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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.

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.

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.

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.

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

Complement activation is largely implicated in the pathogenesis of several clinical conditions and its therapeutic neutralization has proven effective in preventing tissue and organ damage. A problem that still needs to be solved in the therapeutic control of complement-mediated diseases is how to avoid side effects associated with chronic neutralization of the complement system, in particular, the increased risk of infections. We addressed this issue developing a strategy based on the preferential delivery of a C5 complement inhibitor to the organ involved in the pathologic process. To this end, we generated Ergidina, a neutralizing recombinant anti-C5 human antibody coupled with a cyclic-RGD peptide, with a distinctive homing property for ischemic endothelial cells and effective in controlling tissue damage in a rat model of renal ischemia/reperfusion injury (IRI). As a result of its preferential localization on renal endothelium, the molecule induced complete inhibition of complement activation at tissue level, and local protection from complement-mediated tissue damage without affecting circulating C5. The ex vivo binding of Ergidina to surgically removed kidney exposed to cold ischemia supports its therapeutic use to prevent posttransplant IRI leading to delay of graft function. Moreover, the finding that the ex vivo binding of Ergidina was not restricted to the kidney, but was also seen on ischemic heart, suggests that this RGD-targeted anti-C5 antibody may represent a useful tool to treat organs prior to transplantation. Based on this evidence, we propose preliminary data showing that Ergidina is a novel targeted drug to prevent complement activation on the endothelium of ischemic kidney.

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.

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.

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.

Objective To show that a new recombinant protein (MT07) obtained by fusing a synovial-homing peptide to a neutralizing antibody to C5 can be selectively delivered to inflamed synovium and can effectively control joint inflammation in experimental models of arthritis. Methods Binding of MT07 to human, rat, and mouse synovial tissue was evaluated in vitro by immunofluorescence, and selective localization in the inflamed joints of rats was documented in vivo using time-domain optical imaging. The antiinflammatory effect of MT07 was tested in a rat model of antigen-induced arthritis (AIA) and in a mouse model of collagen antibody–induced arthritis (CAIA). Results MT07 was able to bind to samples of inflamed synovium from humans, mice, and rats while failing to recognize uninflamed synovium as well as inflamed mouse lung or rat kidney. In vivo analysis of the biodistribution of MT07 confirmed its preferential homing to inflamed joints, with negligible inhibition of circulating C5 levels. MT07 prevented and resolved established inflammation in a rat model of AIA, as demonstrated by changes in joint swelling, polymorphonuclear cell counts in synovial washes, release of interleukin-6 and tumor necrosis factor α, and tissue damage. A similar therapeutic effect was obtained testing MT07 in a CAIA model. Conclusion Our findings show that the novel recombinant molecule MT07 has the unique ability to selectively target inflamed joints and to exert local control of the inflammatory process by neutralizing the complement system without interfering with circulating C5 levels. We believe that this approach can be extended to other antiinflammatory drugs currently used to treat patients with rheumatoid arthritis.

Tissue transglutaminase (tTG) plays an important role in celiac disease pathogenesis and antibodies to tTG are a diagnostic marker of gluten-sensitive enteropathy. The aim of this study was to investigate the localization of tTG in the duodenal mucosa in control tissues and in different histological stages of celiac disease by using a commercial and a novel set of anti-tTG monoclonal antibodies, to see whether this assessment can be useful for diagnostic purpose. The distribution of tTG was firstly evaluated in 18 untreated celiac patients by using a commercial monoclonal antibody (CUB7402) against tissue transglutaminase enzyme and directed against the loop-core region of the enzyme. Thereafter, in further 30 untreated celiac patients we employed three newly characterized anti-tTG monoclonal antibodies produced against recombinant human-tTG. The epitopes recognized are located in three distinct domains of the protein corresponding to the core, C1 and C2 protein structure. Eleven age- and sex-matched patients with chronic duodenitis acted as controls. All subjects underwent upper endoscopy to obtain biopsy samples from the duodenum. Overall, we found that (i) tTG is equally expressed in CD at different stages of disease; (ii) tTG is expressed, at similar level, in CD and controls with duodenitis. Assessment of tTG level in biopsy samples by immunohistochemical methods is not useful in the clinical diagnostic work-up of CD., Facultad de Ciencias Exactas

Gluten sensitivity is an autoimmune disease that usually causes intestinal atrophy resulting in a malabsorption syndrome known as celiac disease. However, gluten sensitivity may involve several organs and is often associated with extraintestinal manifestations. Typically, patients with celiac disease have circulating anti-tissue transglutaminase and anti-gliadin antibodies. When patients with gluten sensitivity are affected by other autoimmune diseases, other autoantibodies may arise like anti-epidermal transglutaminase in dermatitis herpetiformis, anti-thyroid peroxidase antibodies in thyroiditis, and anti-islet cells antibodies in type 1 diabetes. The most common neurological manifestation of gluten sensitivity is ataxia, the so-called gluten ataxia (GA). In patients with GA we have demonstrated that anti-gliadin and anti-tissue transglutaminase antibodies cross-react with neurons but that additional anti-neural antibodies are present. The aim of the present article is to review the knowledge on animal models of gluten sensitivity, as well as reviewing the role of anti-neural antibodies in GA.

