B-cell Mediated Immune Responses Research Articles

HPV16 Intratypic Variants in Head and Neck Cancers: A North American Perspective.

Human papillomavirus (HPV) is the major causative agent for cervical and many head and neck cancers (HNCs). HPVs randomly acquire single nucleotide polymorphisms (SNPs) that may become established via positive selection. Within an HPV type, viral isolates differing by <2% in the L1 region are termed "variants" and classified based on combinations of SNPs. Studies in cervical cancer demonstrate clear differences between HPV16 intratypic variants in terms of persistence of infection, tumor histology, cancer risk, and death. Much less is known about the frequency of HPV16 variants in HNC, and their effects on clinical outcomes. We combined HPV16 positive (HPV16+) HNC samples from a local Southwestern Ontario, Canada cohort with those from the Cancer Genome Atlas to create a larger North American cohort of 149 cases with clinical data and determined the distribution of intratypic variants and their impact on clinical outcomes. Most isolates were lineage A, sublineage A1, or A2, with roughly half exhibiting the T350G polymorphism in E6. Univariable analysis identified significant differences between 350T and 350G intratypic variants in clinical T, N, and O staging, as well as disease-free survival. Multivariable analysis failed to identify any clinical factor as a statistically significant covariate for disease-free survival differences between 350T and 350G. Significant differences in several measures of B-cell mediated immune response were also observed between 350T and 350G intratypic variants. We suggest that HPV genetic variation may be associated with HNC clinical characteristics and may have prognostic value.

Immune response to SARS-CoV-2 mRNA vaccination in multiple sclerosis patients after rituximab treatment interruption.

Peripheral B cell depletion via anti-CD20 treatment is a highly effective disease-modifying treatment for reducing new relapses in multiple sclerosis (MS) patients. A drawback of rituximab (RTX) and other anti-CD20 antibodies is a poor immune response to vaccination. While this can be mitigated by treatment interruption of at least six months prior to vaccination, the timing to resume treatment while maintaining subsequent vaccine responses remains undetermined. Here, we characterized SARS-CoV-2 S-directed antibody and B cell responses throughout three BNT162b2 mRNA vaccine doses in RTX-treated MS patients, with the first two doses given during treatment interruption. We examined B-cell mediated immune responses in blood samples from patients with RTX-treated MS throughout three BNT162b2 vaccine doses, compared to an age- and sex-matched healthy control group. The first vaccine dose was given 1.3 years (median) after the last RTX infusion, the second dose one month after the first, and the third dose four weeks after treatment re-initiation. We analyzed SARS-CoV-2 S-directed antibody levels using enzyme-linked immunosorbent assay (ELISA), and the neutralization capacity of patient serum against SARS-CoV-2 S-pseudotyped lentivirus using luciferase reporter assay. In addition, we assessed switched memory (CD19+CD20+CD27+IgD-), unswitched memory (CD19+CD20+CD27+IgD+), naïve (CD19+CD20+CD27-IgD+), and double negative (DN, CD19+CD20+CD27-IgD-) B cell frequencies, as well as their SARS-CoV-2 S-specific (CoV+) and Decay Accelerating Factor-negative (DAF-) subpopulations, using flow cytometry. After two vaccine doses, S-binding antibody levels and neutralization capacity in SARS-CoV-2-naïve MS patients were comparable to vaccinated healthy controls, albeit with greater variation. Higher antibody response levels and CoV+-DN B cell frequencies after the second vaccine dose were predictive of a boost effect after the third dose, even after re-initiation of rituximab treatment. MS patients also exhibited lower frequencies of DAF- memory B cells, a suggested proxy for germinal centre activity, than control individuals. S-binding antibody levels in RTX-treated MS patients after two vaccine doses could help determine which individuals would need to move up their next vaccine booster dose or postpone their next RTX infusion. Our findings also offer first indications on the potential importance of antigenic stimulation of DN B cells and long-term impairment of germinal centre activity in rituximab-treated MS patients.

