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High-Throughput Sequencing technologies are transforming many research fields, including the analysis of phage display libraries. The phage display technology coupled with deep sequencing was introduced more than a decade ago and holds the potential to circumvent the traditional laborious picking and testing of individual phage rescued clones. However, from a bioinformatics point of view, the analysis of this kind of data was always performed by adapting tools designed for other purposes, thus not considering the noise background typical of the ‘interactome sequencing’ approach and the heterogeneity of the data. InteractomeSeq is a web server allowing data analysis of protein domains (‘domainome’) or epitopes (‘epitome’) from either Eukaryotic or Prokaryotic genomic phage libraries generated and selected by following an Interactome sequencing approach. InteractomeSeq allows users to upload raw sequencing data and to obtain an accurate characterization of domainome/epitome profiles after setting the parameters required to tune the analysis. The release of this tool is relevant for the scientific and clinical community, because InteractomeSeq will fill an existing gap in the field of large-scale biomarkers profiling, reverse vaccinology, and structural/functional studies, thus contributing essential information for gene annotation or antigen identification. InteractomeSeq is freely available at https://InteractomeSeq.ba.itb.cnr.it/

Background Recently published paediatric guidelines for diagnosing coeliac disease do not include recommendations on the follow-up of coeliac disease patients. Goal The aim of this study was to assess the management practices and experience of coeliac disease patients with their follow-up appointments in Central Europe. Study Gastroenterologists and coeliac disease patients in five Central European countries were asked to complete the web-based questionnaire focusing on coeliac disease management practices. Results Answers from 147 gastroenterologists and 2041 coeliac disease patients were available for the analysis. More than half of the gastroenterologists (58.5%) schedule the first follow-up visit within 3 months after the diagnosis. At follow-up, tissue transglutaminase antibodies are checked in almost all patients (95.9%). Approximately two-thirds (60.7%) of gastroenterologists refer all of their patients to the dietitian at diagnosis. Similarly, 42.8% of coeliac disease patients reported that they had not been appointed to a dietitian. Almost one-third of coeliac disease patients (30.8%) reported that they had no follow-up appointments with gastroenterologist at all. Conclusions Follow-up of coeliac disease patients is suboptimal in Central Europe. Many patients are not followed regularly. A lot of patients are not referred to a dietitian. The recommendations on the optimal follow-up of coeliac disease patients are needed in order to improve patient care.

Background: the neoplastic B cells of the Helicobacter pylori-related low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma proliferate in response to H. pylori, however, the nature of the H. pylori antigen responsible for proliferation is still unknown. The purpose of the study was to dissect whether CagY might be the H. pylori antigen able to drive B cell proliferation. Methods: the B cells and the clonal progeny of T cells from the gastric mucosa of five patients with MALT lymphoma were compared with those of T cell clones obtained from five H. pylori–infected patients with chronic gastritis. The T cell clones were assessed for their specificity to H. pylori CagY, cytokine profile and helper function for B cell proliferation. Results: 22 of 158 CD4+ (13.9%) gastric clones from MALT lymphoma and three of 179 CD4+ (1.7%) clones from chronic gastritis recognized CagY. CagY predominantly drives Interferon-gamma (IFN-γ) and Interleukin-17 (IL-17) secretion by gastric CD4+ T cells from H. pylori-infected patients with low-grade gastric MALT lymphoma. All MALT lymphoma-derived clones dose dependently increased their B cell help, whereas clones from chronic gastritis lost helper activity at T-to-B-cell ratios greater than 1. Conclusion: the results obtained indicate that CagY drives both B cell proliferation and T cell activation in gastric MALT lymphomas.

Biologic drugs, especially anti-TNF, are considered as the gold standard therapy in rheumatoid arthritis. However, non-uniform efficacy, incidence of infections, and high costs are major concerns. Novel tissue-specific agents may overcome the current limitations of systemic administration, providing improved potency, and safety. We developed a bispecific antibody (BsAb), combining human arthritic joint targeting, via the synovial-specific single-chain variable fragment (scFv)-A7 antibody, and TNFα neutralization, via the scFv-anti-TNFα of adalimumab, with the binding/blocking capacity comparable to adalimumab -immunoglobulin G (IgG). Tissue-targeting capacity of the BsAb was confirmed on the human arthritic synovium in vitro and in a synovium xenograft Severe combined immune deficient (SCID) mouse model. Peak graft accumulation occurred at 48 h after injection with sustained levels over adalimumab-IgG for 7 days and increased therapeutic effect, efficiently decreasing tissue cellularity, and markers of inflammation with higher potency compared to the standard treatment. This study provides the first description of a BsAb capable of drug delivery, specifically to the disease tissue, and a strong evidence of improved therapeutic effect on the human arthritic synovium, with applications to other existing biologics.

OBJECTIVES: Celiac disease (CD) remains undiagnosed for a long time in many adult and pediatric patients. We assessed the knowledge about CD among healthcare professionals (HCPs) and CD patients in Central Europe (CE). METHODS: HCPs and CD patients from 5 CE countries were asked to complete the web-based questionnaire about CD. The questions were divided into subsections on epidemiology, clinical presentation, diagnostics, treatment, and follow-up. Achieved scores of different specialists managing patients with CD were compared and regional differences in patients' knowledge were analyzed. RESULTS: Questionnaire was completed by 1381 HCPs and 2262 CD patients or their caregivers from Croatia, Hungary, Germany, Italy, and Slovenia. Mean score achieved by HCPs was 50.9%, and by CD patients 56.4%. Pediatric gastroenterologists scored the highest (69.4%; P