The efficacy of antibody-based immunotherapy is due to the activation of apoptosis, the engagement of antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity (CDC). We developed a novel strategy to enhance CDC using bispecific antibodies (bsAbs) that neutralize the C-regulators CD55 and CD59 to enhance C-mediated functions. Two bsAbs (MB20/55 and MB20/59) were designed to recognize CD20 on one side. The other side neutralizes CD55 or CD59. Analysis of CDC revealed that bsAbs could kill 4-25 times more cells than anti-CD20 recombinant antibody in cell lines or cells isolated from patients with chronic lymphocytic leukemia. The pharmacokinetics of the bsAbs was evaluated in a human-SCID model of Burkitt lymphoma. The distribution profile of bsAbs mimics the data obtained by studying the pharmacokinetics of anti-CD20 antibodies, showing a peak in the tumor mass 3-4 days after injection. The treatment with bsAbs completely prevented the development of human/SCID lymphoma. The tumor growth was blocked by the activation of the C cascade and by the recruitment of macrophages, polymorphonuclear and natural killer cells. This strategy can easily be applied to the other anti-tumor C-fixing antibodies currently used in the clinic or tested in preclinical studies using the same vector with the appropriate modifications.

Background Intestinal permeability is determined by measuring nonmetabolized sugars. In animals, intestinal permeability is determined in urine, using cumbersome and expensive metabolic cages. We developed an HPLC method for determining concentrations of lactulose (L) and l -rhamnose (R) in blood-drop of rabbits and mice, and we compared these results with the procedure based on sugars excreted in urine. We measured the intestinal permeability induced by a fragment (ΔG) of the zonula occludens toxin which opens the paracellular pathway. Methods The animals received sugar solution and later received the same solution+ΔG. Five-hour urine collection and timed blood tests were performed after ingestion of sugars. Sugars were measured with HPLC, and the percentage of recovered sugars was expressed as L/R ratio. Results At 60 min after administration of sugars, the mean L/R ratio for rabbits and mice was 0.026 and 0.052, respectively. At 60 min after administration of sugars+ΔG, the mean L/R ratio for rabbits and mice was 0.22 and 0.53. The mean L/R ratio in the urine was 0.023 at basal condition and 0.25 after ΔG ingestion. Conclusions Testing small serum samples for sugar permeability is effective for monitoring changes in permeability of the gut in animals. This cheap simple method allows us to measure in vivo the biological activity of other molecules which modulate the paracellular pathway.

A single-chain fragment variable (scFv) recognizing β2-glycoprotein 1 (β2GPI) from humans and other species was isolated from a human phage display library and engineered to contain an IgG1 hinge-CH2-CH3 domain. The scFv-Fc directed against β2GPI domain I-induced thrombosis and fetal loss, thus mimicking the effect of antibodies from patients with antiphospholipid syndrome (APS). Complement is involved in the biological effect of anti-β2GPI scFv-Fc, as demonstrated by its ability to promote in vitro and in vivo complement deposition and the failure to induce vascular thrombosis in C6-deficient rats and fetal loss in C5-depleted mice. A critical role for complement was also supported by the inability of the CH2-deleted scFv-Fc to cause vessel occlusion and pregnancy failure. This antibody prevented the pathological effects of anti-β2GPI antibodies from APS patients and displaced β2GPI-bound patient antibodies. The CH2-deleted antibody represents an innovative approach potentially useful to treat APS patients refractory to standard therapy.

Treatment of patients suffering from chronic diseases such as rheumatoid arthritis with recombinant antibodies is time consuming and fairly expensive and can be associated with side effects due to generalized depletion of the target molecule. We have addressed these issues by developing an alternative approach consisting of the intraarticular injection of a DNA vector encoding for the anti-C5 neutralizing recombinant miniantibody MB12/22. This method allows local production of the antibody in sufficient amount to be effective in preventing joint inflammation in a rat model of antigen-induced arthritis. Injection of the DNA vector in a right knee of normal rats resulted in the production of the minibody detected in the synovial washes by western blot with a strong signal peaking at 3 days after administration. DNA encoding for the minibody was shown for 14 days in the synovial tissue and was undetectable in the controlateral knee and in other organs. The preventive effect of this approach was evaluated in rats receiving a single injection of the vector 3 days before the induction of antigen-induced arthritis and analyzed 3 days later. The treated rats exhibited a lower increase in swelling, associated with a lower number of PMN in the articular washes and reduced deposition of C9 in synovial tissue compared to control rats. These results suggest that treating the inflamed joints with a vector that induces a local production of a neutralizing anti-C5 antibody may represent a useful strategy to inhibit in situ complement activation and to treat patients with monoarthritis. Moreover, this approach may be adopted as a novel therapeutic strategy to prevent monoarthritis as an alternative to local treatment with antibodies commonly used in this form of arthritis, with the advantages of the lower cost and the longer persistence of antibody production.

