Sangeetha Thanaraj

John Hamlin

Dr Alexander C Ford

P John Hamlin

Granulocyte / Monocyte Adsorptive Apheresis for Ulcerative Colitis

Keywords: Granulocyte / Monocyte Adsorptive Apheresis for Ulcerative Colitis Inflammatory bowel disease < Disease-based, Large intestine < Organ-based, Devices < Topics, Inflammation < ulcerative colitis blood component removal granulocyte monocyte

come

and usually a variable extent of the colon. The condition is relatively common, with an incidence of 7 to 9 per 100,000 people, [START_REF] Loftus | Ulcerative colitis in Olmsted County, Minnesota, 1940-1993: incidence, prevalence, and survival[END_REF][START_REF] Loftus | Update on the incidence and prevalence of Crohn's disease and ulcerative colitis in Olmsted County, Minnesota, 1940-2000[END_REF] and a prevalence of between 200 and 250 per 100,000 people. [START_REF] Loftus | Ulcerative colitis in Olmsted County, Minnesota, 1940-1993: incidence, prevalence, and survival[END_REF][START_REF] Loftus | Update on the incidence and prevalence of Crohn's disease and ulcerative colitis in Olmsted County, Minnesota, 1940-2000[END_REF][START_REF] Kappelman | The prevalence and geographic distribution of Crohn's disease and ulcerative colitis in the United States[END_REF] It is a lifelong disease, whose natural history is characterised by a relapsing and remitting course, leading to chronic symptoms that affect quality of life and ability to function. [START_REF] Bernklev | Relationship between sick leave, unemployment, disability, and health-related quality of life in patients with inflammatory bowel disease[END_REF][START_REF] Boonen | The impact of inflammatory bowel disease on labor force participation: results of a population sampled casecontrol study[END_REF] As the precise aetiology of UC remains unknown, curative therapy is not possible. However, there are pharmacological agents that are of proven benefit for the condition. Both corticosteroids and 5-aminosalicylic acids (5-ASAs) are more effective than placebo in inducing remission of flare ups of disease activity. [START_REF] Longmore | An assessment of prednisone, salazopyrin, and topical hydrocortisone hemisuccinate used as out-patient treatment for ulcerative colitis[END_REF][START_REF] Truelove | Cortisone in ulcerative colits: final report on a therapeutic trial[END_REF][START_REF] Sutherland | Oral 5-aminosalicylic acid for induction of remission in ulcerative colitis[END_REF] In addition, 5-ASAs and immunosuppressant drugs, such as azathioprine, reduce the risk of subsequent relapse of activity in quiescent disease. [START_REF] Jewell | Azathioprine in ulcerative colitis: final report on controlled therapeutic trial[END_REF][START_REF] Sood | The beneficial effect of azathioprine on maintenace of remission in severe ulcerative colitis[END_REF][START_REF] Sutherland | Oral 5-aminosalicylic acid for maintenance of remission in ulcerative colitis (Review)[END_REF][START_REF] Timmer | Azathioprine and 6mercaptopurine for maintenance of remission in ulcerative colitis[END_REF] Despite this, a proportion of sufferers will go on to develop disease that is resistant to conventional medical therapies, and become corticosteroid-dependent . [START_REF] Jr | The natural history of corticosteroid therapy for inflammatory bowel disease: a population-based study[END_REF][START_REF] Ho | The efficacy of corticosteroid therapy in inflammatory bowel disease: analysis of a 5-year UK inception cohort[END_REF] These individuals may ultimately require panproctocolectomy, with either end ileostomy or ileal pouch anal anastamosis (IPAA) formation.