Background: the neoplastic B cells of the Helicobacter pylori-related low-grade gastric mucosa-associated lymphoid tissue (MALT) lymphoma proliferate in response to H. pylori, however, the nature of the H. pylori antigen responsible for proliferation is still unknown. The purpose of the study was to dissect whether CagY might be the H. pylori antigen able to drive B cell proliferation. Methods: the B cells and the clonal progeny of T cells from the gastric mucosa of five patients with MALT lymphoma were compared with those of T cell clones obtained from five H. pylori–infected patients with chronic gastritis. The T cell clones were assessed for their specificity to H. pylori CagY, cytokine profile and helper function for B cell proliferation. Results: 22 of 158 CD4+ (13.9%) gastric clones from MALT lymphoma and three of 179 CD4+ (1.7%) clones from chronic gastritis recognized CagY. CagY predominantly drives Interferon-gamma (IFN-γ) and Interleukin-17 (IL-17) secretion by gastric CD4+ T cells from H. pylori-infected patients with low-grade gastric MALT lymphoma. All MALT lymphoma-derived clones dose dependently increased their B cell help, whereas clones from chronic gastritis lost helper activity at T-to-B-cell ratios greater than 1. Conclusion: the results obtained indicate that CagY drives both B cell proliferation and T cell activation in gastric MALT lymphomas.

OBJECTIVES: During the past decades, there has been a shift in the clinical presentation of coeliac disease (CD) to nonclassical, oligosymptomatic, and asymptomatic forms. We assessed clinical presentation of CD in children and adolescents in Central Europe. METHODS: Paediatric gastroenterologists in 5 countries retrospectively reported data of their patients diagnosed with CD. Clinical presentation was analyzed and the differences among very young (

Background: Dermatitis Herpetiformis (DH) diagnosis relies on direct immunofluorescence (DIF) on skin biopsy. Serology for anti-epidermal transglutaminase antibodies (Anti-TG3 Abs) has attracted interest, being inexpensive and less invasive. However, the available test has variable sensitivity and low specificity in Coeliac Disease (CD) patients without DH. Objective: To evaluate the diagnostic accuracy and reproducibility of a novel quantitative ELISA for the measurement of Anti-TG3 Abs. Methods: ELISA was set-up with recombinant TG3 and a standard curve based on a human recombinant monoclonal IgA anti-TG3. Sera of 57 DH patients, 212 CD patients, 120 patients with non-gluten related gastrointestinal diseases (CTRL) and 168 healthy controls (HC) were tested. Results: The test was reproducible with high sensitivity (91%). Test specificity was high when considering HC (99%) as well as CTRL (98%). Interestingly in CD patients test specificity was agedependent and was higher (97%) in patients ≤ 25 years of age. Limitations: Not all DH were confirmed by DIF. Conclusion: The ELISA test could be used as a screening tool when DH is clinically suspected and may serve as a sole diagnostic test in younger patients. The quantification of Anti-TG3 Abs levels will facilitate the comparison of results from different laboratories.

Biofilms are aggregates of microbial cells encased in a highly hydrated matrix made up of self-produced extracellular polymeric substances (EPS) which consist of polysaccharides, proteins, nucleic acids, and lipids. While biofilm matrix polysaccharides are unraveled, there is still poor knowledge about the identity and function of matrix-associated proteins. With this work, we performed a comprehensive proteomic approach to disclose the identity of proteins associated with the matrix of biofilm-growing Burkholderia multivorans C1576 reference strain, a cystic fibrosis clinical isolate. Transmission electron microscopy showed that B. multivorans C1576 also releases outer membrane vesicles (OMVs) in the biofilm matrix, as already demonstrated for other Gram-negative species. The proteomic analysis revealed that cytoplasmic and membrane-bound proteins are widely represented in the matrix, while OMVs are highly enriched in outer membrane proteins and siderophores. Our data suggest that cell lysis and OMVs production are the most important sources of proteins for the B. multivorans C1576 biofilm matrix. Of note, some of the identified proteins are lytic enzymes, siderophores, and proteins involved in reactive oxygen species (ROS) scavenging. These proteins might help B. multivorans C1576 in host tissue invasion and defense towards immune system assaults.

West Nile virus is a widespread mosquito-borne human and animal pathogenic virus of increasing importance. The E protein of the viral envelope is critical for attachment and entry into the host cell and has been the target for vaccine design and small molecule inhibitors. Furthermore, the detection of anti-E IgM and IgG antibodies is widely used in serology to diagnose these infections. Here we describe a strategy for the production of recombinant antibodies against the E protein of West Nile virus for research and immunodiagnostic purposes. Initially the fast and easy protocol previously developed for the similar Tick-borne encephalitis virus has been adapted to West Nile virus E antigen production and purification. A human naive scFv phage library has been selected on the produced antigen, identifying a panel of highly specific anti-E protein antibodies. Once produced as scFv-Fc recombinant proteins, the selected antibodies have been characterized by mapping their binding sites and by defining their affinity for the target. The impact on neutralizing virus attachment and entry has been also evaluated. The obtained results demonstrate the potential of the produced reagents for research and diagnostic applications.