Development of a chimeric vaccine candidate based on Toxoplasma gondii major surface antigen 1 and apicoplast proteins using comprehensive immunoinformatics approaches

This study was aimed at designing and evaluation of a multimeric vaccine construct against Toxoplasma gondii via utilization of SAG1 along with apicoplast ribosomal proteins (S2, S5 and L11). Top-ranked MHC-I and MHC-II binding as well as shared, immunodominant linear B-cell epitopes were predicted and joined together via appropriate linkers. Also, TLR-4 agonist (RS-09 synthetic protein) and His-tag were added to the N- and C-terminal of the vaccine sequence. The finally-engineered chimeric vaccine had a length of 291 amino acids with a molecular weight of 31.46 kDa. Physico-chemical features showed a soluble, highly-antigenic and non-allergenic candidate. Secondary and tertiary structures were predicted, and subsequent analyses confirmed the construct stability that was capable to properly interact with human TLR-4. Immunoinformatics-based simulation displayed potent stimulation of T- and B-cell mediated immune responses upon vaccination with the proposed multi-epitope candidate. In conclusion, obtained information demonstrated a highly antigenic vaccine candidate, which could develop high levels of IFN-γ and other components of cellular immune profile, and can be directed for toxoplasmosis prophylactic purposes.

EZH2 Inhibition Interferes With the Activation of Type I Interferon Signaling Pathway and Ameliorates Lupus Nephritis in NZB/NZW F1 Mice.

Enhancer of zeste homolog 2 (EZH2) is a histone-lysine N-methyltransferase mediating trimethylation of H3K27, which represses gene expression and is critical to immune regulation. Inhibition of EZH2 is proved to have the potential of treating many diseases. However, whether inhibition of EZH2 affects type I interferon (IFN-I) signaling pathway, the abnormality of which is an important pathogenic mechanism for SLE, is still elusive. Here, we report, unexpectedly, a positive regulatory function of EZH2 in IFN-I signaling pathway, which contributes to the overactivation of IFN-I signaling pathway in SLE. We show that the expression of EZH2 was upregulated and positively correlated with the overexpression of interferon stimulated genes (ISGs) in both peripheral blood mononuclear cells and renal tissues of SLE patients. In vitro inhibition of EZH2 by either siRNAs or chemical inhibitors reduced the phosphorylation of STAT1 and the induction of ISGs stimulated by IFN-I. Additionally, inhibition of EZH2 interfered with the in vivo and ex vivo activation of IFN-I signaling pathway elicited by intravenous injection of adenovirus vector expressing mouse IFN-α5 and exogeneous stimulation with IFN-α, respectively. We evaluated the therapeutic effects of EZH2 inhibitor in NZB/NZW F1 mice which depend on IFN-I signaling pathway for the lupus-like disease development. Administration of EZH2 inhibitor prolonged the survival, reduced the levels of anti-dsDNA autoantibodies, and improved lupus nephritis of the mice. What’s more, EZH2 inhibitor attenuated the expression of ISGs in the kidneys of these mice. In summary, we show that excessive EZH2 contributes to the overactivation of IFN-I signaling pathway in SLE. EZH2 inhibitor has the potential to inhibit IFN-I signaling pathway and alleviate lupus nephritis. Additionally, diverse disease driving pathways exist among systemic lupus erythematosus (SLE) patient, and even in the same patients. Common regulators of different pathogenic pathways can be multivalent therapeutic targets. Together with previous studies showing EZH2 is involved in T-cell and B-cell mediated immune responses, EZH2 could be a potent multivalent therapeutic target for SLE.

Applying high throughput and comprehensive immunoinformatics approaches to design a trivalent subunit vaccine for induction of immune response against emerging human coronaviruses SARS-CoV, MERS-CoV and SARS-CoV-2

