Investigation of the deregulation of interleukin-6 in two non-Hodgkin's lymphoma cell lines

Increased serum interleukin-6 (IL-6) is a poor prognostic factor for patients with lymphoma. This may be related to the fact that IL-6 has been shown to be an autocrine and paracrine growth factor for lymphoma cells. We have investigated the regulation of IL-6 in two lymphoma cell lines which produce IL-6 as an autocrine growth factor. The cell lines, LY3 and LY12, were established from two patients with non-Hodgkin's lymphoma. One patient had diffuse large cell lymphoma (LY3), whereas the other had small noncleaved cell lymphoma (LY12). There was no rearrangement or amplification of the IL-6 gene, but we detected IL-1 alpha and TNF production in addition to IL-6. We investigated the effect of inhibitors of IL-1 and TNF on IL-6 production in LY3 and LY12. Our results show that IL-6 production is mainly secondary to endogenous IL-1 production in LY3 cells, however LY12 cells produce IL-6 via a different mechanism since neither anti-IL-1 nor anti-TNF significantly inhibited IL-6 production.^ Transfection of LY12 cells with wildtype and mutant IL-6 promoter-chloramphenicol acetyl transferase constructs, showed increased activity of a trans-acting factor that binds to the NF-kB motif. Therefore, we determined whether there were abnormalities in members of the NF-kB family of transcription factors, such as p65, p50, p52/lyt-10 or rel, which bind to kB motifs. We found increased expression of the p52/lyt-10 transcription factor and activation of the NF-kB pathway in LY12. However, expression of p50, p65 and rel was not increased in LY12 cells. Future investigations could be aimed at determining the effect of inhibitors of NF-kB on IL-6 production. ^

Genetic susceptibility to increased bacterial translocation influences the response to biological therapy in patients with Crohn's disease

OBJECTIVE: The aetiology of Crohn's disease (CD) has been related to nucleotide-binding oligomerisation domain containing 2 (NOD2) and ATG16L1 gene variants. The observation of bacterial DNA translocation in patients with CD led us to hypothesise that this process may be facilitated in patients with NOD2/ATG16L1-variant genotypes, affecting the efficacy of anti-tumour necrosis factor (TNF) therapies. DESIGN: 179 patients with Crohn's disease were included. CD-related NOD2 and ATG16L1 variants were genotyped. Phagocytic and bactericidal activities were evaluated in blood neutrophils. Bacterial DNA, TNFα, IFNγ, IL-12p40, free serum infliximab/adalimumab levels and antidrug antibodies were measured. RESULTS: Bacterial DNA was found in 44% of patients with active disease versus 23% of patients with remitting disease (p=0.01). A NOD2-variant or ATG16L1-variant genotype was associated with bacterial DNA presence (OR 4.8; 95% CI 1.1 to 13.2; p=0.001; and OR 2.4; 95% CI 1.4 to 4.7; p=0.01, respectively). This OR was 12.6 (95% CI 4.2 to 37.8; p=0.001) for patients with a double-variant genotype. Bacterial DNA was associated with disease activity (OR 2.6; 95% CI 1.3 to 5.4; p=0.005). Single and double-gene variants were not associated with disease activity (p=0.19). Patients with a NOD2-variant genotype showed decreased phagocytic and bactericidal activities in blood neutrophils, increased TNFα levels in response to bacterial DNA and decreased trough levels of free anti-TNFα. The proportion of patients on an intensified biological therapy was significantly higher in the NOD2-variant groups. CONCLUSIONS: Our results characterise a subgroup of patients with CD who may require a more aggressive therapy to reduce the extent of inflammation and the risk of relapse

Treatment with tumor necrosis factor inhibitors in axial spondyloarthritis: comparison between private rheumatology practices and academic centers in a large observational cohort