Objectives. The current European Society for Paediatric Gastroenterology, Hepatology, and Nutrition (ESPGHAN) guidelines introduced the option to diagnose coeliac disease (CD) in children and adolescents without upper endoscopy if the defined criteria are met. The aim of our study was to evaluate how frequently paediatric gastroenterologists in Central Europe used the “no-biopsy” approach and how often the duodenal biopsy could have been omitted. Methods. Medical records of patients aged<19 years diagnosed with CD in 2016 from five European countries were analysed, focusing on levels of transglutaminase antibodies (TGA) at the time of diagnosis and on whether the diagnosis was confirmed using duodenal biopsy or “no-biopsy” approach. Clinical presentation and delays until final diagnosis were analysed according to diagnostic approach. Results. Data from 653 children (63.9% female, median age: 7 years, range: 7 months-18.5 years) from Croatia, Hungary, Germany, Italy, and Slovenia were analysed. One fifth (n=134) of included children were asymptomatic at diagnosis. Of 519 symptomatic children, 107 (20.6%) were diagnosed by the “no-biopsy” approach. Out of the remaining 412 children who underwent duodenal biopsies, 214 (51.9%) had TGA≥10 times upper level of normal (ULN) and would have been eligible for the “no-biopsy” approach. Signs and symptoms of malabsorption were more frequent in children diagnosed without duodenal biopsies. There were no differences in diagnostic delays with respect to the diagnostic approach. Conclusion. In this cohort, about 60% of symptomatic CD patients could have been diagnosed without duodenal biopsies. The aim of the “no-biopsy” approach was to make the diagnostic procedure less challenging without compromising its reliability. However, this option was applied only in 20%, in spite of fewer burdens to the family and reduced costs. The reasons for this discrepancy are unknown. Physicians should be made more aware about the reliability of CD diagnosis without biopsies when the ESPGHAN guidelines for CD diagnosis are followed.

Transposable elements (TEs) compose about half of the mammalian genome and, as embedded sequences, up to 40% of long noncoding RNA (lncRNA) transcripts. Embedded TEs may represent functional domains within lncRNAs, providing a structured RNA platform for protein interaction. Here we show the interactome profile of the mouse inverted short interspersed nuclear element (SINE) of subfamily B2 (invSINEB2) alone and embedded in antisense (AS) ubiquitin C-terminal hydrolase L1 (Uchl1), an lncRNA that is AS to Uchl1 gene. AS Uchl1 is the representative member of a functional class of AS lncRNAs, named SINEUPs, in which the invSINEB2 acts as effector domain (ED)-enhancing translation of sense protein-coding mRNAs. By using RNA-interacting domainome technology, we identify the IL enhancer-binding factor 3 (ILF3) as a protein partner of AS Uchl1 RNA. We determine that this interaction is mediated by the RNA-binding motif 2 of ILF3 and the invSINEB2. Furthermore, we show that ILF3 is able to bind a free right Arthrobacter luteus (Alu) monomer sequence, the embedded TE acting as ED in human SINEUPs. Bioinformatic analysis of Encyclopedia of DNA Elements-enhanced cross-linking immunoprecipitation data reveals that ILF3 binds transcribed human SINE sequences at transcriptome-wide levels. We then demonstrate that the embedded TEs modulate AS Uchl1 RNA nuclear localization to an extent moderately influenced by ILF3. This work unveils the existence of a specific interaction between embedded TEs and an RNA-binding protein, strengthening the model of TEs as functional modules in lncRNAs.-Fasolo, F., Patrucco, L., Volpe, M., Bon, C., Peano, C., Mignone, F., Carninci, P., Persichetti, F., Santoro, C., Zucchelli, S., Sblattero, D., Sanges, R., Cotella, D., Gustincich, S. The RNA-binding protein ILF3 binds to transposable element sequences in SINEUP lncRNAs.

Despite the advances in the treatment of rheumatoid arthritis (RA) achieved in the last few years, several patients are diagnosed late, do not respond to or have to stop therapy because of inefficacy and/or toxicity, leaving still a huge unmet need. Tissue-specific strategies have the potential to address some of these issues. The aim of the study is the development of a safe nanotechnology approach for tissue-specific delivery of drugs and diagnostic probes. CD34

Tick-borne encephalitis virus (TBEV) is a member of the Flavivirus genus and is the main pathogenic arbovirus circulating in Europe, Russia and China. The envelope (E) protein is exposed on the viral surface and is the main antigen that is employed in diagnostic tests based on the detection of protein-specific antibodies from serum samples of infected individuals. The high degree of similarity among the E proteins of flaviviruses can, in some cases, lead to cross-reactivity and false-positive results in serological tests. Increased specificity in the detection of positive sera for different Flavivirus infections is often obtained by using a portion of the E protein, namely, the DIII domain. Different strategies and expression systems have been described for E and DIII protein production. Here, we present the optimization of an easy and fast method for TBEV E and DIII antigen production and partial purification from E. coli inclusion bodies. The antigenic properties of the produced antigens are retained, as validated by ELISAs with anti-TBEV murine sera as well as sera from infected human patients. The potential applications of both proteins as diagnostic reagents were confirmed.

Despite the advances in the treatment of rheumatoid arthritis (RA) achieved in the last few years, several patients are diagnosed late, do not respond to or have to stop therapy because of inefficacy and/or toxicity, leaving still a huge unmet need. Tissue-specific strategies have the potential to address some of these issues. The aim of the study is the development of a safe nanotechnology approach for tissue-specific delivery of drugs and diagnostic probes. CD34 + endothelial precursors were addressed in inflamed synovium using targeted biodegradable nanoparticles (tBNPs). These nanostructures were made of poly-lactic acid, poly-caprolactone, and PEG and then coated with a synovial homing peptide. Immunofluorescence analysis clearly demonstrated their capacity to selectively address CD34 + endothelial cells in synovial tissue obtained from human, mouse, and rat. Biodistribution studies in two different animal models of rheumatoid arthritis (antigen-induced arthritis/AIA and collagen-induced arthritis/CIA) confirmed the selective accumulation in inflamed joints but also evidenced the capacity of tBNP to detect early phases of the disease and the preferential liver elimination. The therapeutic effect of methotrexate (MTX)-loaded tBNPs were studied in comparison with conventional MTX doses. MTX-loaded tBNPs prevented and treated CIA and AIA at a lower dose and reduced administration frequency than MTX. Moreover, MTX-loaded tBNP showed a novel mechanism of action, in which the particles target and kill CD34 + endothelial progenitors, preventing neo-angiogenesis and, consequently, synovial inflammation. tBNPs represent a stable and safe platform to develop highly-sensitive imaging and therapeutic approaches in RA targeting specifically synovial neo-angiogenesis to reduce local inflammation.