Coronaviruses (CoVs) cause diseases such as severe acute respiratory syndrome (SARS), Middle East respiratory syndrome (MERS), and coronavirus disease 2019 (COVID-19). Therefore, this study was conducted to combat major CoVs via a trivalent subunit vaccine, which was engineered by implementing sequences of spike (S) protein, nucleocapsid (N), envelope (E), membrane (M) protein, non-structural protein (nsp) 3, and nsp8 antigens. The CTL, HTL, MHC I, and IFN-γ epitopes were predicted via CTLPRED, IEDB, and IFN epitope servers, respectively. Also, to stimulate strong helper T lymphocytes (HTLs) responses, Pan HLA DR-binding epitope (PADRE) was used. Also, for boosting the immune response, β-defensin 2 was added to the construct as an adjuvant. Furthermore, TAT was applied to the vaccine to facilitate the intracellular delivery. Finally, TAT, adjuvant, PADRE, and selected epitopes were appropriately assembled. Based on the predicted epitopes, a trivalent multi-epitope vaccine with a molecular weight of 74.8 kDa was constructed. Further analyses predicted the molecule to be a strong antigen, and a non-allergenic and soluble protein. Secondary and tertiary structures were predicted. Additionally, analyses validated the stability of the proposed vaccine. Molecular docking and molecular dynamics simulation (MDS) showed binding affinity and stability of the vaccine-TLR3 complex was favorable. The predicted epitopes demonstrated a strong potential to stimulate T and B-cell mediated immune responses. Furthermore, codon optimization and in silico cloning guaranteed increased expression. In summary, investigations demonstrated that this next-generation approach might provide a new horizon for the development of a highly immunogenic vaccine against SARS‐CoV, MERS‐CoV, and SARS-CoV-2. Communicated by Ramaswamy H. Sarma

The global transcriptomic signature in sinonasal tissues reveals roles for tissue type and chronic rhinosinusitis disease phenotype.

RNA sequencing (RNA-Seq) allows the characterization of a global transcriptomic signature in a least-biased fashion, but few studies have applied this method to investigate the pathophysiology of CRS. We collected mucosal tissue samples from 6 CRS without nasal polyps (CRSsNP), 6 CRS with nasal polyps (CRSwNP), and 6 control patients. Additional matched polyp samples were collected from the 6 CRSwNP patients. RNA was extracted and sequenced on the Illumina HiSeq-2500. Differential gene expression and pathway analyses were performed. CRSsNP showed evidence of upregulated interferon-mediated immunity, MHC-class-I mediated antigen presentation, CXCR3 binding, neutrophil chemotaxis and degranulation, and potential downregulation of genes related to cilia movement and production. CRSwNP polyp tissue showed upregulation of B-cell mediated immune responses, but reduced expression of genes related to epithelial morphogenesis and haemostasis. Polyps also showed a generalized reduction of positive gene regulation. The sinonasal transcriptomic signature was largely determined by tissue type (polyp versus mucosa) and disease phenotype, with minimal signal originating from the individual patient. RNA-Seq is a useful tool to explore the complex pathophysiology of CRS. Our findings stress the importance of tissue selection in molecular research utilizing sinonasal tissue, and demonstrate the limitation of the sNP/wNP paradigm (and the importance of endotyping). On the other hand, classical CRSsNP/wNP disease phenotypes played some role in determining the global transcriptomic signature, and should not be hastily discarded. The value of RNA-Seq-described transcriptomic signatures in exploring endotypes is yet to be explored in future studies.

Development of a conserved chimeric vaccine based on helper T-cell and CTL epitopes for induction of strong immune response against Schistosoma mansoni using immunoinformatics approaches

Currently, three recombinant antigens based vaccines are under clinical trials against Schistosomiasis, but there is no vaccine available for prophylaxis or therapeutic. This study was conducted to construct a multi-epitope based vaccine against Schistosoma mansoni via utilizing Sm14, Sm21.7, Sm23, Sm29, Smp80, Sm-CB and SM-TSP-2 antigens. Helper T lymphocyte (HTL), cytotoxic T lymphocyte (CTL) and IFN-γ epitopes were predicted. Furthermore, Pan HLA DR-binding epitope was added to the vaccine. Moreover, 50S ribosomal protein L7/L12 of Mycobacterium tuberculosis as a novel TLR4 agonist was applied. The TAT peptide was added to the vaccine to augment intracellular delivery. The selected epitopes were linked together through appropriate linkers and chimeric vaccine was constructed with 617 amino acids with molecular weight of 65.43 kDa. Physico-chemical properties revealed a soluble protein with antigenic and non-allergic properties. Further analyses validated the stability of the construct that was able to interact with TLR4. Immunoinformatics analysis demonstrated the strong potential of constructed vaccine to stimulate T and B-cell mediated immune responses. In summary, obtained data indicated that the proposed vaccine can properly induce both T and B cells immune responses and could possibly be utilized for prophylactic or therapeutic aims in response to infection caused by S. mansoni.