OBJECTIVE To evaluate the initiation of and response to tumor necrosis factor (TNF) inhibitors for axial spondyloarthritis (axSpA) in private rheumatology practices versus academic centers. METHODS We compared newly initiated TNF inhibition for axSpA in 363 patients enrolled in private practices with 100 patients recruited in 6 university hospitals within the Swiss Clinical Quality Management (SCQM) cohort. RESULTS All patients had been treated with ≥ 1 nonsteroidal antiinflammatory drug and > 70% of patients had a baseline Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) ≥ 4 before anti-TNF agent initiation. The proportion of patients with nonradiographic axSpA (nr-axSpA) treated with TNF inhibitors was higher in hospitals versus private practices (30.4% vs 18.7%, p = 0.02). The burden of disease as assessed by patient-reported outcomes at baseline was slightly higher in the hospital setting. Mean levels (± SD) of the Ankylosing Spondylitis Disease Activity Score were, however, virtually identical in private practices and academic centers (3.4 ± 1.0 vs 3.4 ± 0.9, p = 0.68). An Assessment of SpondyloArthritis international Society (ASAS40) response at 1 year was reached for ankylosing spondylitis in 51.7% in private practices and 52.9% in university hospitals (p = 1.0) and for nr-axSpA in 27.5% versus 25.0%, respectively (p = 1.0). CONCLUSION With the exception of a lower proportion of patients with nr-axSpA newly treated with anti-TNF agents in private practices in comparison to academic centers, adherence to ASAS treatment recommendations for TNF inhibition was equally high, and similar response rates to TNF blockers were achieved in both clinical settings.

Appropriateness and long-term discontinuation rate of biological therapies in ulcerative colitis

BACKGROUND Anti-TNFα agents are commonly used for ulcerative colitis (UC) therapy in the event of non-response to conventional strategies or as colon-salvaging therapy. The objectives were to assess the appropriateness of biological therapies for UC patients and to study treatment discontinuation over time, according to appropriateness of treatment, as a measure of outcome. METHODS We selected adult ulcerative colitis patients from the Swiss IBD cohort who had been treated with anti-TNFα agents. Appropriateness of the first-line anti-TNFα treatment was assessed using detailed criteria developed during the European Panel on the Appropriateness of Therapy for UC. Treatment discontinuation as an outcome was assessed for categories of appropriateness. RESULTS Appropriateness of the first-line biological treatment was determined in 186 UC patients. For 64% of them, this treatment was considered appropriate. During follow-up, 37% of all patients discontinued biological treatment, 17% specifically because of failure. Time-to-failure of treatment was significantly different among patients on an appropriate biological treatment compared to those for whom the treatment was considered not appropriate (p=0.0007). Discontinuation rate after 2years was 26% compared to 54% between those two groups. Patients on inappropriate biological treatment were more likely to have severe disease, concomitant steroids and/or immunomodulators. They were also consistently more likely to suffer a failure of efficacy and to stop therapy during follow-up. CONCLUSION Appropriateness of first-line anti-TNFα therapy results in a greater likelihood of continuing with the therapy. In situations where biological treatment is uncertain or inappropriate, physicians should consider other options instead of prescribing anti-TNFα agents.

CD4 T cells are required for both development and maintenance of disease in a new mouse model of reversible colitis

Current therapies to treat inflammatory bowel diseases have limited efficacy, significant side effects, and often wane over time. Little is known about the cellular and molecular mechanisms operative in the process of mucosal healing from colitis. To study such events, we developed a new model of reversible colitis in which adoptive transfer of CD4(+)CD45RB(hi) T cells into Helicobacter typhlonius-colonized lymphopenic mice resulted in a rapid onset of colonic inflammation that was reversible through depletion of colitogenic T cells. Remission was associated with an improved clinical and histopathological score, reduced immune cell infiltration to the intestinal mucosa, altered intestinal gene expression profiles, regeneration of the colonic mucus layer, and the restoration of epithelial barrier integrity. Notably, colitogenic T cells were not only critical for induction of colitis but also for maintenance of disease. Depletion of colitogenic T cells resulted in a rapid drop in tumor necrosis factor α (TNFα) levels associated with reduced infiltration of inflammatory immune cells to sites of inflammation. Although neutralization of TNFα prevented the onset of colitis, anti-TNFα treatment of mice with established disease failed to resolve colonic inflammation. Collectively, this new model of reversible colitis provides an important research tool to study the dynamics of mucosal healing in chronic intestinal remitting-relapsing disorders.Mucosal Immunology advance online publication 16 September 2015; doi:10.1038/mi.2015.93.

