silence macrophages by misusing the ImageStreamX instrument. LC3-positive autophagosomes were enu- merated in viable, non-apoptotic cells using the Bright Detail Intensityalgorithm implemented in IDEAS 6. Autophagic flux was determined P. Crauwels,* S. Gottwalt,* F. Ja¨ckel,* M. Thomas,* E. Bank,* by incubation with chloroquine. As an alternative measure of P. Walther,  M. Bastianà & G. van Zandbergen* autophagy, uptake of the novel autophagosomotropic dye CytoID *Immunology, Paul-Ehlich-Institut, Langen, Germany,  Electronmicros- (Enzo) was analysed using conventional flow cytometry. Autophagy copy Facility, University Ulm, Ulm, Germany, Veterinary Medicine, was assayed in negatively selected B cells stimulated with combinations of anti-IgM and anti-CD40 antibodies, and interferon-a.
Results: Autophagy was significantly increased in the CD19+ B cells of Purpose/Objective: An appropriate T cell response to Leishmania patients with SLE compared with healthy controls (Lm) infection is critical for an effective immune response. Human (P = <0.001, n = 22 patients, 15 controls), and there was a positive macrophages (MF) can present antigen to T lymphocytes and at the correlation with SLEDAI score (r = 0.67, P = <0.002). There was same time serve as host cells. Upon macrophage infection the virulent however, no association in CD4+ T cells (P = 0.49). There was no inoculum of Lm promastigotes consists of apoptotic and viable statistical evidence of confounding due to patient age or medication promastigotes. The viable promastigotes enter a maturing phagolyso- use. Assessment of autophagic flux using the autophagosome-lysosome some were they can survive and grow as amastigotes; the fate of fusion inhibitor chloroquine revealed an accumulation of autophag- Materials and methods: In this study, we hypothesize that the Analysis of ex vivo viable, annexin V negative human B cells apoptotic promastigotes use the MFs«autophagy machinery to down demonstrated a significant increase in autophagy in unstimulated regulate MF antigen presentation and T cell activation.
compared with anti-IgM stimulated cells, with further decreases Results: Upon promastigote uptake by human primary MFs, we observed with the addition of anti-CD40 and interferon-a.
found apoptotic promastigotes to enter a compartment positive for the Conclusions: The process of autophagy has not been previously autophagy marker LC3. This LC3 compartment matured over time and examined in ex vivo human B cells from patients with systemic became LAMP positive. 24 h later the compartment resolved after autoimmune disease. We demonstrate that autophagy is enhanced in highly efficient parasite degradation. When co-incubated with autol- this context. Given our in vitro data, we may advance the hypothesis ogous T lymphocytes, MFs infected with viable promastigotes induced that autophagy is acting as a survival mechanism for auto reactive B a strong CD4-positive T cell proliferation. Compared to viable cells lacking adequate survival signals. An alternative explanation parasites a significantly lower T cell reactivity was observed in response requiring further investigation is that autophagy is acting to promote to MFs inoculated with apoptotic or a mixed population of apoptotic presentation of self-antigens by B cells. Autophagy is readily inhibited and viable parasites. Subsequently, preliminary results suggest that only by many common pharmaceutical agents and may therefore represent in the presence of apoptotic promastigotes and human T cells Lm infection could be sustained in human MF over a period of 7 days.
Conclusions: We found that apoptotic promastigotes enter a matur-ing LC3 compartment. Our data suggest that degradation of parasites in this compartment could be involved in a down regulation of T cell Role of autophagy in the immunopathogenesis of leprosy activation. We now further investigate and characterize the prolifer-ating T cell subsets and how the autophagy machinery and apoptotic B. J. Andrade Silva,* P. R. Andrade,* T. P. Amadeu,* V. Diniz,  promastigotes may dampen immune responses in human primary S. Coˆrte-Real,  V. C. Valentim,* H. Ferreira,* J. A. C. Nery,* E. N. Sarno* & R. Olmo Pinheiro**Leprosy Laboratory, Oswaldo Cruz Institute, Rio de Janeiro, Brazil,  Structural Biology Laboratory, Oswaldo Cruz Institute, Rio de Janeiro, Autophagy is activated in the B cells of patients with SLE andcorrelates with disease activity Purpose/Objective: Leprosy is a chronic infectious disease that canpresent different clinical forms and there is evidence that the estab- A. J. Clarke, U. Ellinghaus & T. J. Vyse lishment of different clinical forms is driven by host innate mecha- Medical and Molecular Genetics, King’s College London, London, UK nisms. Macrophages from tuberculoid (BT) and lepromatous (LL)patients seem to have a different behavior in relation to the myco- Purpose/Objective: Autophagy is increasingly appreciated as an bacteria. While in LL patients there are highly infected macrophages, in important immune surveillance and effector mechanism, but under- BT rare or few bacilli are found. Electron microscopy studies showed standing of its dynamic function in human autoimmune disease is the presence of phagosomes with double membrane in macrophages limited. We sought to evaluate its role in the B and T lymphocytes of exposed to M. leprae (ML), suggesting a possible involvement of patients with SLE compared with healthy controls.