However, surgery for UC that is not responsive to conventional medical therapy is not without morbidity, and may carry a mortality in excess of 2%, [START_REF]UK IBD audit 2nd round (2008) report: National results for the organisation and process of adult IBD care in the UK (Generic Hospital Report)[END_REF] and quality of life outcomes in patients undergoing IPAA surgery are variable. [START_REF] Lichtenstein | Quality of life after proctocolectomy with ileoanal anastamosis for patients with ulcerative colitis[END_REF] In addition, surgery may not be suitable for all patients, due to either co-morbidities that make such an approach high-risk, or because individual patients do not find this treatment option acceptable. In this situation the clinician is left with a difficult decision to make. Long-term corticosteroid use is clearly not desirable, due to the potentially severe side-effects associated with these drugs, and infliximab, a monoclonal antibody to tumour necrosis factor-α, is not approved for use in subacute manifestations of UC by the National Institute for Health and Clinical Excellence in the UK. [START_REF]Infliximab for subacute manifestations of ulcerative colitis[END_REF] Therefore, alternative medical therapies are required, both to reduce the potential for adverse events associated with long-term corticosteroid use, and to provide non-surgical alternatives for such patients.

Whilst the exact factors that cause UC are incompletely understood, they are likely to include environmental triggers in a genetically susceptible individual, leading to a dysregulation of the immune system of the GI tract. Leukocytes, including granulocytes and monocytes, are recruited into the GI mucosa in UC, [START_REF] Williams | Phagocytes, toxic oxygen metabolites and inflammatory bowel disease: implications for treatment[END_REF] and have the potential to initiate and amplify inflammation by releasing a cascade of proinflammatory cytokines, proteases, and oxygen derivatives, leading to extensive tissue injury. These observations have led to the use of granulocyte / monocyte adsorptive apheresis (GMAA), a technique that removes such activated cells from the peripheral blood. [START_REF] Kashiwagi | Immunomodulatory effects of granulocyte and monocyte adsorption apheresis as a treatment for patients with ulcerative colitis[END_REF] There have been numerous studies published in the literature that have reported the efficacy of this technique in patients with UC, [START_REF] Shimoyama | Safety and efficacy of granulocyte and monocyte adsorption apheresis in patients with active ulcerative colitis: a multicenter study[END_REF][START_REF] Yamamoto | Granulocyte and monocyte adsorptive apheresis in the treatment of active distal ulcerative colitis: a prospective, pilot study[END_REF][START_REF] Suzuki | Selective granulocyte and monocyte adsorptive apheresis as a first-line treatment for steroid naïve patients with active ulcerative colitis: a prospective uncontrolled study[END_REF] but only a limited number of randomised controlled trials (RCTs). We have performed a systematic review of RCTs to assess the efficacy of GMAA in the treatment of UC. UC were eligible for inclusion. The control arms of these trials were required to receive any of conventional medical therapy, sham apheresis procedure, or an alternative regimen using an active GMAA column. Trials that permitted other concomitant therapies were eligible, as long as these were administered to both the intervention and control arms. The primary outcome studied was remission of disease activity. Secondary outcomes, where reported, included response to therapy, if remission rates were not described, effect of GMAA on endoscopic healing and quality of life, evidence of any corticosteroid-sparing effects, and adverse events arising as a result of GMAA.

Studies of GMAA in UC were identified with the terms ulcerative colitis, inflammatory bowel disease, proctitis, or colitis (both as medical subject headings (MeSH) and free text terms). These were combined using the set operator AND with studies identified with the terms: blood component removal, leukapheresis, monocytes, or granulocytes (both as MeSH and free text terms), or granulocyte apheresis, monocyte apheresis, or adacolumn (as free text terms).

Abstracts of the papers identified by the initial search were evaluated by the lead investigator for appropriateness to the study question. All potentially relevant papers were obtained and evaluated in detail.

Assessment of Risk of Bias

This was performed independently by two investigators, with disagreements resolved by discussion with a third investigator. Risk of bias was assessed as described in the Cochrane handbook, [START_REF] Higgins | Cochrane handbook for systematic reviews of interventions: Version 5.0.0. www[END_REF] by recording the method used to generate the randomization schedule, the method used to conceal allocation, whether blinding was implemented, what proportion of patients completed follow-up, whether an intentionto-treat analysis was extractable, and whether there was evidence of selective reporting of outcomes.