Objectives: Coeliac disease (CD) is a systemic autoimmune disorder affecting about 1% of the population. Many patients remain undiagnosed or are diagnosed with substantial delay. We assessed diagnostic delays in symptomatic CD children in Central Europe (CE). Methods: Paediatric gastroenterologists in 5 CE countries retrospectively reported data of their patients diagnosed in 2016. Age at first CD-related symptom(s), first visit to paediatric gastroenterologist and confirmed diagnosis were used to determine diagnostic delays. Results: Data from 393 children (65% girls, median age 7 years, range 7 months to 18.5 years) from Croatia, Hungary, Germany, Italy, and Slovenia were analysed. Median duration from first symptom(s) to visit to paediatric gastroenterologist was 5 months (range 0–10 years ; preschool 4 months, school-aged 5 months), and further duration until final diagnosis was 1 month (range 0–5 years) with significant regional differences (P < 0.001). Median diagnostic delay was 6 months (range 0–10 years ; preschool 5 months, school-aged 7 months). Type of clinical presentation had little, however, significant effect on delays. Reduced body mass in delays longer than 3 years compared with delays shorter than 1 year was found (z score −0.93 vs −0.39, P < 0.05). Conclusions: Time from first symptoms to CD diagnosis in children in 5 CE countries is slightly shorter compared with few other small paediatric studies, and significantly shorter than reported for adults. Nevertheless, delays of more than 3 years in 6.6% of children are worrisome. Raising awareness about the variable symptoms and implementation of reliable diagnostic tools will further reduce diagnostic delays

During the last 20 years in vitro technologies opened powerful routes to combine the generation of large libraries together with fast selection and screening procedures to identify lead candidates. One of the most successful methods is based on the use of filamentous phages. Functional Antibodies (Abs) fragments can be displayed on the surface of phages by fusing the coding sequence of the antibody variable (V) regions to the phage minor coat protein pIII. By creating large libraries, antibodies with affinities comparable to those obtained using traditional hybridoma technology can be isolated by a series of cycles of selection on the antigen of interest. In this system, antibody genes can be recovered simultaneously with selection and can be easily further engineered, for example by increasing their affinity to levels unobtainable in the immune system, or by modulating their specificity and their effector functions (by recloning into a full-length immunoglobulin scaffold). This chapter describes the basic protocols for antibody library construction and selection of binder with desired specificity.

During the last 20 years in vitro technologies opened powerful routes to combine the generation of large libraries together with fast selection and screening procedures to identify lead candidates. One of the most successful methods is based on the use of filamentous phages. Functional Antibodies (Abs) fragments can be displayed on the surface of phages by fusing the coding sequence of the antibody variable (V) regions to the phage minor coat protein pIII. By creating large libraries, antibodies with affinities comparable to those obtained using traditional hybridoma technology can be isolated by a series of cycles of selection on the antigen of interest. In this system, antibody genes can be recovered simultaneously with selection and can be easily further engineered, for example by increasing their affinity to levels unobtainable in the immune system, or by modulating their specificity and their effector functions (by recloning into a full-length immunoglobulin scaffold). This chapter describes the basic protocols for antibody library construction and selection of binder with desired specificity.

In vitro display technologies have put together the generation of large antibody libraries with selection and screening procedures to identify lead candidates. Phage display antibody libraries allow selecting and identifying binders for a variety of antigens. Nonetheless, the procedure is limited by the possibility to quantitatively follow the enrichment during selection cycles and tune up the clones for specific binding proprieties (i.e., affinity). Yeast display allows the expression of thousands of copies of the antibody on each cell, simultaneously carrying the plasmid encoding that antibody, moreover the selection parameters can be accurately controlled by flow cytometry-based analysis and sorting.The combination of phage and yeast display takes advantage of both platforms by starting with a vast number of antibodies in the phage display selections followed by the precise sorting of the clones specifically recognizing the target of interest.In the present chapter, we illustrate protocols to generate and enrich - using fluorescence-activated cell sorting (FACS) - yeast display antibody libraries, using selection outputs obtained from phage antibody display libraries as starting material. The present methods can be easily applicable for the identification of monoclonal antibodies with desired binding properties.

n vitro display technologies have put together the generation of large antibody libraries with selection and screening procedures to identify lead candidates. Phage display antibody libraries allow selecting and identifying binders for a variety of antigens. Nonetheless, the procedure is limited by the possibility to quantitatively follow the enrichment during selection cycles and tune up the clones for specific binding proprieties (i.e., affinity). Yeast display allows the expression of thousands of copies of the antibody on each cell, simultaneously carrying the plasmid encoding that antibody, moreover the selection parameters can be accurately controlled by flow cytometry-based analysis and sorting. The combination of phage and yeast display takes advantage of both platforms by starting with a vast number of antibodies in the phage display selections followed by the precise sorting of the clones specifically recognizing the target of interest. In the present chapter, we illustrate protocols to generate and enrich - using fluorescence-activated cell sorting (FACS) - yeast display antibody libraries, using selection outputs obtained from phage antibody display libraries as starting material. The present methods can be easily applicable for the identification of monoclonal antibodies with desired binding properties.