Regulation of peripheral classical and non-classical monocytes on infliximab treatment in patients with rheumatoid arthritis and ankylosing spondylitis.

OBJECTIVE To investigate the regulatory effect of tumour necrosis factor (TNF) blockade with infliximab on the distribution of peripheral blood monocyte subpopulations in patients with rheumatoid arthritis (RA) and ankylosing spondylitis (AS). METHODS Purified CD11b+CD14+ monocytes from 5 patients with RA and 5 AS were analysed ex vivo before and after infliximab treatment by flow cytometry for CD16, CD163, CD11b, C-C chemokine receptor type 2 (CCR2) and CXC chemokine receptor 4 (CXCR4) at baseline and at days 2, 14, 84 and 168 after the first infliximab administration. Serum levels of the stromal cell-derived factor (SDF)-1 and monocyte chemotactic peptide (MCP)-1 at different time points were measured in either patient group before and on infliximab treatment. RESULTS Anti-TNF treatment with infliximab led to a significant increase of circulating CD11b+ non-classical and a concomitantly decrease of CD11b+ classical monocytes, to a decline in SDF-1 levels and reduced expression of CCR2 and CXCR4 on non-classical monocyte subpopulation. CONCLUSIONS Our study shows, that TNFα blockade by infliximab resulted in a dichotomy of the regulation of classical and non-classical monocytes that might have substantial impact on inhibition of osteoclastogenesis and of subsequent juxta-articular bone destruction and systemic bone loss in RA and AS.

Dissection of antitumor activity by lymphokine -activated killer cells: Role of FcR+ precursors and obligatory role of tumor necrosis factor-$\alpha$ in antibody -dependent cellular cytotoxicity

Human peripheral blood lymphocytes (PBL) cultured for varying lengths of time in IL-2 are able to mediate antibody independent cellular cytotoxicity (AICC) as well as antibody dependent cellular cytotoxicity (ADCC) against a wide range of tumor targets. The objective of our study is to determine the cytotoxic potential of the subset of LAK cells involved in ADCC, the tumor recognition mechanism in ADCC, the kinetics of ADCC mediated by PBL cultured under various conditions and the role of TNF-$\alpha$ in the development and maturation of ADCC effectors in the LAK population.^ The model system in this study for ADCC used a monoclonal antibody 14G2a (IgG2a), that recognizes the GD2 epitope on human melanoma cell line, SK-Mel-1. The target recognition mechanism operative in AICC (traditionally known as lymphokine activated killing or LAK) is an acquired property of these IL-2 activated cells which confers on them the unique ability to distinguish between tumor and normal cells. This recognition probably involves the presence of a trypsin sensitive N-linked glycoprotein epitope on tumor cells. Proteolytic treatment of the tumor cells with trypsin renders them resistant to AICC by PBL cultured in IL-2. However, ADCC is unaffected. This ADCC, mediated by the relatively small population of cells that are positive for the Fc receptor for IgG (FcR), is an indication that this subset of "LAK" cells does not require the trypsin sensitive epitope on tumor cells to mediate killing. Enriching PBL for FcR+ cells markedly enhanced both AICC and ADCC and also reduced the IL-2 requirement of these cells.^ The stoichiometry of Fc receptor (FcR) expression on the cytotoxic effectors does not correlate with ADCC lytic activity. Although FcRs are necessary to mediate ADCC, other factors, appear to regulate the magnitude of cytolytic activity. In order to investigate these putative factors, the kinetics of ADCC development was studied under various conditions (in IL-2 (10u/ml) and 100u/ml), in IL-2(10u/ml) + TNF$\alpha$ (500u/ml) and in TNF-$\alpha$ (500u/ml) alone). Addition of exogenous TNF-$\alpha$ into the four hour cytotoxicity assay did not increase ADCC, nor did anti-TNF antibodies result in inhibition. On the other hand, addition of anti-TNF antibodies to PBL and IL-2 for 24 hours, resulted in a marked inhibition of the ADCC, suggesting that endogenous TNF-$\alpha$ is obligatory for the maturation and differentiation of ADCC effectors. ^