Antitransglutaminase (anti-TG2) antibodies are synthesised in the intestine and their presence seems predictive of future coeliac disease (CD). This study investigates whether mucosal antibodies represent an early stage of gluten intolerance even in the absence of intestinal damage and serum anti-TG2 antibodies.This study investigated 22 relatives of patients with CD genetically predisposed to gluten intolerance but negative for both serum anti-TG2 antibodies and intestinal abnormalities. Fifteen subjects were symptomatic and seven were asymptomatic. The presence of immunoglobulin A anti-TG2 antibodies in the intestine was studied by creating phage-antibody libraries against TG-2. The presence of intestinal anti-TG2 antibodies was compared with the serum concentration of the intestinal fatty acid-binding protein (I-FABP), a marker for early intestinal mucosal damage. The effects of a 12-month gluten-free diet on anti-TG2 antibody production and the subjects' clinical condition was monitored. Twelve subjects entered the study as controls.The intestinal mucosa appeared normal in 18/22; 4 had a slight increase in intraepithelial lymphocytes. Mucosal anti-TG2 antibodies were isolated in 15/22 subjects (68%); in particular symptomatic subjects were positive in 13/15 cases and asymptomatic subjects in 2/7 cases (p=0.01). No mucosal antibodies were selected from the controls' biopsies. There was significant correlation between the presence of intestinal anti-TG2 antibodies and positive concentrations of I-FABP (p=0.0008). After a gluten-free diet, 19/22 subjects underwent a second intestinal biopsy, which showed that anti-TG2 antibodies had disappeared in 12/15 (p=0.002), while I-FABP decreased significantly (p0.0001). The diet resolved both extraintestinal and intestinal symptoms.A new form of genetic-dependent gluten intolerance has been described in which none of the usual diagnostic markers is present. Symptoms and intestinal anti-TG2 antibodies respond to a gluten free-diet. The detection of intestinal anti-TG2 antibodies by the phage-antibody libraries has an important diagnostic and therapeutic impact for the subjects with gluten-dependent intestinal or extraintestinal symptoms. Clinical trial number NCT00677495.

Objective. Earlier work has demonstrated that serum autoantibodies from coeliac patients targeted against transglutaminase 2 (TG2) inhibit in vitro angiogenesis. The aim of this study was to establish whether coeliac patient-derived monoclonal TG2-targeted antibodies produced by recombination technology exert similar anti-angiogenic effects to serum-derived coeliac autoantibodies. In addition, we studied whether the monoclonal patient autoantibodies modulate endothelial cell TG2 activity and whether such modulation is related to the anti-angiogenic effects. Material and methods. The influence of coeliac patient-derived monoclonal TG2-targeted antibodies on endothelial cell tubule formation was studied using a three-dimensional angiogenic cell culture model. Endothelial cell TG2 enzymatic activity was determined by means of a live-cell enzyme-linked immunosorbent assay. Results. Coeliac patient-derived monoclonal TG2-targeted antibodies produced by recombination technology inhibited endothelial tubule formation and enhanced the crosslinking activity of TG2. When this enzymatic activity was inhibited using site-directed irreversible TG2 inhibitors in the presence of autoantibodies, in vitro angiogenesis reverted to the control level. Conclusions. Since we found a significant negative correlation between endothelial cell angiogenesis and TG2 activity, we suggest that the anti-angiogenic effects of coeliac patient-derived TG2-targeted autoantibodies are exerted by enhanced enzymatic activity of TG2.

OBJECTIVE Anti-tissue transglutaminase (TG2) antibodies are the serological marker of celiac disease. Given the close association between celiac disease and type 1 diabetes, we investigated the production and deposition of anti-TG2 antibodies in the jejunal mucosa of type 1 diabetic children. RESEARCH DESIGN AND METHODS Intestinal biopsies were performed in 33 type 1 diabetic patients with a normal mucosal architecture: 14 had high levels (potential celiac disease patients) and 19 had normal levels of serum anti-TG2 antibodies. All biopsy specimens were investigated for intestinal deposits of IgA anti-TG2 antibodies by double immunofluorescence. In addition, an antibody analysis using the phage display technique was performed on the intestinal biopsy specimens from seven type 1 diabetic patients, of whom four had elevated and three had normal levels of serum anti-TG2 antibodies. RESULTS Immunofluorescence studies showed that 11 of 14 type 1 diabetic children with elevated levels and 11 of 19 with normal serum levels of anti-TG2 antibodies presented with mucosal deposits of such autoantibodies. The phage display analysis technique confirmed the intestinal production of the anti-TG2 antibodies; however, whereas the serum-positive type 1 diabetic patients showed a preferential use of the VH5 antibody gene family, in the serum-negative patients the anti-TG2 antibodies belonged to the VH1 and VH3 families, with a preferential use of the latter. CONCLUSIONS Our findings demonstrate that there is intestinal production and deposition of anti-TG2 antibodies in the jejunal mucosa of the majority of type 1 diabetic patients. However, only those with elevated serum levels of anti-TG2 antibodies showed the VH usage that is typical of the anti-TG2 antibodies that are produced in patients with celiac disease.

