5^th European Immunology Conference July 21-23, 2016 Berlin, Germany

Daniela Ramos Rodrigues has completed her PhD in 2013 from the Universidade Estadual Paulista. She is currently a Postdoctoral fellow from the same university and immunology paracoccidioidomycosis is her main area of research.

Paracoccidioidomycosis is a systemic mycosis, endemic to most Latin American countries, especially in Brazil, whose etiologic agent is the thermo dimorphic fungus of genus Paracoccidioides, comprising cryptic species of Paracoccidioides brasiliensis (Pb), S1, PS2, PS3 and Paracoccidioides lutzii. Studies aiming to characterize the immune response of the infected host have focused on the role of different subsets of CD4+ cells with special interest for the mechanisms involved in the preferential induction of either of them, which are largely dependent on the initial interaction of the fungus with cells of the innate immune response. Neutrophils were recently shown to influence the activation of different leukocyte types including NK cells, B cells and DCs. Therefore, neutrophils by regulating DCs recruitment and activation may have an important role in the development of adaptive immune response. Here, we aimed to evaluate whether human PMNs sensitized with the fungus are able to modulate DCs capacity to induce naïve CD4+ T cells proliferation. Peripheral blood PMNs obtained from 9 healthy donors were sensitized with Pb18 and/or Pb265 for 48 hours, co-cultured with autologous DCs by 48 hours followed by co-culture with autologous CD4+ cells by 120 hours. Phytohemaglutinin (PHA) was used as positive control to CD4+ cells proliferation. We found that neutrophils sensitized with Pb18 or Pb265 activate DCs to induce an intense CD4+ cells proliferation. We concluded that a crosstalk between neutrophils and DCs may be very important for induction of the host adaptive immune response against P. brasiliensis.

Reginaldo Keller Fernandes has completed his Master’s degree from Universidade Estadual Paulista and currently pursuing his Doctoral studies at the same university. He is studying Immunology of Paracoccidioidomycosis. He has also published 5 papers in reputed journals.

Paracoccidioidomycosis is a systemic mycosis, endemic to most Latin American countries, especially in Brazil, whose etiologic agent is the thermo dimorphic fungus of the genus Paracoccidioides, comprising cryptic species of Paracoccidioides brasiliensis (Pb), S1, PS2, PS3 and Paracoccidioides lutzii. The consequences of the fungus interaction with cells of the innate immune response, such as dendritic cells (DCs), highlighting the ability of these cells to instruct the adaptive immune response are not fully understood. In a previous study we found that human monocytes derived DCs fail to adequately mature in response to Pb. Thus, we aimed to evaluate whether these cells are able to induce CD4 proliferation and what is the predominant profile of the proliferated cells. Human DCs obtained from in vitro differentiation of monocytes were challenged with more (Pb18) and less virulent (Pb265) fungus strains during 48 hours, followed by co culture with CD4+ cells by 48 hours to 120 hours. Phytohemaglutinin (PHA) was used as positive control to CD4+ cells proliferation. Our results showed which dendritic cells challenged with Paracoccidioides brasiliensis induce Treg response. These findings may provide novel information for the understanding of the complex interplay between the host and this fungus.

Ching-Len Liao is currently the Director of National Institute of Infectious Diseases and Vaccinology, National Health Research Institutes from Taiwan. He was trained as a Virologist from University of Southern California, Keck School of Medicine.

The positive-sense RNA genome of mosquito-borne flaviviruses appears to be flexible in terms of accommodating extra insertions of heterologous antigens into their virus genes. With assistance from inclusion of designed flanking sequences derived from the junction between E-NS1 and NS2B-NS3, such insertions can be introduced into junction of E-NS1 or NS2BNS3 to separate inserts from the Flavivirus polyprotein. In contrast, flaviviral E and NS1 can directly house foreign epitopes to become fusion proteins without significantly disturbing normal virus replication. Herein, we illustrate that the newly identified C-terminal of the core β-ladder domain in NS1 could be readily inserted into entities such as EV-71 epitopes and the resulting NS1-epitope fusion proteins appeared to maintain normal virus replication, secretion ability and multimeric formation from infected cells. Nonetheless, such an insertion attenuated the recombinant JEV in mice, despite having retained the brain replication ability observed in wild-type JEV. Mother dams immunized with recombinant JEV expressing EV71 epitope-NS1 fused proteins elicited neutralizing antibodies that protected the newborn mice against lethal EV71 challenge. Together, our results implied a potential application of using JEV NS1 as a viral carrier protein to express a heterologous epitope to stimulate dual/multiple protective immunity concurrently against several pathogens.