Microglia at brain stab wounds express connexin 43 and in vitro form functional gap junctions after treatment with interferon-γ and tumor necrosis factor-α

Gap junctional communication between microglia was investigated at rat brain stab wounds and in primary cultures of rat and mouse cells. Under resting conditions, rat microglia (FITC-isolectin-B4-reactive cells) were sparsely distributed in the neocortex, and most (95%) were not immunoreactive for Cx43, a gap junction protein subunit. At brain stab wounds, microglia progressively accumulated over several days and formed aggregates that frequently showed Cx43 immunoreactivity at interfaces between cells. In primary culture, microglia showed low levels of Cx43 determined by Western blotting, diffuse intracellular Cx43 immunoreactivity, and a low incidence of dye coupling. Treatment with the immunostimulant bacterial lipopolysaccharide (LPS) or the cytokines interferon-γ (INF-γ) or tumor necrosis factor-α (TNF-α) one at a time did not increase the incidence of dye coupling. However, microglia treated with INF-γ plus LPS showed a dramatic increase in dye coupling that was prevented by coapplication of an anti-TNF-α antibody, suggesting the release and autocrine action of TNF-α. Treatment with INF-γ plus TNF-α also greatly increased the incidence of dye coupling and the Cx43 levels with translocation of Cx43 to cell–cell contacts. The cytokine-induced dye coupling was reversibly inhibited by 18α-glycyrrhetinic acid, a gap junction blocker. Cultured mouse microglia also expressed Cx43 and developed dye coupling upon treatment with cytokines, but microglia from homozygous Cx43-deficient mice did not develop significant dye coupling after treatment with either INF-γ plus LPS or INF-γ plus TNF-α. This report demonstrates that microglia can communicate with each other through gap junctions that are induced by inflammatory cytokines, a process that may be important in the elaboration of the inflammatory response.

Biologic therapies and non-melanoma skin cancer rates in three chronic immune-mediated diseases

Trabalho Final do Curso de Mestrado Integrado em Medicina, Faculdade de Medicina, Universidade de Lisboa, 2014

Characterization of tumour necrosis factor alpha release by human granulocytes in response to procainamide challenge