An unbalance between Abs that recognize an autoantigen (idiotypes; IDs) and Igs that bind such Abs (anti-IDs) is considered a functional event in autoimmune disorders. We investigated the presence of an ID/anti-ID network in celiac disease (CD), a condition in which antitissue transglutaminase 2 (TG2) Abs are suspected to contribute to CD pathogenesis. To characterize the ID side, we reproduced by in vitro yeast display the intestine-resident Abs from CD and control patients. These TG2-specific IDs were used to identify potential anti-IDs in the serum. We observed elevated titers of anti-IDs in asymptomatic patients with predisposition to CD and demonstrated that anti-ID depletion from the serum restores a detectable humoral response against TG2. Our study provides an alternative approach to quantify CD-related autoantibodies in cases that would be defined “negative serology” with current diagnostic applications. Therefore, we suggest that developments of this technology could be designed for perspective routine tests.

The interaction between the enzyme transglutaminase 2 (TG2) and fibronectin (FN) is involved in the cell-matrix interactions that regulate cell signaling, adhesion, and migration and play central roles in pathologic conditions, particularly fibrosis and cancer. A precise definition of the exact interaction domains on both proteins could provide a tool to design novel molecules with potential therapeutic applications. Although specific residues involved in the interaction within TG2 have been analyzed, little is known regarding the TG2 binding site on FN. This site has been mapped to a large internal 45-kDa protein fragment coincident with the gelatin binding domain (GBD). With the goal of defining the minimal FN interacting domain for TG2, we produced several expression constructs encoding different portions or modules of the GBD and tested their binding and functional properties. The results demonstrate that the I8 module is necessary and sufficient for TG2-binding in vitro, but does not have functional effects on TG2-expressing cells. Modules I7 and I9 increase the strength of the binding and are required for cell adhesion. A 15-kDa fragment encompassing modules I7-9 behaves as the whole 45-kDa GBD and mediates signaling, adhesion, spreading, and migration of TG2+ cells. This study provides new insights into the mechanism for TG2 binding to FN.-Soluri, M. F., Boccafoschi, F., Cotella, D., Moro, L., Forestieri, G., Autiero, I., Cavallo, L., Oliva, R., Griffin, M., Wang, Z., Santoro, C., Sblattero, D. Mapping the minimum domain of the fibronectin binding site on transglutaminase 2 (TG2) and its importance in mediating signaling, adhesion, and migration in TG2-expressing cells.

Folding reporters are proteins with easily identifiable phenotypes, such as antibiotic resistance, whose folding and function is compromised when fused to poorly folding proteins or random open reading frames. We have developed a strategy where, by using TEM-1 β-lactamase (the enzyme conferring ampicillin resistance) on a genomic scale, we can select collections of correctly folded protein domains from the coding portion of the DNA of any intronless genome. The protein fragments obtained by this approach, the so called "domainome", will be well expressed and soluble, making them suitable for structural/functional studies. By cloning and displaying the "domainome" directly in a phage display system, we have showed that it is possible to select specific protein domains with the desired binding properties (e.g., to other proteins or to antibodies), thus providing essential experimental information for gene annotation or antigen identification. The identification of the most enriched clones in a selected polyclonal population can be achieved by using novel next-generation sequencing technologies (NGS). For these reasons, we introduce deep sequencing analysis of the library itself and the selection outputs to provide complete information on diversity, abundance and precise mapping of each of the selected fragment. The protocols presented here show the key steps for library construction, characterization, and validation.

Coeliac disease is hallmarked by an abnormal immune reaction against ingested wheat-, rye- and barley-derived gluten and the presence of transglutaminase 2 (TG2)-targeted autoantibodies. The small-bowel mucosal damage characteristic of the disorder develops gradually from normal villus morphology to inflammation and finally to villus atrophy with crypt hyperplasia. Patients with early-stage coeliac disease have TG2-autoantibodies present in serum and small-intestinal mucosa and they may already suffer from abdominal symptoms before the development of villus atrophy. Previously, we have shown that intraperitoneal injections of coeliac patient-derived sera or purified immunoglobulin fraction into mice induce a condition mimicking early-stage coeliac disease. In the current study, we sought to establish whether recombinantly produced patient-derived TG2-targeted autoantibodies are by themselves sufficient for the development of such an experimentally induced condition in immune-compromised mice. Interestingly, mice injected with coeliac patient TG2-antibodies had altered small-intestinal mucosal morphology, increased lamina propria cellular infiltration and disease-specific autoantibodies deposited in the small bowel, but did not evince clinical features of the disease. Thus, coeliac patient-derived TG2-specific autoantibodies seem to be sufficient for the induction of subtle small-bowel mucosal alterations in mice, but the development of clinical features probably requires additional factors such as other antibody populations relevant in coeliac disease.