Ferah Genel is currently an Associate Professor in Dr. Behçet Uz Children’s Hospital, Department of Pediatrics, Division of Pediatric Immunology. He/She is interested in Congenital Immune Deficiencies, Flow cytometry and he/she has 52 papers published in reputed journals to his/her credit.

Introduction: Hyper immunoglobulin E syndrome (HIES) is a rare systemic disease characterized by recurrent skin and lung infections, eczema and serum total IgE elevation. It can easily be confused with atopic dermatitis and other causes of rare immune deficiencies in clinical practice. We presented a case which was followed with atopic dermatitis diagnosis for a long time and received the diagnosis of HIES associated with STAT3 mutation during the follow-up. Case: A 10-year old girl was referred to our clinic for atopic dermatitis resistant to medical treatment and extremely high total IgE levels (18223 IU/L). In her past history, she had omphalitis during neonatal period; eczematous lesions developed in hairy and hairless skin, white plaques developed in the mucosa of mouth and tongue continuously since the infancy period, recurrent purulent otitis. She did not have a family history of immunodeficiency. She was begun to be followed up with the diagnosis of atopic dermatitis when she was a 16-month-old baby and atopic dermatitis symptoms did not respond to treatment sufficiently. Asthma and allergic rhinitis symptoms developed during the follow-up period. On admission, physical examination revealed prevalent eczematous eruptions, oropharyngeal candidiasis, changes in the nails suggesting mycotic infection. In the laboratory analysis, IgA, M and IgG levels, C3 and C4 levels of the patient were determined to be normal and tetanus, diphtheria and hepatitis B vaccine responses of the case emerged positive. In lymphocyte subgroups, total lymphocyte and CD8+ cells had 5-10 percentile value numerically in terms of age; CD19+ and NK cells were detected below the value of 5 percentile. Candida albicans multiplied in a culture medium received from oral mucosa. With possible diagnosis of HIES, the patient was started to be administered IV immunoglobulin replacement, trimethoprim-sulfamethoxazole and flukonazole prophylaxis. Genetic analysis performed at CeMM-Research Centre for Molecular Medicine of the Austrian Academy of Sciences revealed STAT3 mutation. Conclusion: In such cases with atopic dermatitis symptoms and elevated total IgE levels, who do not adequately respond to the treatment, HIES associated with STAT3 mutation should be remembered during differential diagnosis.

1996-2000 B.Sc. in Biology/ Microbiology from Al-Mustansiria University-Iraq. 2007-2009 M.Sc. in Microbiology from Pune University-India. 2013 Started my PhD study at Strathclyde University (SIPBS) with the project of “Developing a vaccine against Leishmania Spp.

Leishmaniasis is a disease that causes significant mortality and morbidity. In the last five years, 1 million cases of cutaneous leishmaniasis (CL) have been reported and 300,000 cases of visceral leishmaniasis (VL). There have been 20,000 deaths/ year and 310 million people are at risk of infection. At present there is no clinical vaccine for this disease. We have shown that vaccination with recombinant (γGCS) produced in Eschericheria coli can protect against CL and VL in murine models. However in this expression vector truncated recombinant protein was produced. Therefore, we carried out studies to determine if we could produce better quality γGCS in L. tarentolae, which is phylogenetically more related to Leishmania. We successfully produced a construct that allowed transfection of L. tarnetolae with the gene sequence of γGCS from 3 different Leishmania species. Western blot studies showed that full-length protein was produced indicating that L. tarentolae may be a better expression vector than E. coli.

Konstantinos Kotzamanis has completed his Bachelor’s studies in Chemistry/Biochemistry at the Aristotle University of Thessaloniki. He was awarded a scholarship from IKY/EU (Greek State Scholarship Foundation) for his MSc at the University of Edinburgh, which he received in 2012. He was, in addition, awarded a scholarship from IKY/EU and the University of Edinburgh for his PhD studies in the lab of Professor Peter Ghazal. He has one publication and has been awarded a prize for best poster at the BSI summer school in 2014.