New Discoveries Regarding Endotypes of Chronic Rhinosinusitis with Nasal Polyp

Chronic rhinosinusitis (CRS) is a common chronic inflammatory disease of the nasal and paranasal mucosa and it usually affects substantial impaired quality of life. CRS is a highly heterogeneous disease and currently defined as subgroups of patients based on nasal endoscopic findings, accompanied either by CRS with nasal polyps (CRSwNP) or CRS without nasal polyps (CRSsNP). However, clinical phenotypes does not adequately reflect the pathophysiologic diversity within patients with CRS. Thus, CRS was also classified according to the inflammatory endotypes, which defined as subtypes of disease with an immunologically different from others by the involvement of a specific molecule or cell. To date, it has been well known that CRSsNP patients characterized by a predominant T helper cell type 1 inflammatory response, whereas CRSwNP patients from Western driven by a T helper cell type 2 inflammation and increased eosinophil infiltration. Meanwhile, CRSwNP patients from Asian displayed a mixed T cell profile with a non-eosinophilic inflammation pattern. However, recent some studies have reported the new discoveries regarding immunologic different endotypes of CRSwNP, such as innate lymphoid cells and activated B-cell mediated immune response. Therefore, herein we describe concepts and new discoveries of immunologic endotype in patients with CRSwNP. Key words: B-cell ㆍ Chronic rhinosinusitis ㆍ Innate immunity ㆍ Nasal polyp

Development of a multi-epitope peptide vaccine inducing robust T cell responses against brucellosis using immunoinformatics based approaches

Current investigations have demonstrated that a multi-epitope peptide vaccine targeting multiple antigens could be considered as an ideal approach for prevention and treatment of brucellosis. According to the latest findings, the most effective immunogenic antigens of brucella to induce immune responses are included Omp31, BP26, BLS, DnaK and L7–L12. Therefore, in the present study, an in silico approach was used to design a novel multi-epitope vaccine to elicit a desirable immune response against brucellosis. First, five novel T-cell epitopes were selected from Omp31, BP26, BLS, DnaK and L7–L12 proteins using different servers. In addition, helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied to induce CD4+ helper T lymphocytes (HTLs) responses. Selected epitopes were fused together by GPGPG linkers to facilitate the immune processing and epitope presentation. Moreover, cholera toxin B (CTB) was linked to N terminal of vaccine construct as an adjuvant by using EAAAK linker. A multi-epitope vaccine was designed based on predicted epitopes which was 377 amino acid residues in length. Then, the physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility and allergenicity of this multi-epitope vaccine were assessed using immunoinformatics tools and servers. Based on obtained results, a soluble, and non-allergic protein with 40.59kDa molecular weight was constructed. Expasy ProtParam classified this chimeric protein as a stable protein and also 89.8% residues of constructed vaccine were located in favored regions of the Ramachandran plot. Furthermore, this multi-epitope peptide vaccine was able to strongly induce T cell and B-cell mediated immune responses. In conclusion, immunoinformatics analysis indicated that this multi-epitope peptide vaccine can be effectively expressed and potentially be used for prophylactic or therapeutic usages against brucellosis.