Osteopontin (OPN) is highly expressed in demyelinating lesions in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE). OPN is cleaved by thrombin into N- (OPN-N) and C-terminal (OPN-C) fragments with different ligands and functions. In EAE, administering recombinant OPN induces relapses, whereas treatment with anti-OPN antibodies ameliorates the disease. Anti-OPN auto-antibodies (autoAbs) are spontaneously produced during EAE but have never been detected in MS. The aim of the study was to evaluate anti-OPN autoAbs in the serum of MS patients, correlate them with disease course, and recapitulate the human findings in EAE. We performed ELISA in the serum of 122 patients collected cross-sectionally, and 50 patients with relapsing-remitting (RR) disease collected at diagnosis and followed longitudinally for 10 years. In the cross-sectional patients, the autoAb levels were higher in the RR patients than in the primary and secondary progressive MS and healthy control groups, and they were highest in the initial stages of the disease. In the longitudinal group, the levels at diagnosis directly correlated with the number of relapses during the following 10 years. Moreover, in patients with active disease, who underwent disease-modifying treatments, autoAbs were higher than in untreated patients and were associated with low MS severity score. The autoAb displayed neutralizing activity and mainly recognized OPN-C rather than OPN-N. To confirm the clinical effect of these autoAbs in vivo, EAE was induced using myelin oligodendrocyte glycoprotein MOG35-55 in C57BL/6 mice pre-vaccinated with ovalbumin (OVA)-linked OPN or OVA alone. We then evaluated the titer of antibodies to OPN, the clinical scores and in vitro cytokine secretion by spleen lymphocytes. Vaccination significantly induced antibodies against OPN during EAE, decreased disease severity, and the protective effect was correlated with decreased T cell secretion of interleukin 17 and interferon -γ ex vivo. The best effect was obtained with OPN-C, which induced significantly faster and more complete remission than other OPN vaccines. In conclusion, these data suggest that production of anti-OPN autoAbs may favor remission in both MS and EAE. Novel strategies boosting their levels, such as vaccination or passive immunization, may be proposed as a future strategy in personalized MS therapy.

Type 2 transglutaminase (TG2) has an important pathogenic role in celiac disease (CD), an inflammatory intestinal disease that is caused by the ingestion of gluten-containing cereals. Indeed, TG2 deamidates specific gliadin peptides, thus enhancing their immunogenicity. Moreover, the transamidating activity seems to provoke an autoimmune response, where TG2 is the main autoantigen. Many studies have highlighted a possible pathogenetic role of anti-TG2 antibodies, because they modulate TG2 enzymatic activity and they can interact with cell-surface TG2, triggering a wide range of intracellular responses. Autoantibodies also alter the uptake of the alpha-gliadin peptide 31-43 (p31-43), responsible of the innate immune response in CD, thus partially protecting cells from p31-43 damaging effects in an intestinal cell line. Here, we investigated whether anti-TG2 antibodies protect cells from p31-43-induced damage in a CD model consisting of primary dermal fibroblasts. We found that the antibodies specifically reduced the uptake of p31-43 by fibroblasts derived from healthy subjects but not in those derived from CD patients. Analyses of TG2 expression and enzymatic activity did not reveal any significant difference between fibroblasts from healthy and celiac subjects, suggesting that other features related to TG2 may be responsible of such different behaviors, e.g., trafficking or subcellular distribution. Our findings are in line with the concept that a "celiac cellular phenotype" exists and that TG2 may contribute to this phenotype. Moreover, they suggest that the autoimmune response to TG2, which alone may damage the celiac mucosa, also fails in its protective role in celiac cells.

Several serological diagnostics rely on enzyme-linked immunosorbent assay (ELISA) to detect bacterial infections. However, for some pathogens, including Bartonella henselae, diagnosis still depends on manually intensive, time-consuming assays including micro-immunofluorescence, Western blotting or indirect immunofluorescence. For such pathogens, there is obviously still a need to identify antigens to establish a reliable, fast and high-throughput assay (Dupon et al. 8). We evaluated two B. henselae proteins to develop a novel serological ELISA: a well-known antigen, the 17-kDa protein, and GroEL, identified during this study by a proteomic approach. When serum IgG were tested, the specificity and sensitivity were 76 and 65·7% for 17-kDa, respectively, and 82 and 42·9% for GroEL, respectively. IgM were found to be more sensitive and specific for both proteins: 17-kDa protein, specificity 86·2% and sensitivity 75%; GroEL, specificity 97·7% and sensitivity 45·3%. IgM antibodies were also measured in lymphoma patients and patients with Mycobacterium tuberculosis infection to assess the usefulness of our ELISA to distinguish them from B. henselae infected patients. The resulting specificities were 89·1 and 93·5% for 17-kDa protein and GroEL, respectively. Combining the results from the two tests, we obtained a sensitivity of 82·8% and a specificity of 83·9%. Our work described and validated a proteomic approach suitable to identify immunogenic proteins useful for developing a serological test of B. henselae infection. Significance and Impact of the Study A reliable serological assay for the diagnosis of Cat Scratch Disease (CSD) – a pathological condition caused by Bartonella henselae infection – has not yet been developed. Such an assay would be extremely useful to discriminate between CSD and other pathologies with similar symptoms but different aetiologies, for example lymphoma or tuberculosis. We investigate the use of two B. henselae proteins – GroEL and 17-kDa – to develop a serological-based ELISA, showing promising results with the potential for further development as an effective tool for the differential diagnosing of B. henselae infection.

The identification of effective biomarkers for early diagnosis, prognosis, and response to treatments remains a challenge in ovarian cancer (OC) research. Here, we present an unbiased high-throughput approach to profile ascitic fluid autoantibodies in order to obtain a tumor-specific antigen signature in OC. We first reported the reactivity of immunoglobulins (Igs) purified from OC patient ascites towards two different OC cell lines. Using a discovery set of Igs, we selected tumor-specific antigens from a phage display cDNA library. After biopanning, 700 proteins were expressed as fusion protein and used in protein array to enable large-scale immunoscreening with independent sets of cancer and noncancerous control. Finally, the selected antigens were validated by ELISA. The initial screening identified eight antigenic clones: CREB3, MRPL46, EXOSC10, BCOR, HMGN2, HIP1R, OLFM4, and KIAA1755. These antigens were all validated by ELISA in a study involving ascitic Igs from 153 patients (69 with OC, 34 with other cancers and 50 without cancer), with CREB3 showing the highest sensitivity (86.95%) and specificity (98%). Notably, we were able to identify an association between the tumor-associated (TA) antibody response and the response to a first-line tumor treatment (platinum-based chemotherapy). A stronger association was found by combining three antigens (BCOR, CREB3, and MRLP46) as a single antibody signature. Measurement of an ascitic fluid antibody response to multiple TA antigens may aid in the identification of new prognostic signatures in OC patients and shift attention to new potentially relevant targets.