Viruses are obligatory parasites that both exploit and counter selective host pathways, including metabolism, to effectively propagate. Recent studies from our group and others have revealed a central connection between infection and metabolism in regulating the host response. In particular macrophages, among other immune cells, reprogram and tightly regulate their metabolism upon activation from IFN and/or TLRs. Here, we employ transcriptomics and metabolomics analyses of wild type and mutant Cytomegalovirus infection of wild type and IFNB-/- or IFNAR-/- macrophages. These investigations enable the unraveling of host directed versus virus driven responses and we find unexpectedly that Cytomegalovirus opportunistically co-opts early pro-inflammatory changes in glycolysis for establishing infection of macrophages.

Gerard Pasternak was graduated from the Medical University Wroclaw in 2008. Since 2015, he has been the Assistant at the Department and Clinic of Pediatrics, Immunology and Rheumatology of Developmental Age MU Wroclaw and the Department of Immunology and Pediatrics, Provincial Hospital J. Gromkowski, Wrocław (since 2010). He serves as a didactic Assistant Professor in the Department. He participates in conducting activities in the field of primary immunodeficiency for students of III-VI of the year and takes part in the preparation and conducting of workshops for patients with PID and their families. His professional interests and research are concentrated on the diagnosis and treatment of primary and secondary immunodeficiencies with particular emphasis on deficit of the IgG subclasses.

Neutrophil function is an essential component of innate immunity which depends on many factors, both internal and outside of the organism. Deficits in disorders of humoral immunity associated with a deficit of antibodies are the most common primary immunodeficiency in children with recurrent respiratory tract infections and bronchial asthma. Study included 100 children (aged 6 months to 18 years) referred to the Department of Clinical Immunology and Pediatrics purpose of diagnosis of recurrent respiratory tract infections. All children screening for hemolytic activity of the complement, the concentration of the major classes of immunoglobulins (IgG, IgA, IgM, IgE) of the IgG subclasses (IgG1, IgG2, IgG3, IgG4), the number of lymphocytes with a phenotype of CD3+, CD4+, CD8+, CD19+ and CD56+ were examined and properties of phagocytic neutrophils. It was evaluated neutrophils phagocytosis efficiency relative to Stapylococcus aureus 209P, phagocytic index and the percentage of phagocytic neutrophils. Examined children were divided into two groups: Those with normal concentrations of the IgG subclasses or a deficit of them. It has been demonstrated a number of abnormalities of the neutrophil function in children with a deficit of the IgG subclasses as well as normal concentrations of of the IgG subclasses.

Aleksandra Lewandowicz-Uszyńska is currently acting as Manager of the 3rd Department and Clinic of Pediatrics, Immunology and Rheumatology of Developmental Age and Head Physician of the Department of Clinical Immunology and Pediatrics Provincial Hospital J. Gromkowski in Wroclaw. She was graduated from the Medical University of Wroclaw in 1988 and she is a Specialist in Pediatrics and Clinical Immunology. She has participated in numerous scientific internships in the leading Immunological Centers. Her research interests are focused on the phenomena of humoral and cellular immunity (with particular emphasis on the role of neutrophils) in primary and secondary immunodeficiencies (PID). She is also a Provincial Consultant in Pediatrics for Lower Silesia, Member of Board of the Polish Society of Experimental and Clinical Immunology, the Committee of Immunology and Etiology of infections Human Polish Academy of Sciences, Polish Working Group, PIDs, Polish Federation of Pediatric, the Scientific Committee of Standards of Medical and OSOZ. She is the President of the Section of Developmental Immunology Since 1997. She is the author of 102 scientific publications, approximately 100 congress reports, 13 textbook chapters in the field of pediatrics and clinical immunology.