Background Despite the obvious success of anti-TNFα agents in Rheumatoid Arthritis (RA) treatment, high number of non-responders and systemic adverse effects, 1 including reactivation of latent infections, highlight the need of broader efficacy and improved safety profiles. Recently the importance of angiogenesis in the progression of arthritis has been unveiled. 2 Angiogenesis can be exploited for tissue selective drug delivery approaches to drive in situ drug accumulation reducing systemic side effects. Objectives Develop a bispecific antibody prodrug with vascular specificity to deliver an anti-TNFα payload to the arthritic tissue, allowing anti-TNFα activation selectively in the arthritic synovium and avoiding systemic TNFα engagement. Methods The anti-ICAM1 variable region was cloned in fusion with the anti-TNFα antibody Adalimumab in a dual variable domain (DVD) bispecific format. Linker between the outer and inner variable domain was engineered to be short and bear an MMP1 cleavable site forming an activatable DVD (aDVD) antibody. Reactivity with RA human synovium was determined using immunohistochemistry on tissue sections. MMP1-mediated antibody activation was tested using RA synovial fluids and RA synovial fibroblasts. Activity and biological functionality of the anti-TNFα moiety was assessed in vitro using ELISA, Biacore and L-929 TNFα-cytotoxicity assays. Human TNF transgenic mouse model of arthritis (tg197) was used to assess in vivo therapeutic activity. Results The aDVD bispecific antibody was able to retain synovial microvascular targeting, conveyed by the anti-ICAM1 variable domain, in RA synovial tissue sections. The intact molecule however, showed a reduced capacity to bind TNFα with 500-fold affinity decrease caused by steric hindrance of the outer variable region. Digestion with physiologically relevant concentrations of MMP1 was sufficient to restore 100% anti-TNFα activity (EC50 53 pM) comparable to the parent Adalimumab antibody (49 pM). The un-processed aDVD antibody was significantly impaired in rescuing the L-929 cell line from TNFα-induced cytotoxicity. Digestion with MMP1 and activation of the anti-TNFα domain restored the anti-TNFα biological activity with an IC50 of 0.1 nM equal to Adalimumab IgG (IC50 0.1 nM). The antibody was also able to localise in the arthritic joint and reduce bone degradation in the tg197 mouse model in vivo . Conclusions Our results demonstrate the development of a synovium-specific pro-drug delivery system for RA. The impaired TNFα binding capacity of the intact molecule ensures a decreased systemic TNFα engagement. Presence of metalloproteinases in RA synovium 3 would activate and deliver the anti-TNFα activity locally in the site of disease. This strategy may allow greater therapeutically-relevant doses to be delivered in the synovium, increasing pharmacological potency, potentially allowing reduction in the dosage and/or administration frequency with the ultimate goal of reducing systemic exposure, achieve better therapeutic index and decreasing healthcare costs. 4 References Taylor, P.C. & Feldmann, M. Nat Rev Rheumatol 5, 578-82 (2009). Paleolog, E. International journal of experimental pathology 90, 249-310 (2009). Manicourt, D.H., et al. Arthritis Rheum 38, 1031-9 (1995). Ferrari, M. et al. Nat Rev Rheumatol (in press) Disclosure of Interest None declared

Background and objective Despite the obvious success of current anti-TNFα agents in Rheumatoid Arthritis (RA) treatment, high number of non-responders and systemic adverse effects (e.g. reactivation of tuberculosis) highlight the need of broader efficacy and improved safety profiles. Recently the importance of angiogenesis in the progression of arthritis has been unveiled. Angiogenesis can be exploited for tissue selective drug delivery approaches to drive in-situ drug accumulation, reducing systemic side effects. Towards this end we developed a bispecific antibody pro-drug platform with ICAM1 specificity to deliver an anti-TNFα payload to the arthritic tissue, allowing anti-TNFα activation selectively in the arthritic synovium and avoiding systemic TNFα engagement. Materials and methods The anti-ICAM1 variable region was subcloned in fusion with the anti-TNFα antibody Adalimumab in dual variable domain (DVD) bispecific format. The linker between the outer and inner variable domain was engineered to be short and bear an MMP1 cleavable site, forming an activatable DVD (aDVD) antibody. Reactivity with RA human synovium was determined using immunohistochemistry on tissue sections. MMP1-mediated antibody activation was tested using RA synovial fluids and RA synovial fibroblasts. Activity and biological functionality of the anti-TNFα moiety was assessed in-vitro using ELISA, Biacore and L-929 TNFα-cytotoxicity assays. Results The aDVD bispecific antibody was able to maintain synovial microvascular targeting, conveyed by the anti-ICAM1 variable domain, in RA synovial tissue sections. The intact molecule however, showed a reduced capacity to bind TNFα with 500-fold affinity decrease caused by steric hindrance of the outer variable region. Digestion with physiologically relevant concentrations of MMP1 was sufficient to restore 100% anti-TNFα activity (EC50 53 pM), comparable to the parent Adalimumab antibody (49 pM). Further, the un-processed aDVD antibody was significantly impaired in rescuing the L-929 cell line from TNFα-induced cytotoxicity. Digestion with MMP1 and activation of the anti-TNFα domain restored the anti-TNFα biological activity with an IC50 of 0.1 nM comparable to Adalimumab IgG (IC50 0.1 nM). Conclusions Our results demonstrate the development of a synovium-specific pro-drug delivery system for RA. The impaired TNFα binding capacity of the intact molecule ensures a decreased systemic TNFα engagement. Presence of metalloproteinases in RA synovium would activate and deliver the anti-TNFα activity locally in the site of disease. This strategy may allow greater therapeutically-relevant doses to be delivered in the synovium, increasing pharmacological potency, allowing reduction in the dosage and/or administration frequency, with the ultimate goal of reducing systemic exposure, achieve better therapeutic index and decreasing healthcare costs.