The role of human granulocytes in the promotion of procainamide (PA) toxicity in vitro has been studied and one of the agents responsible for DNA strand scission and cell death in human target cells has been characterized. Crude peripheral blood mononuclear cells (cPBMNs) isolated by density centrifugation, and the lymphocyte cell lines--CCRF-HSB2 and WIL-2NS--were exposed to PA, and DNA strand breaks were quantified by fluorescent analysis of DNA unwinding. Therapeutic plasma concentrations of PA (0-50 microM) caused dose-dependent cytotoxicity, determined by dye exclusion, and strand breaks in cPBMNs incubated for 3 and 1.5 hr at 37 degrees, respectively. Using 50 microM PA a five-fold increase in DNA strand breaks was observed after 1.5 hr, with significant induction of strand breaks also being observed for 10 and 25 microM concentrations. Toxicity was much reduced in lymphocyte cell lines (maximal killing = 3.0% at 50 microM PA compared with 13.2% in cPBMNs). A similar decrease in toxicity was observed where N-acetyl procainamide (NAPA) was substituted for PA (less than 50% of strand breaks at all concentrations). Further investigations showed that the presence of a contaminating granulocyte population in the cPBMN fraction was responsible for the induction of PA toxicity. Incubation of a highly enriched granulocyte population with PA for 1 hr prior to exposure to purified peripheral blood mononuclear cells (pPBMNs) led to the complete restoration of the toxic effects. The resulting cyto- and genotoxicity were not significantly different to levels observed in cPBMNs. Significantly, incubation of granulocytes with NAPA did not induce toxicity in target pPBMNs. Ultrafiltration of granulocyte supernatants led to the identification of two toxic fractions of < 3000 and > 30,000 Da. Temporal studies showed that the toxicity associated with the < 3000 Da fraction appeared during the first 10-15 min incubation with PA whereas the > 30,000 Da fraction did not display significant toxicity until the 40-60 min period. Further assessment of the nature of these agents indicated that the 30,000 Da fraction was a protein. SDS-PAGE analysis showed an inducible 17,800 Da species appearing in granulocyte supernatants after 40 min incubation with PA. Dot blot analysis indicated that tumour necrosis factor alpha (TNF alpha) was present in the > 30,000 Da fraction. Evidence that TNF alpha was the high-molecular weight species responsible for PA-induced toxicity was obtained from neutralization assays employing an anti-TNF alpha antibody.(ABSTRACT TRUNCATED AT 400 WORDS)

Bioengineered Approaches to Prevent Hypertrophic Scar Contraction

Burn injuries in the United States account for over one million hospital admissions per year, with treatment estimated at four billion dollars. Of severe burn patients, 30-90% will develop hypertrophic scars (HSc). Current burn therapies rely upon the use of bioengineered skin equivalents (BSEs), which assist in wound healing but do not prevent HSc. HSc contraction occurs of 6-18 months and results in the formation of a fixed, inelastic skin deformity, with 60% of cases occurring across a joint. HSc contraction is characterized by abnormally high presence of contractile myofibroblasts which normally apoptose at the completion of the proliferative phase of wound healing. Additionally, clinical observation suggests that the likelihood of HSc is increased in injuries with a prolonged immune response. Given the pathogenesis of HSc, we hypothesize that BSEs should be designed with two key anti-scarring characterizes: (1) 3D architecture and surface chemistry to mitigate the inflammatory microenvironment and decrease myofibroblast transition; and (2) using materials which persist in the wound bed throughout the remodeling phase of repair. We employed electrospinning and 3D printing to generate scaffolds with well-controlled degradation rate, surface coatings, and 3D architecture to explore our hypothesis through four aims.

In the first aim, we evaluate the impact of elastomeric, randomly-oriented biostable polyurethane (PU) scaffold on HSc-related outcomes. In unwounded skin, native collagen is arranged randomly, elastin fibers are abundant, and myofibroblasts are absent. Conversely, in scar contractures, collagen is arranged in linear arrays and elastin fibers are few, while myofibroblast density is high. Randomly oriented collagen fibers native to the uninjured dermis encourage random cell alignment through contact guidance and do not transmit as much force as aligned collagen fibers. However, the linear ECM serves as a system for mechanotransduction between cells in a feed-forward mechanism, which perpetuates ECM remodeling and myofibroblast contraction. The electrospinning process allowed us to create scaffolds with randomly-oriented fibers that promote random collagen deposition and decrease myofibroblast formation. Compared to an in vitro HSc contraction model, fibroblast-seeded PU scaffolds significantly decreased matrix and myofibroblast formation. In a murine HSc model, collagen coated PU (ccPU) scaffolds significantly reduced HSc contraction as compared to untreated control wounds and wounds treated with the clinical standard of care. The data from this study suggest that electrospun ccPU scaffolds meet the requirements to mitigate HSc contraction including: reduction of in vitro HSc related outcomes, diminished scar stiffness, and reduced scar contraction. While clinical dogma suggests treating severe burn patients with rapidly biodegrading skin equivalents, these data suggest that a more long-term scaffold may possess merit in reducing HSc.