RESULTS

The search strategy identified a total of 1729 citations, 16 of which appeared to be relevant to the study question, and were therefore retrieved and evaluated in more detail (Figure 1). Nine articles, [START_REF] Hanai | Adsorptive granulocyte and monocyte apheresis versus prednisolone in patients with corticosteroiddependent moderately severe ulcerative colitis[END_REF][START_REF] Hanai | Intensive granulocyte and monocyte adsorption versus intravenous prednisolone in patients with severe ulcerative colitis: an unblinded randomised multi-centre controlled study[END_REF][START_REF] Sands | A randomized, double-blind, sham-controlled study of granulocyte/monocyte apheresis for active ulcerative colitis[END_REF][START_REF] Sakuraba | An open-label prospective randomized multicenter study shows very rapid remission of ulcerative colitis by intensive granulocyte and monocyte adsorptive apheresis as compared with routine weekly treatment[END_REF][START_REF] Sakuraba | A pilot open-labeled prospective randomized study between weekly and intensive treatment of granulocyte and monocyte adsorption apheresis for active ulcerative colitis[END_REF][START_REF] Ricart | Evaluation of 5 versus 10 granulocyteaphaeresis sessions in steroid-dependent ulcerative colitis: a pilot, prospective, multicenter, randomized study[END_REF][START_REF] Dignass | Clinical trial: five or ten cycles of granulocyte-monocyte apheresis show equivalent efficacy and safety in ulcerative colitis[END_REF][START_REF] Nakamura | Effect of granulocyte and monocyte adsorption apheresis on urinary albumin excretion and plasma endothelin-1 concentration in patients with active ulcerative colitis[END_REF][START_REF] Shimoyama | Granulocyte and monocyte apheresis with the G-1 column in the treatment of patients with active ulcerative colitis[END_REF] reporting the results of ten separate RCTs, were eligible for inclusion in the systematic review. We did not perform a formal metaanalysis due to concerns about the high risk of bias among the RCTs we identified.