In silico analyses of heat shock protein 60 and calreticulin to designing a novel vaccine shifting immune response toward T helper 2 in atherosclerosis

Recent experiments demonstrated that atherosclerosis is a Th1 dominant autoimmune condition, whereas Th2 cells are rarely detected within the atherosclerotic lesions. Several studies have indicated that Th2 type cytokines could be effective in the reduction and stabilization of atherosclerotic plaque. Therefore, the modulation of the adaptive immune response by shifting immune responses toward Th2 cells by a novel vaccine could represent a promising approach to prevent from progression and thromboembolic events in coronary artery disease. In the present study, an in silico approach was applied to design a novel multi-epitope vaccine to elicit a desirable immune response against atherosclerosis. Six novel IL-4 inducing epitopes were selected from HSP60 and calreticulin proteins. To enhance epitope presentation, IL-4 inducing epitopes were linked together by AAY and HEYGAEALERAG linkers. In addition, helper epitopes selected from Tetanus toxin fragment C (TTFrC) were applied to induce CD4+ helper T lymphocytes (HTLs) responses. Moreover, cholera toxin B (CTB) was employed as an adjuvant. A multi-epitope construct was designed based on predicted epitopes which was 320 residues in length. Then, the physico-chemical properties, secondary and tertiary structures, stability, intrinsic protein disorder, solubility and allergenicity of this chimeric protein were analyzed using bioinformatics tools and servers. Based on bioinformatics analysis, a soluble, and non-allergic protein with 35.405kDa molecular weight was designed. Expasy ProtParam classified this chimeric protein as a stable protein. In addition, predicted epitopes in the chimeric vaccine indicated strong potential to induce B-cell mediated immune response and shift immune responses toward protective Th2 immune response. Various in silico analyses indicate that this vaccine is a qualified candidate for improvement of atherosclerosis by inducing immune responses toward T helper 2.

Novel Humanised ROR1 Chimeric Antigen Receptors for the Treatment of Haematological Malignancies

Introduction:Receptor Tyrosine Kinase Like Orphan Receptor 1 (ROR1) is a surface antigen expressed on a range of haematological and solid malignancies including Chronic Lymphocytic Leukaemia (CLL). Although expressed during embryogenesis, its virtual absence on normal adult tissues makes it an attractive target for immunotherapy, especially with Chimeric Antigen Receptor modified T-cells (CAR T-cells). We have generated novel fully humanised ROR1CAR constructs for the treatment of CLL and other ROR1 positive malignancies.Results:Following a rat immunisation programme 38 oligloclonal hybridoma clones were single cell sorted and subjected to 5'RACE. Of 13 novel anti-ROR1 antibodies isolated, 10 retained specific binding when cloned into a heavy-linker-light single chain variable fragment (scFv) format. These scFvs in combination with a second generation CAR architecture comprising CD3zeta and 4-1BB demonstrated specific toxicity against ROR1 positive cell lines after T-cell transduction using lentiviral vectors.We found cytotoxicity with ROR1CAR T-cells was dependent on target cell ROR1 density. In order to ensure our screening assays allowed us to select which of the 10 binders was most suitable for targeting primary CLL, we assessed the antigen density of ROR1 and CD19 on CLL cells. Median expression of ROR1 was 2304 molecules/cell (Range 800-4828), compared to CD19, which had a much higher density of 12,583 (Range 5894-23,652). In view of this, subsequent functional assessment was focused on SKW and Jeko1 cell lines with constitutive ROR1 expression at levels similar to CLL cells, as opposed to those transduced to express supra-physiological levels.Our initial optimisation focused on modifying the CAR extracellular spacer region. We demonstrated a reciprocal relationship between cytotoxicity and the distance between T-cells and target cells. This was assessed by using clones that bound either the membrane-distal immunoglobulin domain or a more membrane-proximal frizzled domain of ROR1. The use of an optimum spacer enhanced cytotoxicity of all scFv constructs but yielded two lead candidates: Clones A & F. These showed consistently superior cytotoxicity against target cell lines compared to the other isolated clones. In addition epitope mapping revealed binding sites unique from the previously described rabbit R12 and murine 4A5 anti-ROR1 CAR T-cells.One of the advantages of targeting ROR1 as opposed to CD19 is sparing the normal B-cell compartment from CAR mediated eradication. However this comes with the consequent risk of B-cell mediated immune responses against rat-derived scFvs. To minimise immunogenicity we undertook a humanisation programme and grafted the complementary determining regions (CDR) of the heavy and light chains of Clone A and F into 5 acceptor human germline VH and VL sequences, generating 25 potential scFvs for each. Binding assessment showed seventeen successfully humanised binders for Clone A and three for Clone F. Of these, 5/17 and 3/3 showed activity in a CAR format against target cells. A final selection was made based on specific cytotoxicity, enhanced cytokine secretion (Interleukin-2 and Interferon gamma) and proliferation compared to the parental clones resulting in 2 unique constructs targeting different extracellular domains of ROR1. In addition, we have demonstrated cytotoxicity against a panel of ROR1 positive solid cancer cell lines to demonstrate their wider applicability.Conclusion:ROR1CAR T-cells have the potential to be an effective therapeutic not just for CLL but also Acute Lymphoblastic Leukaemia, Mantle Cell Lymphoma and solid malignancies. We have described the first humanised ROR1 CARs, which target novel epitopes and have proved effective in relevant pre-clinical assays. Although other ROR1 CARs have been described, we believe the unique properties of these constructs merits further investigation and comparison in the preclinical and clinical setting. DisclosuresGohil:UCL Business: Patents & Royalties: ROR1 based immunotherapies. Della Peruta:UCL Business: Patents & Royalties: ROR1 based immunotherapies. Paredes-Moscosso:UCL Business: Patents & Royalties: ROR1 based immunotherapies. Pule:Roche: Honoraria; UCL Business: Patents & Royalties; Autolus Ltd: Employment, Equity Ownership, Research Funding; Amgen: Honoraria. Nathwani:UCL Business: Patents & Royalties: ROR1 based Immunotherapies.