Malignant pleural mesothelioma (MPM) is an aggressive tumor linked to environmental/occupational exposure to asbestos, characterized by the presence of significant areas of hypoxia. In this study, we firstly explored the expression and the role of transglutaminase 2 (TG2) in MPM cell adaptation to hypoxia. We demonstrated that cells derived from biphasic MPM express the full-length TG2 variant at higher levels than cells derived from epithelioid MPM and normal mesothelium. We observed a significant induction of TG2 expression and activity when cells from biphasic MPM were grown as a monolayer in chronic hypoxia or packed in spheroids, where the presence of a hypoxic core was demonstrated. We described that the hypoxic induction of TG2 was HIF-2 dependent. Importantly, TGM2-v1 silencing caused a marked and significant reduction of MPM cell viability in hypoxic conditions when compared with normoxia. Notably, a TG2-selective irreversible inhibitor that reacts with the intracellular active form of TG2, but not a non-cell-permeable inhibitor, significantly compromised cell viability in MPM spheroids. Understanding the expression and function of TG2 in the adaptation to the hypoxic environment may provide useful information for novel promising therapeutic options for MPM treatment.

Osteoblasts, osteocytes, and osteoclasts (OCs) are involved in the bone production and resorption, which are crucial in bone homeostasis. OC hyperactivation plays a role in the exaggerated bone resorption of diseases such as osteoporosis, rheumatoid arthritis, and osteolytic tumor metastases. This work stems from the finding that OCs can express B7h (ICOS-Ligand), which is the ligand of the ICOS T cell costimulatory molecule. Because recent reports have shown that, in endothelial, dendritic, and tumor cells, B7h triggering modulates several activities of these cells, we analyzed the effect of B7h triggering by recombinant ICOS-Fc on OC differentiation and function. The results showed that ICOS-Fc inhibits RANKL-mediated differentiation of human monocyte-derived OC-like cells (MDOCs) by inhibiting the acquirement of the OC morphology, the CD14− cathepsin K+ phenotype, and the expression of tartrate-resistant acid phosphatase, OSCAR, NFATc1, and DC-STAMP. Moreover, ICOS-Fc induces a reversible decrease in the sizes of cells and nuclei and cathepsin K expression in mature MDOCs. Finally, ICOS-Fc inhibits the osteolytic activities of MDOCs in vitro and the development of bone loss in ovariectomized or soluble RANKL-treated mice. These findings open a novel field in the pharmacological use of agonists and antagonists of the ICOS–B7h system.

Background TNF-α neutralizing molecules represent one of the most efficient therapeutic approaches to control inflammation in rheumatoid arthritis (RA). The widespread distribution in the body induces the inhibition of TNF-α in all the tissues, requesting the use of high dose of this expensive drug. Another problem that has not yet been solved in the management of RA patients is how to reduce and possibly avoid the side effects, particularly the increased risk of common and opportunistic infections, which may be associated with long-term administration of these therapeutic drugs. Objectives The aim of the present investigation was to show that a recombinant protein obtained by fusing a synovial targeting peptide to an anti-TNF-α neutralizing antibody has a distinctive homing property for inflamed synovium and is effective in controlling joint inflammation in experimental models of arthritis. Methods The modified anti-TNF-α antibody specific for the synovial tissue (MC13) was generated by cloning the sequence for a synovial homing peptide (CKSTHDRLC) downstream of the cDNA for the variable regions of anti- TNF-α antibody adalimumab in the context of a miniantibody (scFv-Hinge-CH2-CH3 domains). Immunofluorescence technique was used to initially assess the ability of fluorescent-MC13 to target only inflamed synovial tissue. The biodistribution and the efficacy of the treatment with MC13 were evaluated in a rat model of antigen-induced arthritis (AIA). Results In vitro studies have shown that adalimumab and MC13 maintain the ability to neutralize human TNF-α and bind to rat and mouse TNF-α. Based on these findings, we used a rat model of arthritis to evaluate their in vivo therapeutic effect. The ability of MC13 to selectively target inflamed synovial tissues was evaluated by immunofluorescence analysis. A rat model of antigen-induced mono-arthritis was used to analyse the in vivo distribution of MC13 labelled with the near-infrared probe cyanine 5.5 using time-domain near infrared optical imaging. The results showed that the labeled-MC13 preferentially localized in the inflamed synovium with a peak at 4 days and persisted for more than 3 weeks, while failing to bind to healthy synovial tissue. MC13 was able to prevent inflammation in the rat model of antigen-induced arthritis, as evaluated by the measurement of joint swelling, the count of PMN number and the level of IL6 in synovial tissue and the histologic assessment of tissue damage. Intravenous injection of MC13 prevents acute inflammation in all the animals while the same dose of adalimumab was almost ineffective. MC13, administred i.v. 4 days after arthritis induction, was also able to cure joints analysed 7 days after the induction of inflammation. Conclusions Our results demonstrated that MC13 can selectively target inflamed and abrogate the inflammation in a model of antigen-induced arthritis with no sign of toxicity. Disclosure of Interest None declared