Individual trial characteristics are provided in Table 1. Four trials compared GMAA with conventional medical therapy, [START_REF] Hanai | Adsorptive granulocyte and monocyte apheresis versus prednisolone in patients with corticosteroiddependent moderately severe ulcerative colitis[END_REF][START_REF] Hanai | Intensive granulocyte and monocyte adsorption versus intravenous prednisolone in patients with severe ulcerative colitis: an unblinded randomised multi-centre controlled study[END_REF][START_REF] Nakamura | Effect of granulocyte and monocyte adsorption apheresis on urinary albumin excretion and plasma endothelin-1 concentration in patients with active ulcerative colitis[END_REF][START_REF] Shimoyama | Granulocyte and monocyte apheresis with the G-1 column in the treatment of patients with active ulcerative colitis[END_REF] two separate trials (reported in a single article) compared GMAA with sham apheresis, [START_REF] Sands | A randomized, double-blind, sham-controlled study of granulocyte/monocyte apheresis for active ulcerative colitis[END_REF] and four trials compared "intensive" versus "conventional" GMAA regimens. [START_REF] Sakuraba | An open-label prospective randomized multicenter study shows very rapid remission of ulcerative colitis by intensive granulocyte and monocyte adsorptive apheresis as compared with routine weekly treatment[END_REF][START_REF] Sakuraba | A pilot open-labeled prospective randomized study between weekly and intensive treatment of granulocyte and monocyte adsorption apheresis for active ulcerative colitis[END_REF][START_REF] Ricart | Evaluation of 5 versus 10 granulocyteaphaeresis sessions in steroid-dependent ulcerative colitis: a pilot, prospective, multicenter, randomized study[END_REF][START_REF] Dignass | Clinical trial: five or ten cycles of granulocyte-monocyte apheresis show equivalent efficacy and safety in ulcerative colitis[END_REF] Only two trials were at lowrisk of bias according to our assessment. [START_REF] Sands | A randomized, double-blind, sham-controlled study of granulocyte/monocyte apheresis for active ulcerative colitis[END_REF] Six studies were conducted entirely in Japan, [START_REF] Hanai | Adsorptive granulocyte and monocyte apheresis versus prednisolone in patients with corticosteroiddependent moderately severe ulcerative colitis[END_REF][START_REF] Hanai | Intensive granulocyte and monocyte adsorption versus intravenous prednisolone in patients with severe ulcerative colitis: an unblinded randomised multi-centre controlled study[END_REF][START_REF] Sakuraba | An open-label prospective randomized multicenter study shows very rapid remission of ulcerative colitis by intensive granulocyte and monocyte adsorptive apheresis as compared with routine weekly treatment[END_REF][START_REF] Sakuraba | A pilot open-labeled prospective randomized study between weekly and intensive treatment of granulocyte and monocyte adsorption apheresis for active ulcerative colitis[END_REF][START_REF] Nakamura | Effect of granulocyte and monocyte adsorption apheresis on urinary albumin excretion and plasma endothelin-1 concentration in patients with active ulcerative colitis[END_REF][START_REF] Shimoyama | Granulocyte and monocyte apheresis with the G-1 column in the treatment of patients with active ulcerative colitis[END_REF] two in Europe, [START_REF] Ricart | Evaluation of 5 versus 10 granulocyteaphaeresis sessions in steroid-dependent ulcerative colitis: a pilot, prospective, multicenter, randomized study[END_REF][START_REF] Dignass | Clinical trial: five or ten cycles of granulocyte-monocyte apheresis show equivalent efficacy and safety in ulcerative colitis[END_REF] one in the USA and Canada, [START_REF] Sands | A randomized, double-blind, sham-controlled study of granulocyte/monocyte apheresis for active ulcerative colitis[END_REF] and one in both Europe and Japan. [START_REF] Sands | A randomized, double-blind, sham-controlled study of granulocyte/monocyte apheresis for active ulcerative colitis[END_REF] Eight of the trials reported remission rates, [START_REF] Hanai | Adsorptive granulocyte and monocyte apheresis versus prednisolone in patients with corticosteroiddependent moderately severe ulcerative colitis[END_REF][START_REF] Hanai | Intensive granulocyte and monocyte adsorption versus intravenous prednisolone in patients with severe ulcerative colitis: an unblinded randomised multi-centre controlled study[END_REF][START_REF] Sands | A randomized, double-blind, sham-controlled study of granulocyte/monocyte apheresis for active ulcerative colitis[END_REF][START_REF] Sakuraba | An open-label prospective randomized multicenter study shows very rapid remission of ulcerative colitis by intensive granulocyte and monocyte adsorptive apheresis as compared with routine weekly treatment[END_REF][START_REF] Sakuraba | A pilot open-labeled prospective randomized study between weekly and intensive treatment of granulocyte and monocyte adsorption apheresis for active ulcerative colitis[END_REF][START_REF] Ricart | Evaluation of 5 versus 10 granulocyteaphaeresis sessions in steroid-dependent ulcerative colitis: a pilot, prospective, multicenter, randomized study[END_REF][START_REF] Dignass | Clinical trial: five or ten cycles of granulocyte-monocyte apheresis show equivalent efficacy and safety in ulcerative colitis[END_REF] while the remaining two reported response rates to therapy. [START_REF] Nakamura | Effect of granulocyte and monocyte adsorption apheresis on urinary albumin excretion and plasma endothelin-1 concentration in patients with active ulcerative colitis[END_REF][START_REF] Shimoyama | Granulocyte and monocyte apheresis with the G-1 column in the treatment of patients with active ulcerative colitis[END_REF] Six RCTs performed a true intention-to-treat analysis, with dropouts assumed to be treatment failures. 24-26, 28, 31 Efficacy of GMAA compared with conventional medical therapy in achieving remission or response in UC