Involvement of MyD88 in B-cell mediated immune response in a mouse model of LPS-induced fetal death

Involvement of MyD88 in B-cell mediated immune response in a mouse model of LPS-induced fetal death

Cytokine profiles in multifocal motor neuropathy and progressive muscular atrophy

Multifocal motor neuropathy (MMN) and progressive muscular atrophy (PMA) are associated with IgM monoclonal gammopathy or the presence IgM anti-GM1-antibodies. To further investigate the pathophysiology of MMN and PMA we determined concentrations of 16 mainly B-cell associated inflammatory markers in serum from 25 patients with MMN, 55 patients with PMA, 25 patients with amyotrophic lateral sclerosis (ALS) and 50 healthy controls. Median serum concentrations of the 16 tested cytokines and chemokines were not significantly increased in patients with MMN or patients with PMA, irrespective of the presence of IgM monoclonal gammopathy or high IgM anti-GM1 antibodies. These results argue against a systemic B-cell mediated immune response underlying the pathogenesis of MMN and PMA.

Semaphorin 4A is dynamically regulated during thymocyte development in mice

Semaphorins are important regulators of peripheral T and B-cell mediated immune responses in mice and humans. Modulatory roles of semaphorins in T cell development are also being characterized. We carefully analyzed the gene expression and protein levels of semaphorins 4A, 4D, and 7A at various developmental stages of T cell maturation in the thymus of C57BL/6 mice. Sema7a was expressed at very low levels, while Sema4d was abundant at all developmental stages of mouse thymocytes. We found the most interesting pattern of gene regulation and protein localization for semaphorin 4A. Both semaphorin 4A mRNA and protein were clearly detected on the earliest progenitors and were downregulated through thymic development. SEMA4A protein also showed a distinct cortico-medullary pattern of localization. Our findings contribute to an understanding of the complex roles played by semaphorins in the network of spatially and temporally regulated cues underpinning T cell development in the thymus.

592 Evaluation of Genome-Wide Loci of Iron Metabolism in Hereditary Hemochromatosis Identifies Pcsk7 as a Predictor of Liver Cirrhosis