Long term exposure to gluten in coeliacs,1 and coeliac disease (CD) diagnosis after 16 years of age2 may induce type 1 diabetes (T1D) and other autoimmune disorders. Increased prevalence of CD among diabetics and their relatives is well documented.3 Early introduction of gluten to children at high risk for T1D produces T1D associated islet autoantibodies.4 Similarly, in the absence of overt clinical symptoms of T1D, some coeliac children produce diabetes autoantibodies in a gluten dependent manner.5 In diabetics, intestinal challenge with gluten produces mucosal recruitment of lymphocytes,6 similar to that in CD patients.7 In diabetics, however, there is no production of CD related anti-tissue transglutaminase antibodies (anti-tTG).6 We have used a phage display assay8 to show that in CD patients, production of anti-tTG is limited to the intestine. Here, we …

BACKGROUND: Early diagnosis and treatment with gluten-free diet reduces mortality and the prevalence of associated disorders in celiac disease (CD). A simple "in the office" test of anti-transglutaminase antibodies might be of great help in first-line screening for CD. AIMS: We evaluated the sensitivity and specificity of two commercial kits based, respectively, on rapid detection of IgA-IgG anti-human-transglutaminase antibodies (anti-h-tTG) in serum and IgA anti-h-tTG antibody in one drop of whole blood. These assays were compared to a well-established enzyme-linked immunosorbent assay technique. METHODS: Serum samples were analyzed from 114 biopsy-confirmed celiacs, 120 healthy controls, 20 first-degree relatives of celiacs, and 75 diseased controls. The whole blood samples were analyzed from 51 biopsy-confirmed celiacs and 100 controls. RESULTS: The serum-based test was positive in all 114 celiacs (sensitivity 100%). Among the controls there were seven healthy blood donors, one first-degree relative, and three diseased controls who tested positive (specificity 94.9%). The blood drop-based assay testing IgA antibodies was positive in 46 of 51 (sensitivity 90.2%), and since three of the five patients testing negative had total IgA deficiency, the sensitivity value can be increased to 95.8%. All 100 controls tested negative (specificity 100%). CONCLUSIONS: The commercial kits described here produce high values of sensitivity and specificity, offering the general practitioner who suspects a possible case of CD the real possibility to look for anti-h-tTG antibodies in his own medical office during a standard visit at a satisfyingly low cost.

We monitored the number of intravascular platelet-leukocyte aggregates (PLAs) and thrombotic occlusions (TOs) by intravascular microscopy in the mesentery of rats receiving antiphospholipid (aPL) immunoglobulin G (IgG) purified from the sera of patients with antiphospholipid syndrome. aPL IgG had no procoagulant effect, but it caused rapid endothelial deposition of fibrinogen, followed by PLA and TO in rats receiving an intraperitoneal injection of bacterial lipopolysaccharide 3 hours before IgG infusion. Anti-β2-glycoprotein I-depleted aPL IgG failed to induce PLAs and TOs. C3 and C9 colocalized with aPL IgG on the mesenteric vessels. The number of PLAs and TOs was markedly reduced in C6-deficient rats and in animals treated with anti-C5 miniantibody, suggesting the contribution of the terminal complement (C) complex to the aPL antibody-mediated intravascular thrombosis. In conclusion, our data indicate that antibodies to β2-glycoprotein I trigger coagulation subsequent to a priming proinflammatory factor and that the terminal C complex is the main mediator of the coagulation process.