Background Rheumatoid arthritis (RA) is a chronic, systemic autoimmune disease affecting joints due to the persistent synovial tissue inflammation. RA treatment has dramatically evolved in the last 20 years due to the production of biological Disease Modifying Antirheumatic Drugs (bDMARDs). However, side effects and the high costs of biological drugs are holding back their widespread usage. Moreover, some patients fail to respond to bDMARDs, for all of these reasons, DMARDs remains the main desired strategy for the treatment of RA, and Methotrexate (MTX) is still the “anchor” drug to treat RA. A successful treatment depends also on the early diagnosis, treating patients as soon as possible to prevent the late stages of the pathology. Objectives The aim of this work is to develop targeted biodegradable nanoparticles (tBNPs) that could be used as a platform suitable to delivery drugs or diagnostic tracers specifically into the inflamed synovial tissue. This approach usually allows to enhance the efficacy and to reduce the side effects. Methods We used polymeric biodegradable nanoparticles, made of polylactic acid, polycaprolactone and polyethylene glycol and coated with a peptide characterized for its ability to target only inflamed synovial tissue. The tBNPs diameter was 170 nm with a low negative charge. Toxicity was studied in-vitro assay and in-vivo. Immunofluorescence technique was used to initially assess the ability of fluorescent-tBNPs to preferentially target inflamed synovial tissue. The biodistribution of the nanoparticles and the efficacy of the treatment with tBNPs loaded with MTX were studied in a rat model of antigen-induced arthritis (AIA). Finally, tBNPs efficacy was also studied in a mouse model of collagen-induced arthritis (CIA). Results Targeted BNPs evidenced the ability to specifically bind inflamed synovial tissue both in vitro and in vivo. The targeting is dependent on the inflammation degree and this highlights the potentiality of the tBNPs as an early diagnostics tools. A single injection of targeted BNPs loaded with MTX completely abrogated the inflammatory process in acute AIA rat model while the same dose of free-MTX was ineffective. Equivalent therapeutic effects were obtained comparing MTX-loaded tBNPs and the same dose of free-MTX in the mouse model of chronic CIA and no toxic effect was documented when MTX was loaded in nanoparticles in these animals. Conclusions Our results demonstrated that tBNPs can efficiently and selectively delivery drugs and diagnostic probes to inflamed synovial tissue, providing a new platform for an early detection and efficient chronic treatment of inflammatory arthritis, with minimal side effects. Disclosure of Interest F. Colombo: None declared, L. Nunez Employee of: Biotarget, P. Durigutto: None declared, S. Biffi: None declared, E. Rampazzo: None declared, B. Belmonte: None declared, D. Sblattero: None declared, A. Gulino: None declared, P. L. Meroni: None declared, F. Tedesco: None declared, P. Macor: None declared

// Valentina Griggio 1, 2 , Giorgia Mandili 2, 3 , Candida Vitale 1, 2 , Michela Capello 2, 3 , Paolo Macor 4 , Sara Serra 5 , Barbara Castella 1, 2, 3 , Silvia Peola 2, 3 , Myriam Foglietta 1, 2, 3 , Daniela Drandi 1, 2 , Paola Omede 1 , Daniele Sblattero 4 , Paola Cappello 2, 3, 6 , Roberto Chiarle 2, 3, 7 , Silvia Deaglio 5 , Mario Boccadoro 1, 2 , Francesco Novelli 2, 3, 6, 8 , Massimo Massaia 1, 2, 3 , Marta Coscia 1, 2 1 Division of Hematology, University of Torino, AOU Citta della Salute e della Scienza di Torino, Torino, Italy 2 Department of Molecular Biotechnology and Health Sciences, University of Torino, Torino, Italy 3 Center for Experimental Research and Medical Studies (CeRMS), AOU Citta della Salute e della Scienza di Torino, Torino, Italy 4 Department of Life Sciences – University of Trieste, Trieste, Italy 5 Department of Medical Sciences, University of Torino and Immunogenetics Unit - Human Genetics Foundation (HuGeF), Torino, Italy 6 Molecular Biotechnology Center, Torino, Italy 7 Department of Pathology, Boston Children’s Hospital, Boston, Massachusetts, USA 8 Service of Immunogenetics and Transplantation, AOU Citta della Salute e della Scienza di Torino, Torino, Italy Correspondence to: Marta Coscia, email: marta.coscia@unito.it Keywords: chronic lymphocytic leukemia, humoral responses, tumor antigens, serological proteomics, alpha-enolase Received: July 13, 2016 Accepted: November 22, 2016 Published: November 30, 2016 ABSTRACT In chronic lymphocytic leukemia (CLL) the occurrence and the impact of antibody responses toward tumor-derived antigens are largely unexplored. Our serological proteomic data show that antibodies toward 47 identified antigens are detectable in 29 out of 35 patients (83%) with untreated CLL. The glycolytic enzyme alpha-enolase (ENO1) is the most frequently recognized antigen (i.e. 54% of CLL sera). We show that ENO1 is upregulated in the proliferating B-cell fraction of CLL lymph nodes. In CLL cells of the peripheral blood, ENO1 is exclusively expressed at the intracellular level, whereas it is exposed on the surface of apoptotic leukemic cells. From the clinical standpoint, patients with progressive CLL show a higher number of antigen recognitions compared to patients with stable disease. Consistently, the anti-ENO1 antibodies are prevalent in sera from patients with progressive disease and their presence is predictive of a shorter time to first treatment. This clinical inefficacy associates with the inability of patients’ sera to trigger complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity against leukemic cells. Together, these results indicate that antibody responses toward tumor-derived antigens are frequently detectable in sera from patients with CLL, but they are expression of a disrupted immune system and unable to hamper disease progression.