Background: The etiology of autoimmune diseases is multifactorial but includes a dysregulated self-tolerance and a dysfunctional communication between the sympathetic nervous system (SNS) and the immune system. Regulatory T-cells are required to induce tolerance and prevent autoimmune disease. In cirrhosis, increased SNS-activity may shift cytokineresponse towards Th2-cytokines, suppressing cellular but incrementing humoral, B-cellmediated immune response. These immune modulations, and the peripheral T-cell depletion known to occur in cirrhosis, may support development of autoimmunity. We therefore aimed to investigate whether cirrhosis confers an increased risk of autoimmune disease. Methods: We used the Danish National Patient Registry (NPR) to identify all Danish citizens diagnosed with cirrhosis in 1977-2009. For each of them we sampled five genderand agematched population controls. Competing-risks regression was used to compare the risk of rheumatoid arthritis, multiple sclerosis, or inflammatory bowel disease (identified through NPR) between cirrhosis patients and controls. Results: We included 38,394 cirrhotic patients. During 217,558 total years of follow-up 442 of them developed rheumatoid arthritis (N= 206), multiple sclerosis (N=20), or inflammatory bowel disease (N=216). In the first 10 years after cirrhosis diagnosis their risk did not exceed that of the population controls (Fig. 1), but after that their risk was markedly higher (HR >10 years after cirrhosis was diagnosed). Conclusion: Liver cirrhosis associates with an increased long-term risk for multiple sclerosis, inflammatory bowel disease and/or rheumatoid arthritis. This may indicate a general enhancement in susceptibility for autoimmune phenomena in liver cirrhosis and the underlying mechanisms should be delineated.

544 Liver Cirrhosis Increases Long-Term Risk for Autoimmunity: A Nationwide Population-Based Cohort Study

Background: The etiology of autoimmune diseases is multifactorial but includes a dysregulated self-tolerance and a dysfunctional communication between the sympathetic nervous system (SNS) and the immune system. Regulatory T-cells are required to induce tolerance and prevent autoimmune disease. In cirrhosis, increased SNS-activity may shift cytokineresponse towards Th2-cytokines, suppressing cellular but incrementing humoral, B-cellmediated immune response. These immune modulations, and the peripheral T-cell depletion known to occur in cirrhosis, may support development of autoimmunity. We therefore aimed to investigate whether cirrhosis confers an increased risk of autoimmune disease. Methods: We used the Danish National Patient Registry (NPR) to identify all Danish citizens diagnosed with cirrhosis in 1977-2009. For each of them we sampled five genderand agematched population controls. Competing-risks regression was used to compare the risk of rheumatoid arthritis, multiple sclerosis, or inflammatory bowel disease (identified through NPR) between cirrhosis patients and controls. Results: We included 38,394 cirrhotic patients. During 217,558 total years of follow-up 442 of them developed rheumatoid arthritis (N= 206), multiple sclerosis (N=20), or inflammatory bowel disease (N=216). In the first 10 years after cirrhosis diagnosis their risk did not exceed that of the population controls (Fig. 1), but after that their risk was markedly higher (HR >10 years after cirrhosis was diagnosed). Conclusion: Liver cirrhosis associates with an increased long-term risk for multiple sclerosis, inflammatory bowel disease and/or rheumatoid arthritis. This may indicate a general enhancement in susceptibility for autoimmune phenomena in liver cirrhosis and the underlying mechanisms should be delineated.

P1-01-16: Detecting a Breast Carcinoma-Deriving B-Cell Response: An Immunoproteomics Biomarker Approach.