Primary immunodeficiencies are rare diseases in which the immune abnormalities observed frequently result in the occurrence of persistent infections. Not recognized and the same not treated properly can lead to a number of complications, abnormalities can sometimes finish in premature death. Therefore, where it is stated recurrent infections efforts should be made to confirm or rule out the disease. Studies on the functioning of the immune system in this group of disorders have been carried out for many years and yet there are still many pathophysiological mechanism is unknown. Therefore, it is important to continue to explore these issues. 87 patients were included in the study of the Department of Clinical Immunology and Pediatrics at the age of 6 m to 18 years old, among whom distinguished group of 26 patients with primary immunodeficiency (PID), 43 patients suffering from recurrent respiratory infections and 18 healthy children (without recurrent infections). All patients were evaluated the screening of selected parameters of immunological hemolytic complement activity (CH50) of the concentration immunolglobulin major classes, properties of neutrophils phagocytosis, and a panel of T cells, B cells and NK cells. Additionally the concentration of IFN-γ and TNF-α was examined in serum it was determined polymorphism of the gene encoding TNF-alpha (TNFA, rs1800629, -308 G/A) and IFN-gamma (IFNg, rs2430561, 874 T/A). Genotyping was performed by analysis of melting curves. This method is based on the reaction of the real-time PCR, i.e., PCR in which the detection of amplified DNA may take place during the reaction, without the need for separate detection of the completion of the reaction. As typing kits used are SNiP Light Assay (TIB MOLBIOL Company) and the mixture Probes Master Mix (Roche). Amplifications are performed with the Light Cycler® 480 Multiwell Plate 96 in the camera 480 II Light Cycler (Roche). It has been demonstrated reduced levels of IFN-γ in children with PID and recurrent respiratory infections compared with the controlled group. The observed most frequent polymorphism groups concerned AT base in both the PID and recurrent respiratory infections, as well as in the control group. TNF-α in the PID group were decreased and increased in patients with recurrent respiratory infections compared to the controlled group. Polymorphism is most frequently found in children with PID base pairs of concern GA, in the group of recurrent respiratory tract infections usually observation GG base pairs.

Gerard Pasternak was graduated from the Medical University Wroclaw in 2008. Since 2015, he has been the Assistant at the Department and Clinic of Pediatrics, Immunology and Rheumatology of Developmental Age MU Wroclaw and the Department of Immunology and Pediatrics, Provincial Hospital J. Gromkowski, Wrocław (since 2010). He serves as a didactic Assistant Professor in the Department. He participates in conducting activities in the field of primary immunodeficiency for students of III-VI of the year and takes part in the preparation and conducting of workshops for patients with PID and their families. His professional interests and research are concentrated on the diagnosis and treatment of primary and secondary immunodeficiencies with particular emphasis on deficit of the IgG subclasses.

Ataxia telangiectasia is a very rare primary immunodeficiency with a circle of chromosomal instability occurring around the world with a known genetic defect in the gene ATM. These patients have a number of progressive abnormalities, neurological (ataxia, intention tremor) and the variable irregularities in the immune system (progressive decrease in the number of T cells, the deficit of the major classes of immunoglobulins and IgG subclasses, inability to repair DNA strands), hypersensitivity to ionizing radiation and the huge trend the occurrence of neoplastic diseases. Analysis covered 11 patients (aged 2 to 22 years old, 7 women and 3 men) who are under the care of the Department of Clinical Immunology and Pediatrics who have been identified for the treatment of intravenous immunoglobulins because of recurrent infections and deficit of IgG or IgG subclasses. Analyzed: Clinical course (the first neurological symptoms, the first clinical signs of immunodeficiency) selected immunological tests (the concentration of the major classes of immunoglobulins, IgG subclasses, haemolytic activity of complement, properties of neutrophils phagocytosis, a panel of T cells, B cells and NK cells, and the concentration of AFP) depending on the performance of the individual mutations in the ATM gene. Among the changes detected mutations: c.6754_6754delAfs5X c.434T> G c.6145T> G were not previously reported in the literature. Mutations c.6095G> A c.7630-2A> C are the most commonly detected mutations among patients with ataxia telangiectasia in Poland. Mutations detected in the study group were generally localized in the domain of N-Terminal domain protein and FAT domain. At the two boys in whom the disease manifested itself very early we observed specific mutation and rapid and extremely aggressive course of the disease. These two brothers both carry a novel mutations: c.434T>G, p.L145R, c.6145T>G p.Y2049D. First mutation replaces non-polar, hydrophobic leucine with basic amino arginine at position 145. Second mutation is a substitution of aromatic tyrosine for acidic amino aspartic acid at position 2049 in the protein sequence. To predict the biological effect of two missense changes, in silico analysis was performed using the Protein Variation Effect Analyzer (PROVEAN, J. Craig Venter Institute). The algorithm of analysis predicted c.434T>G (score 3.403) and c.6145T>G (score 6.956) to be pathogenic.