We identified four trials making this comparison, all of which were conducted in Japanese centres. [START_REF] Hanai | Adsorptive granulocyte and monocyte apheresis versus prednisolone in patients with corticosteroiddependent moderately severe ulcerative colitis[END_REF][START_REF] Hanai | Intensive granulocyte and monocyte adsorption versus intravenous prednisolone in patients with severe ulcerative colitis: an unblinded randomised multi-centre controlled study[END_REF][START_REF] Nakamura | Effect of granulocyte and monocyte adsorption apheresis on urinary albumin excretion and plasma endothelin-1 concentration in patients with active ulcerative colitis[END_REF][START_REF] Shimoyama | Granulocyte and monocyte apheresis with the G-1 column in the treatment of patients with active ulcerative colitis[END_REF] The first, [START_REF] Shimoyama | Granulocyte and monocyte apheresis with the G-1 column in the treatment of patients with active ulcerative colitis[END_REF] The most recent RCT making this comparison was conducted at 5 centres in Japan. [START_REF] Hanai | Intensive granulocyte and monocyte adsorption versus intravenous prednisolone in patients with severe ulcerative colitis: an unblinded randomised multi-centre controlled study[END_REF] The investigators randomised 70 patients with severely active UC, most of whom were receiving 5-ASAs in combination with low-dose prednisolone, to receive either twice-weekly GMAA for 3 weeks followed by weekly GMAA, up to a maximum of 11 sessions in total, or intravenous prednisolone at a dose of 40 to 60mg/day for 5 to 10 days, followed by tapered oral prednisolone. Corticosteroiddependent patients in both treatment arms also received mercaptopurine. Follow-up was at 12 weeks, and the primary outcome measure was clinical remission, defined using a clinical activity index of ≤ 4. The authors also reported the numbers of patients achieving a corticosteroid-free remission, and adverse events rates. Overall, 26 (74.3%) of 35 in the GMAA arm achieved remission, compared with 16 (48.6%) of 35 receiving prednisolone (P = 0.02). In addition, 27 (77.1%) of the GMAA patients were corticosteroid-free at 12 weeks, compared with 5 (14.3%) of the patients receiving prednisolone (P = 0.008). Finally, only 5 (14.3%) GMAA patients reported adverse events, compared with 29 (64.4%) of those receiving corticosteroids.

Efficacy of GMAA compared with sham procedure in achieving remission of UC

There were two RCTs comparing GMAA with a sham apheresis procedure, reported in a single article. [START_REF] Sands | A randomized, double-blind, sham-controlled study of granulocyte/monocyte apheresis for active ulcerative colitis[END_REF] The larger of these two trials was conducted in the USA and Canada, and recruited 168 patients with moderate to severely active UC who were already receiving treatment with at least one of 5-ASA, corticosteroids, azathioprine, or mercaptopurine. Participants were treated for 9 weeks, and the primary outcome of were not reported in detail, but were said to be comparable between active intervention and control patients in both trials. Serious adverse events were reported in 14% of all GMAA and 11% of all sham apheresis patients.

Given the perhaps unexpected finding that GMAA was not significantly superior to sham apheresis in achieving clinical remission in active UC, the authors conducted a subgroup analysis, which included only the 63 patients with histological evidence of mucosal erosions or ulceration at baseline. Clinical remission was achieved in 11 (24%) of the 46 of these individuals who received GMAA versus 0 (0%) of the 17 randomised to sham apheresis (P = 0.03). These data suggest that GMAA is of benefit in patients with confirmed endoscopically active disease, and that the treatment effect may have been diluted by the inclusion of patients with much milder disease, or perhaps even functional symptoms.

Efficacy of "intensive" GMAA regimens compared with "conventional" GMAA regimens in achieving remission of UC

There were four RCTs that compared "intensive" GMAA regimens with "conventional" regimens. [START_REF] Sakuraba | An open-label prospective randomized multicenter study shows very rapid remission of ulcerative colitis by intensive granulocyte and monocyte adsorptive apheresis as compared with routine weekly treatment[END_REF][START_REF] Sakuraba | A pilot open-labeled prospective randomized study between weekly and intensive treatment of granulocyte and monocyte adsorption apheresis for active ulcerative colitis[END_REF][START_REF] Ricart | Evaluation of 5 versus 10 granulocyteaphaeresis sessions in steroid-dependent ulcerative colitis: a pilot, prospective, multicenter, randomized study[END_REF][START_REF] Dignass | Clinical trial: five or ten cycles of granulocyte-monocyte apheresis show equivalent efficacy and safety in ulcerative colitis[END_REF] Ricart et al. randomised 20 UC patients with moderately active disease, all of whom were corticosteroid-dependent, to either 10 apheresis sessions over 10 weeks, or five sessions over 5 weeks. [START_REF] Ricart | Evaluation of 5 versus 10 granulocyteaphaeresis sessions in steroid-dependent ulcerative colitis: a pilot, prospective, multicenter, randomized study[END_REF] The primary outcome was clinical remission, according to a clinical activity index of ≤ 4, at week 17. Corticosteroid requirements and quality of life were also recorded. In total, 19 patients completed the study. In the "intensive" arm, 5 (45.5%) of 11 were in remission, compared with 3 (37.5%) of 8 in the "conventional" arm. All those who achieved clinical remission were also in endoscopic remission, and all but one of these patients were corticosteroid-free at week 17. Quality of life scores improved from baseline in both groups, but these differences were not statistically significant.