Aims: To determine coeliac disease prevalence by an anti-transglutaminase antibody assay in a large paediatric population; to evaluate acceptance of the screening programme, dietary compliance, and long term health effects. Methods: Cross-sectional survey of 3188 schoolchildren (aged 6–12) and prospective follow up of diagnosed cases. Main outcome measures were: prevalence of coeliac disease defined by intestinal biopsy or positivity to both human tissue transglutaminase and anti-endomysium antibodies in HLA DQ2-8 positive subjects; percentage of children whose families accepted screening; dietary compliance as defined by negativity for anti-transglutaminase antibodies; and presence of clinical or laboratory abnormalities at 24 month follow up. Results: The families of 3188/3665 children gave their consent (87%). Thirty biopsy proven coeliacs were identified (prevalence 1:106). Three other children testing positive for both coeliac related autoantibodies and HLA DQ2-8 but refusing biopsy were considered as having coeliac disease (prevalence 1:96). Of 33 cases, 12 had coeliac related symptoms. The 30 biopsy proven coeliacs followed a gluten-free diet. Of 28 subjects completing 18–24 months follow up, 20 (71.4%) were negative for anti-transglutaminase antibodies, while eight were slightly positive; symptoms resolved in all 12 symptomatic children. Conclusions: Prevalence of coeliac disease is high in Italian schoolchildren. Two thirds of cases were asymptomatic. Acceptance of the programme was good, as was dietary compliance. Given the high prevalence and possible complications of untreated coeliac disease, the availability of a valid screening method, and evidence of willingness to comply with dietary treatment population mass screening deserves careful consideration.

Celiac disease is characterized by intestinal mucosal injury and malabsorption precipitated by dietary exposure to gluten of some cereals with a prominent role being played by gliadins, specific antigenic determinants found in wheat gluten. Patients suffering from celiac disease have serum antibodies recognizing gliadin, as well as the endomysial autoantigen tissue transglutaminase. Phage display antibody libraries have revealed ectopic production of anti-transglutaminase antibodies by intestinal lymphocytes with a biased use of the VH5 antibody gene family. Here we report a study on the pairing of VH and VL families in the antibodies to transglutaminase. Our results led to the construction of small phage display antibody libraries based on the amplification of the two genes in the VH5 family from intestinal lymphocytes. This method can be used for the rapid characterization of the anti-transglutaminase response in a potentially large number of subjects including asymptomatic patients whose serum antibodies may be undetectable.

Undetected celiac disease (CD) can cause serious complications and excessive mortality (1)(2)(3). Noninvasive tests could be useful in physicians’ offices to identify patients for intestinal biopsies (4). Serum IgA anti-endomysium (AEA) and anti-human transglutaminase (anti-htTG) antibody assays are the most widely used laboratory tests for CD (5). Autoantibody determinations have been simplified by the use of rapid testing (6)(7), but when saliva specimens were evaluated for AEA testing, they were found to be not suitable (8). To evaluate the potential utility of saliva as a sample for anti-htTG testing, we compared salivary IgA anti-htTG (measured by ELISA with a dot-blot method and an AEA test) with serum IgA anti-htTG antibodies (measured by ELISA and an AEA test) in patients with CD and in controls. Salivary IgA anti-htTG activity was analyzed for the IgA secretory chain by use of a monoclonal antibody. The recognition patterns of the saliva samples were compared with those for the serum samples and with the pattern for a gut-derived IgA monoclonal antibody to htTG from one CD patient, using two transglutaminase deletion mutants, one recognized and one not by the patients’ sera (9). We studied 47 untreated CD patients (29 females and 18 males; median age, 19 years; range, 2–52 years) diagnosed in 2002–2003 according to ESPGHAN criteria (10) and 47 patients with celiac disease (26 females and 21 males; median age, 19 years; range, 4–50 years) on a gluten-free diet (GFD) for at least 12 months. We recruited 51 healthy controls with no history of gastrointestinal or autoimmune diseases (25 females and 26 males; median age, 14 years; range, 6–50 years) and identified samples from 49 individuals suffering from various gastrointestinal diseases (18 with Crohn disease, 10 with ulcerative colitis, 10 with milk allergy, 6 with acute …

Summary Context Many human diseases are the result of autoimmune attack, presumably related to a loss of tolerance to self. Autoimmune disease can be divided into either organ-specific illnesses, such as thyroid disease, type 1 diabetes, and mysasthenia gravis, or systemic illnesses, such as rheumatoid arthritis and systemic lupus erythematosus. The pathogenesis of autoimmune damage also segregates autoimmune disease in that some diseases or manifestations are mainly induced by autoantibodies. Pathogenesis may be mainly mediated by autoimmune T lymphocytes. Notwithstanding the underlying mechanism of disease, virtually all autoimmune diseases are associated with circulating autoantibodies, which bind self-protein. Furthermore, for many diseases these autoantibodies are found in serum samples many years before disease onset. Starting point In the past several years a new autoantibody specificity has been identified in the sera of patients with rheumatoid arthritis. These autoantibodies bind citrulline, a post-translational modification of arginine. Markus Nielen and colleagues recently studied the presence of these autoantibodies and rheumatoid factor in blood donors who later developed rheumatoid arthritis ( Arthritis Rheum 2004; 50 : 380–86). About half the patients were positive for at least autoantibody at a median of 4·5 years before the onset of disease. The negative predictive value of these tests was high, while the positive predictive value was very high. Where next? Autoantibodies might not be directly responsible for many of the manifestations of autoimmune disease, but they are markers of future disease in presently healthy individuals. Long-term large studies of outcome are needed to assess the use of assaying autoantibodies for prediction of disease. Such data could lead to intervention trials to prevent autoimmune disease, as are already underway in type 1 diabetes.