In the second aim, we further investigate the impact of scaffold longevity on HSc contraction by studying a degradable, elastomeric, randomly oriented, electrospun micro-fibrous scaffold fabricated from the copolymer poly(l-lactide-co-ε-caprolactone) (PLCL). PLCL scaffolds displayed appropriate elastomeric and tensile characteristics for implantation beneath a human skin graft. In vitro analysis using normal human dermal fibroblasts (NHDF) demonstrated that PLCL scaffolds decreased myofibroblast formation as compared to an in vitro HSc contraction model. Using our murine HSc contraction model, we found that HSc contraction was significantly greater in animals treated with standard of care, Integra, as compared to those treated with collagen coated-PLCL (ccPLCL) scaffolds at d 56 following implantation. Finally, wounds treated with ccPLCL were significantly less stiff than control wounds at d 56 in vivo. Together, these data further solidify our hypothesis that scaffolds which persist throughout the remodeling phase of repair represent a clinically translatable method to prevent HSc contraction.

In the third aim, we attempt to optimize cell-scaffold interactions by employing an anti-inflammatory coating on electrospun PLCL scaffolds. The anti-inflammatory sub-epidermal glycosaminoglycan, hyaluronic acid (HA) was used as a coating material for PLCL scaffolds to encourage a regenerative healing phenotype. To minimize local inflammation, an anti-TNFα monoclonal antibody (mAB) was conjugated to the HA backbone prior to PLCL coating. ELISA analysis confirmed mAB activity following conjugation to HA (HA+mAB), and following adsorption of HA+mAB to the PLCL backbone [(HA+mAB)PLCL]. Alican blue staining demonstrated thorough HA coating of PLCL scaffolds using pressure-driven adsorption. In vitro studies demonstrated that treatment with (HA+mAB)PLCL prevented downstream inflammatory events in mouse macrophages treated with soluble TNFα. In vivo studies using our murine HSc contraction model suggested positive impact of HA coating, which was partiall impeded by the inclusion of the TNFα mAB. Further characterization of the inflammatory microenvironment of our murine model is required prior to conclusions regarding the potential for anti-TNFα therapeutics for HSc. Together, our data demonstrate the development of a complex anti-inflammatory coating for PLCL scaffolds, and the potential impact of altering the ECM coating material on HSc contraction.

In the fourth aim, we investigate how scaffold design, specifically pore dimensions, can influence myofibroblast interactions and subsequent formation of OB-cadherin positive adherens junctions in vitro. We collaborated with Wake Forest University to produce 3D printed (3DP) scaffolds with well-controlled pore sizes we hypothesized that decreasing pore size would mitigate intra-cellular communication via OB-cadherin-positive adherens junctions. PU was 3D printed via pressure extrusion in basket-weave design with feature diameter of ~70 µm and pore sizes of 50, 100, or 150 µm. Tensile elastic moduli of 3DP scaffolds were similar to Integra; however, flexural moduli of 3DP were significantly greater than Integra. 3DP scaffolds demonstrated ~50% porosity. 24 h and 5 d western blot data demonstrated significant increases in OB-cadherin expression in 100 µm pores relative to 50 µm pores, suggesting that pore size may play a role in regulating cell-cell communication. To analyze the impact of pore size in these scaffolds on scarring in vivo, scaffolds were implanted beneath skin graft in a murine HSc model. While flexural stiffness resulted in graft necrosis by d 14, cellular and blood vessel integration into scaffolds was evident, suggesting potential for this design if employed in a less stiff material. In this study, we demonstrate for the first time that pore size alone impacts OB-cadherin protein expression in vitro, suggesting that pore size may play a role on adherens junction formation affiliated with the fibroblast-to-myofibroblast transition. Overall, this work introduces a new bioengineered scaffold design to both study the mechanism behind HSc and prevent the clinical burden of this contractile disease.