Abstract Background: Noninvasive biomarkers for the early detection of breast cancer are crucial due to the fact that the relapse risk of breast cancer is rising with the time point of its detection. Currently, none of the reported molecular biomarkers is established for the clinical use as a diagnostic tool. Previous proteomics-based studies showed the immunogenicity of breast carcinoma and the following B-cell mediated immune response. As a result, several autoantibodies against tumor proteins were detected in the sera of breast cancer patients. However, these putative biomarkers are still lacking of clinically reliable specificity and sensitivity, even of better discrimination of cancer patients when combining different biomarkers. The search for a new antibody biomarker signature remains very important as a potential cancer detection tool. For further investigations we analyzed the antibody pattern in serum samples of diseased patients and healthy controls after incubation with whole protein extract from a native carcinoma and identified the putative tumor-specific immunoreactive antigens. Materials and methods: For our de novo profiling of tumor antigens we used a protein extract from a primary invasive ductal carcinoma. Sera from 20 women, of which 19 were diagnosed with breast carcinoma and one with DCIS (CA), and 20 sera from age-matched healthy donors (CTRL) were obtained and pooled separately. For an optimal separation of tumor antigens a two-dimensional sodium dodecylsulfate gel electrophoresis (2D SDS-PAGE) was applied. Following immunoblots with each serum pool were performed and the immunospecific reactions visualized with a horseradish peroxidase-conjugated anti-IgG antibody. The relevant tumor antigens were identified via Matrix-Assisted Laser Desorption Ionization Time of Flight Mass Spectrometry (MALDI TOF-TOF MS). Results: After the incubation of each serum pool with tumor antigens we obtained different extensive antibody profiles, whereas the visualized immunoreactive components varied in the signal intensity. We identified over 10 tumor antigens which reacted with the corresponding autoantibodies in CA or CTRL approach, showing again the complexity of immune response. Besides of already described breast carcinoma related antigens like alpha enolase, which showed immune reaction also with the healthy serum pool, we identified several potential antigens of interest like peroxiredoxin 6 which showed a strong immune response only after the incubation with cancer sera. Discussion: In our study we visualized a complex immune response pattern showing the autoantibody profiles in cancer and healthy sera against tumor-deriving antigens. Also some of identified breast carcinoma antigens were already described; other novel breast carcinoma-related antigens were detected too. Our next step in the intriguing search for cancer antibody biomarkers is the individual screening of sera to confirm the specificity of the tumor-deriving B-cell responses and the validation of these results by using an independent study population. Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr P1-01-16.

Synthesis and binding analysis of unique AG2 pentasaccharide to human Siglec-2 using NMR techniques

Siglec-2 is a mammalian sialic acid binding protein expressed on B-cell surfaces and is involved in the modulation of B-cell mediated immune response. We synthesized a unique starfish ganglioside, AG2 pentasaccharide Gal fβ(1–3)Gal pα(1–4)Neu5Acα(2–3)Gal pβ(1–4)Glc p, and found that the synthetic pentasaccharide binds to human Siglec-2 by performing 1H NMR experiments. Saturation transfer difference NMR experiments indicated that the C7–C9 side-chain and the acetamide moiety of the central sialic acid residue were located in the binding face of human Siglec-2. We determined the binding epitope of AG2 pentasaccharide to human Siglec-2, as the Gal pα(1–4)Neu5Acα(2–3)Gal p unit.

Epitope Design from Transporter Targets in N. gonorrhoeae

Gonococcal infection and transmission is a global health problem and till date no effective vaccine is available to prevent the disease transmission. Recently, analysing membrane proteome of N. gonorrhoeae we have shown that, several membrane associated proteins of the gonococcus might be important in developing anti-gonorrhoea drugs. Here, we explored the possibility of such 19 essential membrane transporter targets those may be useful in developing peptide vaccines too. Using a classical in silico technique, we have identified four best epitopes from three transporters. All identified epitopes are antigenic and predicted to induce both the T- and B-cell mediated immune responses and transmembrane helix prediction shows that, selected epitopes are mostly outside of the membrane that indicates the suitability of these epitopes to be potential peptide vaccine candidates. Identified epitopes require experimental validation.

Disordered regulation of coagulation and platelet activation in xenotransplantation.

Rejection of xenografts is associated with vascular-based inflammation, thrombocytopenia and the consumption of coagulation factors that may evolve into disseminated intravascular coagulation (DIC). Similarly, bone marrow-derived cellular xenotransplantation procedures are associated with endothelial cell activation and thrombotic microangiopathic injury. These complications generally develop despite the best available measures for depletion of xenoreactive natural antibody, inhibition of complement activation and suppression of T- and B-cell mediated immune responses. The mechanisms underlying the DIC and thrombotic microangiopathy associated with xenotransplantation are unclear. A proposed primary biological dysfunction of xenografts with respect to regulation of clotting could amplify vascular injury, promote immunological responses and independently contribute to graft failure. Disordered thromboregulation could have deleterious effects, comparable to unregulated complement activation, in the pathogenesis of xenograft rejection and may therefore represent a substantive barrier to xenotransplantation.