AIMS: To investigate celiac disease (CD) and related co-morbidity in patients with familial and sporadic cardiomyopathy and in their relatives. METHODS AND RESULTS: We screened anti-human-tissue-transglutaminase (IgA and IgG anti-h-tTG) and anti-endomysial antibodies (AEAs) in 238 consecutive adult patients with inherited or sporadic dilated cardiomyopathy (DCM), 418 relatives, and 2000 healthy blood donors. HLADQ2-DQ8 was tested in tTG-positive subjects. The IgA-tTG-positive patients with cardiomyopathy underwent duodenal biopsy. Twenty-six subjects were tTG-positive: five DCM patients (2.1\%), two of 28 (7.1\%) and three of 390 (0.7\%) relatives with and without echocardiographic abnormalities respectively, and 16 controls (0.8\%). Twenty-two of 26 subjects were AEA-positive, and 25 HLA-positive. Of the five patients with cardiomyopathy and biopsy-proven CD, four suffered iron-deficiency anaemia. Two CD-positive DCM patients and two tTG-positive relatives were from families with inherited disease in which CD did not co-segregate with DCM. CONCLUSIONS; The higher prevalence of CD in patients with sporadic or inherited DCM, and of tTG-positive serology in relatives with echocardiographic abnormalities, suggests that immune-mediated mechanisms are active in subsets of patients/families. However, gluten intolerance cannot be considered causative since CD seems to be associated but not co-segregated with DCM in familial cases.

BACKGROUND: The main autoantigen recognized by the sera of patients with coeliac disease (CD) is tissue transglutaminase (tTG). A human-recombinant form of tTG was used to develop an ELISA to measure anti-tTG serum antibodies for the diagnosis of CD. Preliminary retrospective reports suggest that the human tTG-based ELISA could identify coeliac patients missed by the IgA-anti-endomysium antibody test (AEA). Whether the human recombinant tTG ELISA is sufficiently accurate to become the main diagnostic CD tool in everyday clinical practice is unknown. The objective was to determine, in a prospective study, the sensitivity and specificity of an ELISA test based on the use of human tTG compared with AEA, to analyse the discordant cases for HLA DQ2-8 and for clinical and intestinal biopsy characteristics. METHODS: 1106 patients referred to a gastrointestinal outpatient clinic for symptoms attributable to CD, 52 first-degree relatives of CD patients and 200 healthy controls were tested for both anti-human tTG and AEA antibodies. RESULTS: Out of 1158 subjects, 146 were tested positive for anti-tTG antibodies and 140 were biopsy-proven coeliacs. The AEA test identified 126/1158 coeliacs who also tested positive for anti-tTG antibodies. The 14 patients missed by the AEA test carried the typical HLA-DQ for CD; they had normal levels of total serum IgA and had milder pathology than those with both anti-tTG and AEA positivity (P < 0001). CONCLUSIONS: These results prove that human tTG-based ELISA is an excellent diagnostic tool for CD, for mass screening by both the specialist and the general clinic.

Objectives To evaluate in an in vivo rat experimental model of antigen induced arthritis (AIA) the concentration and persistence of intraarticular (ia) binding of a biocompatible modified polysaccharide, composed of a chitosan backbone compound (Chitlac) (CTL), when administered soon after the induction of arthritis, and analyze any interaction with inflammatory synovial tissue development, in order to assess its possible use as a tool for drug targeting of inflamed synovium. Synovial tissue delivery and the effects on molecular and structural arthritic modifications were compared with those induced by a previously described modified joint tissue-specific anti-C5 recombinant antibody MT07 provided with a homing peptide (Adienne Pharma & Biotech) Methods In 3 groups, each consisting of 4 male wistar rats, monoarthritis (knee joint) was induced by ia injection of 100 μg of methylated bovine serum albumin (mBSA), following previous immunization to mBSA+Freund9s adjuvant. Arthritis development was monitored at least for a 10 days long period by joint swelling (JS) measurements and, further, ex vivo analyses of synovial washing cell counts, TNF-α and IL-6 concentrations, and histomorphology were performed. Soon after (24 hrs) arthritis induction, the 3 groups of rats were iv injected with the fluorophore Cy5.5 (FluoroLink, Amersham Biosciences) 0.05mg/ml alone (CNT), or by the CTL-Cy5.5, or MT07-Cy5.5 bound molecules, respectively, diluted in a 0.1 M sodium carbonate buffer solution. A small-animal time-domain Optix MX preclinical near-infrared imager (Advanced Research Technologies, NL) was used for the in vivo evaluation of Cy5.5-labeled molecules. Two-dimensional scanning regions of interest were selected. The data were recorded as point-spread functions, and the images were reconstructed as fluorescence intensity. Anesthetized rats were checked for imaging of fluorophore distribution at several times before being euthanized for ex vivo joint tissue evaluations, and detection of the tested molecules in other organs Results In both the CNT and CTL treated groups, joint swelling increased to a 25-30% degree from basal levels (p 3 vs 6.7x10 3 in the arthritic joint, at time=10 days; p Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.3652