Ó 2012 The Author(s) Ó 2012 Blackwell Publishing Ltd, Immunology, 137 (Suppl. 1), 185À772 506 Poster Session: Myeloid Cell Development mechanism against liver injury. This study was scheduled to examine Modification of a single lysine in a CYP2E1 epitope induces the role of SMAD7 in liver inflammation, fibrosis and the possible effect of antiviral treatment.
Materials and methods: Liver biopsies from 67 patients with hepatic D. Njoku,* J. Cho,  L. Kim,  L. Strouss,  E. McCarthy,  K. Gilbert,  diseases were studied: (1) 18 with chronic HCV hepatitis (CHC); (2) 19 with chronic HBV hepatitis at diagnosis (CHB/d); (3) four with Johns Hopkins University, Anesthesiology Pathology Pediatrics, Balti- CHB after antiviral treatment and relapse (CHB/non-r) (4) 14 with more, MD, USA,  Johns Hopkins University, Anesthesiology, Baltimore, CHB after antiviral treatment response and remission for >5 years MD, USA, àJohns Hopkins University, Biochemistry, Baltimore, MD, (CHB/r); (5) 12 with non alcoholic fatty liver disease (NAFLD). Three USA, §Johns Hopkins University, Pathology Molecular Microbiology and liver samples with a mild increase of aminotransferases but without histological changes, served as controls. Histological activity index and Purpose/Objective: Key steps in the pathogenesis of immune-medi- staging of fibrosis were also assessed. RNA was extracted and cDNA ated drug-induced liver injury (Im-DILI) have not been identified.
was synthesized using standard protocols. mRNA expression of TGFb After receiving halogenated anesthetics, anti-seizure medications, isoforms (TGFB1, 2, 3), activins (A, B, C, E), ALK4, ALK5, SMAD antibiotics or non-steroidal anti-inflammatory drugs, susceptible pa- molecules (SMAD2, 3, 4, 7), and CTGFwas examined using quanti- tients develop Im-DILI thereby increasing their morbidity and often tative real time PCR. Statistical analysis was performed using SPSS and their mortality. In anesthetic Im-DILI patients, granulocytic hepatitis, P values < 0.05 were considered significant.
trifluoroacetyl chloride (TFA) and IL-4-mediated cytochrome P4502E1 Results: Patients with CHB/r exhibited a significant increase of (CYP2E1) IgG4 antibodies support the diagnosis, while CYP2E1 epi- SMAD7 and ALK4 mRNA expression compared to CHB/d patients, topes responsible for the pathogenesis of Im-DILI are unknown. We and reduced levels of TGFB1, SMAD2, SMAD3, and CTGF. A previously demonstrated a CYP2E1 epitope [Gly113-Leu133 (JHDN5)] significant increase of SMAD7 was also found in NAFLD patients containing a single lysine that was recognized by sera from anesthetic compared to untreated viral hepatitis patients and those who did not DILI patients with specific MHC II haplotypes. We showed that respond to any treatment. Moreover, NAFLD patients were presented JHDN5 was recognized by splenocytes from mice with experimental with elevated levels of TGFB1, TGFB3, INHbC, ALK5, and SMAD4.
Im-DILI induced by immunizations with liver proteins covalently al- Considering the intensity of inflammation, SMAD7, ALK5, and INHbC tered by TFA, a drug hapten formed during metabolism of halogenated exhibited a significant increased expression from absent to minimal anesthetics. We hypothesize that covalent modification of a single inflammation with a gradual reduction as inflammation exacerbates.
lysine in JHDN5 induces IL-4-mediated, Im-DILI in BALB/c mice.