Together, these studies inform the field of critical design parameters in scaffold design for the prevention of HSc contraction. We propose that scaffold 3D architectural design, surface chemistry, and longevity can be employed as key design parameters during the development of next generation, low-cost scaffolds to mitigate post-burn hypertrophic scar contraction. The lessening of post-burn scarring and scar contraction would improve clinical practice by reducing medical expenditures, increasing patient survival, and dramatically improving quality of life for millions of patients worldwide.

Bilateral Panuveitis at Etanercept Initiation for Juvenile Idiopathic Arthritis

Introduction: Uveitis is a well-known extra-rheumatological manifestation of juvenile idiopathic arthritis (JIA). Tumor necrosis factor has been used to treat uveitis associated with inflammatory diseases. A new onset uveitis under anti-TNF therapy is uncommon. Case presentation: A 12-years-old male, affected since the age of six years, by a severe form of polyarticular JIA. When etanercept was started, he presented panuveitis bilaterally, so we switched to infliximab with good response. Conclusions: The TNF soluble receptor could be considered as a possible promoter in inducing endogenous new onset uveitis in JIA.

DNA Damage and Oxidative DNA Damage in Inflammatory Bowel Disease

Background and aims: Inflammation has long been regarded as a major contributor to cellular oxidative damage and to be involved in the promotion of carcinogenesis. Methods: We aimed to investigate the oxidative damage in inflammatory bowel disease [IBD] patients through a case–control and prospective study involving 344 IBD patients and 294 healthy controls. DNA damage and oxidative DNA damage were measured by comet assay techniques, and oxidative stress by plasmatic lipid peroxidation, protein carbonyls, and total antioxidant capacity. Results: Higher DNA damage [p < 0.001] was found both in Crohn’s disease [CD] (9.7 arbitrary units [AU]; interquartile range [IQR]: 6.2–14.0) and ulcerative colitis [UC] [7.1 AU; IQR: 4.4–11.7], when compared with controls [5.4 AU; IQR: 3.8–6.8], and this was also the case with oxidative DNA damage [p < 0.001] [CD: 3.6 AU; IQR: 1.8–6.8; UC: 4.6 AU; IQR: 2.4–8.1], when compared with controls: 2.3 AU; IQR: 1.2–4.2]. Stratifying patients into groups according to therapy (5-aminosalicylic acid [5-ASA], azathioprine, anti-TNF, and combined therapy [azathioprine and anti-TNF]) revealed significant between-group differences in the level of DNA damage, both in CD and UC, with the combined therapy exhibiting the highest DNA damage levels [11.6 AU; IQR: 9.5–14.3, and 12.4 AU; IQR: 10.6–15.0, respectively]. Among CD patients, disease behaviour [B1 and B2], and age at diagnosis over 40 years [A3] stand as risk factors for DNA damage. For UC patients, the risk factors found for DNA damage were disease activity, treatment, age at diagnosis under 40 years [A1 + A2] and disease locations [E2 and E3]. Conclusions: In IBD there is an increase in DNA damage, and treatment, age at diagnosis and inflammatory burden seem to be risk factors.