Background and Objectives Despite the obvious success of current biological agents for treatment of rheumatoid arthritis (RA), achievement of broader efficacy and improved safety profile remains an unmet need in RA therapy. There is significant evidence demonstrating molecular heterogeneity within the vascular endothelium of different tissues, conferring organ tropism to migrating lymphocytes subsets by interaction with specific homing receptors. We have previously reported the isolation of a single-chain Fv (scFvA7) antibody with specificity for synovial arthritic microvasculature, following an in vivo phage display selection on SCID mice model of RA. The aim of the present study was to develop a tissue specific therapeutic for Rheumatoid Arthritis. Materials and Methods The scFvA7 was subcloned in fusion with the CH2-CH3 domain of human IgG (A7 scFv-Fc) and coupled with the anti-TNF antibody Adalimumab via a monocistronic RNA approach, to form a bispecifc antibody (BsAb) using the Knobs-into-Holes technology (A7/Adalimumab). The reactivity in frozen and paraffin embedded tissue sections was investigated using immunohistochemistry and immunofluorescence analysis. In-vitro functionality and biological activity was assessed in Biacore, TNF-ELISA and TNF cytotoxicity assay on L-929 cell line. Results The scFv-Fc fusion protein of Adalimumab showed analogous anti-TNF properties with the parent antibody, EC50 0.04nM (Adalimumab 0.025nM), confirming the validity of the scFv format. The BsAb A7/Adalimumab achieved a high degree of efficient heterodimerisation showing only 3% Adalimumab homodimers and was able to selectively bind TNFα in vitro with similar efficacy to the TNF blocker, EC50 0.06nM, with a KD of 31pM. Despite the monovalency for the anti-TNF activity, the BsAb showed a dose dependent rescue of TNF induced cytotoxicity in L-929 cell line with a 0.5nM IC50 (Adalimumab 0.17nM), demonstrating the biological functionality of the construct. In addition, the A7/Adalimumab antibody proved to efficiently and specifically target the stromal compartment of RA synovial microvasculature, maintaining unaltered the organ tropism of the original scFvA7. Conclusions Our results demonstrate the development of a bispecific antibody showing unaltered TNF blocking capacity and optimal synovial specificity, suggesting that the target molecule for A7 may have applications as a biomarker and immunotherapeutic target, potentially allowing reduction in the dosage and/or administration frequency, with the ultimate goal to reduce the systemic exposure and achieve a better therapeutic index and decreasing health care costs.

BACKGROUND—Coeliac disease is one of the commonest underdiagnosed diseases in general practice. The autoantigen recognised by the sera of patients with coeliac disease has recently been identified as tissue transglutaminase. AIMS—We evaluated a simple non-invasive immunological dot blot assay for coeliac disease, suitable for use by the general physician in the ambulatory setting. The sensitivity and specificity of this dot blot assay based on recognition of recombinant human transglutaminase were compared with those of antiendomysial antibodies and an enzyme linked immunosorbent assay. METHODS—Serum samples were analysed from 64 healthy controls, 58 first degree relatives of coeliacs, 74 diseased controls, and 70 biopsy confirmed untreated patients with coeliac disease. Dot blot assay and enzyme linked immunosorbent assay were performed using recombinant human transglutaminase as antigen. RESULTS—The dot blot assay, which can be performed in 20 minutes, was positive in all 70 untreated coeliacs (sensitivity 100%). Among the three control groups, there were three false positive tests by dot blot (specificity 98%), all belonging to the group of healthy subjects. The antiendomysial antibodies test missed five untreated coeliac patients (sensitivity 93%) and was negative in all three control groups (specificity 100%). The specificity of the immunosorbent assay was 99% for IgA and 98% for IgG, while sensitivity was 93% for IgA, 47% for IgG, and 100% for IgA and IgG combined. CONCLUSIONS—The dot blot assay is highly accurate in detecting untreated subjects with coeliac disease and can be performed in the general physician's medical office during the course of a routine examination. This innovative test is a practical, reliable alternative to both the immunofluorescent based antiendomysial test and immunosorbent assay for detection of transglutaminase antibodies for the diagnosis of coeliac disease. Keywords: coeliac disease; dot blot immunobinding assay; recombinant human tissue transglutaminase

Celiac disease (CD) is an autoimmune enteropathy characterized by intestinal malabsorption and immunological responses to dietary gliadins and an auto antigen located in the endomysium. The latter has recently been identified as the enzyme tissue transglutaminase (tTG). The linkage between gliadins, tTG and the autoimmune response has still to be clarified. In this work we report the production and analysis of a phage antibody library from the peripheral blood lymphocytes (PLB) of a CD patient. The library contained polyreactive and monoreactive antibodies to alpha-gliadin, to the dietary antigen beta-lactoglobulin, but not to tTG. The majority of the VH regions of the anti-alpha-gliadin antibodies belonged to the VH 4 family. The possibility of exploiting phage display antibodies as tools to study the molecular events associated with CD is discussed.