Conclusions: Our data indicate that in cases with low grade fibrosis, Materials and methods: JDN5 was modified by TFA (TFA-JHDN5) as NAFLD (characterized by a lower incidence of severe liver using the methods of Goldberger and Anfinisen. We confirmed 81.5% complications and fibrosis progression) and CHB/r, SMAD7 overex- modification of JHDN5 using the method of Habeeb. BALB/c mice pression might be a mechanism limiting the fibrogenic effect of TGFb were immunized with 100 lg of an unrelated CYP2E1 epitope or suggesting that its induction may provide a target for novel therapeutic JHDN5 ± TFA emulsified in CFA or CFA alone on days 0 and 7 and killed on day 21. IL-4 deficient (KO) mice were similarly treated with This research has been co-financed by the ESF and Greek national CFA ± TFA-JHDN5. Histology scores, antibodies and cytokine levels funds through the Operational Program ‘‘Education and Lifelong were analyzed using MannÀWhitney U-test. A P value <0.05 was Learning’’ of the NSRF À Research Funding Program: Heracleitus II.
Investing in knowledge society through the European Social Fund.
Results: TFA-JHDN5 induced more granulocytic hepatitis (P < 0.01)as well as anti-TFA and anti-CYP2E1 antibodies (P < 0.05) than CFA-immunized BALB/c or KO mice. Granulocyte and macrophage attractants KC, MIP-2, G-CSF, M-CSF, MCP-1, MIP-1a, MIP-1b Peritoneal macrophage inflammatory profile in cirrhosis is and VEGF as well as IL-7 and IL-9 were elevated in BALB/c but not KO dependent on the etiology and is related to ERK phosphorylation livers (P < 0.05). Unmodified epitopes did not induce Im-DILI.
Conclusions: We confirm that covalent modification of a single lysine M. Martı´nez-Esparza,* A. Tapia-Abella´n,* A. J. Ruiz-Alcaraz,* in a CYP2E1 epitope induces Im-DILI in BALB/c mice with features T. Herna´ndez-Caselles,* C. Martı´nez-Pascual,  M. Miras-Lo´pez,  reminiscent of anesthetic Im-DILI in patients. Future studies of this J. Such,à R. France´sà & P. Garcı´a-Pen epitope may uncover unidentified mechanisms of Im-DILI from other *Biochemistry and Molecular Biology (B) and Immunology, University of drugs and help to develop targeted agents to either treat or prevent this Murcia, Murcia, Spain,  Hospital Universitario Virgen de la Arrixaca, Unidad de Trasplante Hepa´tico, Murcia, Spain, àHospital Universitario,Unidad Hepa´tica, Alicante, Spain Purpose/Objective: The aim of this work is to identify functional Overexpression of SMAD7 protects liver from TGFb/Smad-medi- differences in the inflammatory profile of monocyte-derived macro- phages (M-DM) from ascites in cirrhotic patients of different etiolo-gies, alcohol- and hepatitis C virus (HCV)-related cirrhosis, trying to N. Argentou,* G. Germanidis,  E. Apostolou,à T. Vasiliadis,§ extrapolate studies from liver biopsies to immune cells in ascites.
P. Sideras,à A. E. Germenis* & M. Speletas* Materials and methods: We studied 45 patients with cirrhosis and *Medical School, University of Thessaly, Larissa, Greece,  AHEPA non-infected ascites, distributed according to disease etiology, HCV Hospital, Aristotle University of Thessaloniki, Thessaloniki, Greece, (n = 15) or alcohol (n = 30). Cytokines and cellular content in ascites Biomedical Research Foundation, Academy of Athens, Athens, Greece, were assessed by ELISA and flow cytometry, respectively. Cytokines Hippokration Hospital, Aristotle University of Thessaloniki, Thessalo- and ERK phosphorylation level from peritoneal monocyte-derived macrophages isolated and stimulated in vitro were also determined.
Purpose/Objective: SMAD7 is a negative regulator of TGFb/activin Results: A different pattern of leukocyte migration to peritoneal cavity pathway. Recently, animal studies have shown that SMAD7 induction and primed status of macrophages in cirrhosis is observed depending ameliorates TGFb/Smad-mediated fibrogenesis, suggesting a protective on the viral or alcoholic etiology. Whereas no differences in peripheral Ó 2012 The Author(s) Ó 2012 Blackwell Publishing Ltd, Immunology, 137 (Suppl. 1), 185À772

Posters_Abstracts of the European Congress of Immunology,p12, 5-8 September 2012, Glasgow, Scotland

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