Myeloid precursors, osteoclastogenesis, and Spondyloarthropathies

Spondyloarthropathies (or Spondyloarthritides; SpAs) are a group of heterogeneous but genetically related inflammatory disorders in which ankylosing spondylitis (AS) is considered the prototypic form. Among the genes associated with AS, HLA-B27 allele has the strongest association although the cause is still not clear. Rats transgenic for the human HLA-B27 gene (B27 rats) develop a systemic inflammation mirroring the human SpA symptoms and thus provide a useful model to study the contribution of this MHC class I molecule in the disease development. Of particular interest was the observation of absence of arthritis in B27 rats grown in germ-free conditions and a recent theory suggests that microbial dysbiosis and gut inflammation might play a key role in initiating the HLA-B27-associated diseases. Studies in our laboratory have previously demonstrated that HLA-B27 expression alters the development of the myeloid compartment within the bone marrow (BM) in B27 rat and causes loss of a specific dendritic cell (DC) population involved in self-tolerance mechanisms within the gut. The aim of this thesis was to further analyse the myeloid compartment in B27 rats with a particular focus on the osteoclast progenitors and the bone phenotype and to link this to the gut inflammation. In addition, translational studies analysed peripheral monocyte/pre-osteoclasts in AS patients and teased apart the role of cytokines in in vitro human osteoclast differentiation. To understand the dynamics of the myeloid/monocyte compartment within the B27-associated inflammation, monocytes within the bloodstream and BM of B27 rats were characterised via flow cytometry and their ability to differentiate into osteoclast was assessed in vitro. Moreover, an antibiotic regime was used to reduce the B27 ileitis and to evaluate whether this could affect the migration, the phenotype, and the osteoclastogenic potential of B27 monocytes. B27 animals display a systemic and central increase of “inflammatory” CD43low MOs, which are the main contributors to osteoclastogenesis in vitro. Antibiotic treatment reduced ileitis and also reverted the B27 monocyte phenotype. This was also associated with the reduction of the previous described TNFα-enhancement of osteoclast differentiation from B27 BM precursors. These evidences support the idea that in genetically susceptible individuals inflammation in the gut might influence the myeloid compartment within the BM; in other terms, pre-emptively educate precursor cells to acquire specific phenotype end functions after being recruited into the tissue. This might explain the enhanced differentiation of osteoclast from B27 BM progenitors and thus the HLA-B27-associated bone loss. The data shown in this thesis suggest a link between the immunity within the gut and BM haematopoiesis. This provides an attractive and novel research prospective that could help not only to increase the understanding of the HLA-B27-associated aetiopathogenesis but also to unravel the cellular crosstalk that allows the mucosal immunity to program central cell differentiation. Human translational studies on monocyte subsets, cytokines and cytokine network in AS osteoclastogenesis evidenced altered osteoclast differentiation in the presence of IL-22 although no differences in the phenotype and functions of circulating CD14+ monocytes were observed. In addition, studies on the role of TNFα and TNFRs showed a dual role of this inflammatory cytokine in the human OC differentiation. In particular, the activation of TNFR1 in monocytes in early osteoclastogenesis inhibits OC differentiation while TNFα-biasing for TNFR2 on osteoclast precursors mediates the osteoclastogenic effect. Whether similar mechanisms are involved in the TNFα-mediated joint destruction in human rheumatic diseases needs further investigations. This could contribute to the development of novel and more specific anti-TNFα agents for the treatment of bone erosion. In conclusion, taken together my studies support the idea of a crosstalk between the periphery and the central system during the inflammatory response and provide new insights to the mechanisms behind the enhancement of osteoclastogenesis in B27-associated disorders.

Initial experience with golimumab in clinical practice for ulcerative colitis

Background: Golimumab is a TNF-blocking agent indicated as a second-line therapy in ulcerative colitis. Purpose: To research the effectiveness and safety of golimumab in patients with ulcerative colitis in clinical practice. Methods: Retrospective study of the effectiveness and safety of golimumab in patients with ulcerative colitis. All patients received golimumab 200 mg subcutaneously at week 0, and golimumab 100 mg subcutaneously at week 2. After the induction treatment, each patient received 50 mg sc. every 4 weeks in patients with body weight less than 80 kg, and 100 mg every 4 weeks in patients with body weight greater than or equal to 80 kg. Results: Study of a group of 23 ulcerative colitis patients, 7 of whom were naive to any anti-TNF therapy, and 16 patients who had previously been treated with an anti-TNF agent other than golimumab (non-naive patients). The average treatment time with golimumab was 14.3 weeks. Globally, withdrawal of corticosteroids was observed in 74% of cases. Clinical response was observed in 85.5% of patients who had not received biological treatment previously, and in patients who had previously received biological treatment the response rate was 75%. Conclusions: In this short study, golimumab seems to be an alternative treatment in naive and non-naive anti-TNF ulcerative colitis patients. It is also a safe therapy, given that there were no adverse effects in the patients studied.