S Gadiri, Clinical Hospital St Therese Annaba, Algeria

Introduction: Vasculitis associated with anti-neutrophil cytoplasmic antibodies (ANCA) are represented by granulomatosis with polyangiitis (GPA), the Granulomatosis with polyangiitis and eosinophilia (Churg-Strauss) and microscopic polyangiitis (MPA).Their detection uses indirect immunofluorescence tests on human neutrophils fixed in ethanol. With this fixative, three aspects are described: Cytoplasmic (c-ANCA), perinuclear (p-ANCA) and atypical (a-ANCA).The specificity of ANCA (Antiproteinase 3 (PR3) and the specificity of ANCA (Anti-proteinase 3 (PR3) and myeloperoxidase (MPO) is sought by ELISA. C-ANCA-PR3 generally associated with the GPA while MPO p-ANCA is associated with the MPA and the Churg-Strauss. Objective: To study the clinical and immunological characteristics of 18 patients with ANCA vasculitis positive. Materials & Methods: The study includes 18 patients with ANCA vasculitis (12 women and 06 men). ANCA was performed by indirect immunofluorescence, supplemented by ELISA to determine their specificity MPO/PR3. Results: The mean age of patients was 51 years, the diagnosis was: 07 cases of GPA, 08 cases of microscopic polyangiitis (MPA), 03 subjects had signs of overlap between the GPA and MPA. The clinical picture was dominated by renal disease followed by lung disease and ENT. 07 patients had c-ANCA (38.89%), of which 05 were anti-PR3 specificity (27, 77%), 11 patients had p-ANCA (61.11%), including 5 with a specific anti-MPO (27, 77%) and positive ANCA 8 patients showed no 2 searched specificities (44.44%). Conclusion: ANCA vasculitis is rare, clinical and immunological spectrum is very heterogeneous. The demonstration of ANCA directed vis-a-vis PR3 and MPO specific as an aid in the diagnosis of systemic vasculitis.

Camila Pinzan has completed her PhD at the age of 30 years from São Paulo University and her postdoctoral studies from São Paulo University School of Medicine. Currently she is doing her second post doc in University of Cambridge. She has published more than 8 papers in reputed journals.

Toxoplasma gondii actively infect host cells through a dependent process of lectins release, called microneme proteins (MIC), from intracellular organelles. TgMIC1, TgMIC4 and TgMIC6 assembly a complex on T. gondii surface enabling the parasite to bind to host cells via carbohydrate recognition, since TgMIC1 binds to terminal sialic acid and TgMIC4 to terminal galactose. Our aim was to evaluate the carbohydrate-protein interaction between TgMIC1 and TgMIC4 with N-glycans of extracellular TLRs and the innate immune response triggered by this interaction. We observed that TgMIC1 and TgMIC4 (native complex and recombinant proteins) induced proinflammatory cytokines production, especially IL-12, by C57BL/6 mice dendritic cells (DCs) and macrophages similarly to TLR agonists, while TLR2-/-, TLR4-/-or DKO-TLR2-/-/TLR4-/- macrophages produced less IL-12 than wild type (WT) cells. Furthermore, we found that this activation was dependent on carbohydrate recognition since a punctual mutation in carbohydrate recognition domain (CRD) of TgMIC1 abrogated its capacity to induce IL-12 production by WT macrophages. Moreover, during the first hours of T. gondii infection, the absence of TLR2 and TLR4 resulted in lower IL-12 production by DCs. Finally, the infection of WT DCs and macrophages with parasites lacking TgMIC1 or both proteins (DKO-TgMIC1-/-/TgMIC4-/-) also resulted in impaired activation, indicated by lower IL-12 production, compared to infection with WT parasites. Since the decreased IL-12 production was observed in the first hours after infection, we found that TgMIC1 and TgMIC4 trigger the initial response to T. gondii, by recognizing N-glycans of TLRs. The established interactions and the resulting host cell activation may exert relevant biological role during T. gondii infection.