There were no serious adverse events observed in either arm of the trial.

In a small Japanese RCT conducted at a single centre, [START_REF] Sakuraba | A pilot open-labeled prospective randomized study between weekly and intensive treatment of granulocyte and monocyte adsorption apheresis for active ulcerative colitis[END_REF] 30 patients with moderately active UC, 50% of whom were corticosteroid-dependent, were randomised to "intensive" GMAA, consisting of three sessions per week for 2 weeks followed by one session for 4 weeks, or "conventional" GMAA with 10 weekly sessions. The primary endpoint was remission 1 week after the end of therapy ( 7weeks follow-up for the "intensive" arm, versus 11 weeks for the "conventional"), defined using a clinical activity index of ≤ 4, as well as time (in days) to remission.

Changes in quality of life, corticosteroid dose, and endoscopic scores were also reported. Overall, 12 (80.0%) of 15 in the "intensive" GMAA arm were in remission, compared with 10 (66.7%) of 15 in the "conventional" GMAA arm, but this difference was not statistically significant (P = 0.25). However, time to remission was significantly lower in those in the "intensive arm" (10.7 days versus 27.2 days, P = 0.04). Again, there was no significant difference observed in quality of life scores, and there were no significant differences in endoscopic scores between the two arms.

Corticosteroid doses were significantly lower at the end of treatment, compared with baseline, in both arms (P = 0.03 for both). There were four adverse events observed, two in each group.

A multi-centre Japanese RCT compared twice-weekly with weekly GMAA, up to a maximum of 10 sessions, in 163 UC patients with mildly to moderately active disease. [START_REF] Sakuraba | An open-label prospective randomized multicenter study shows very rapid remission of ulcerative colitis by intensive granulocyte and monocyte adsorptive apheresis as compared with routine weekly treatment[END_REF] The primary outcome measure was clinical remission, again defined using a clinical activity index of ≤ 4, at 5 weeks in the "intensive" arm and 10 weeks in the "conventional" arm. Time to remission was also compared between the two treatment regimens. In total, 149 patients provided analysable data at study end. The remission rate was higher in the "intensive" therapy arm of the trial, 71.2% (52 of 73) versus 54.0% (41 of 76) in the "conventional" arm (P = 0.03), and time to remission was significantly lower in the "intensive" therapy arm (14.9 days versus 28.1 days, P < 0.001). Adverse events occurred in similar numbers of patients in each arm of the trial (17 in "intensive" versus 14 in "conventional"), but none were serious.

The largest RCT, to date, to compare "intensive" with "conventional" GMAA was conducted in 42 centres in Europe and recruited 186 patients with moderately to severely active corticosteroid-dependent or resistant UC. [START_REF] Dignass | Clinical trial: five or ten cycles of granulocyte-monocyte apheresis show equivalent efficacy and safety in ulcerative colitis[END_REF] The "intensive" arm received twice-weekly apheresis for 2 weeks followed by a further six sessions over 6 weeks. In the "conventional" arm GMAA was administered weekly for 5 weeks. All patients were already receiving treatment with one or more of 5-ASA, corticosteroids, azathioprine, or mercaptopurine, unless previous intolerance or non-response to these drugs had been demonstrated. [START_REF] Hanai | Intensive granulocyte and monocyte adsorption versus intravenous prednisolone in patients with severe ulcerative colitis: an unblinded randomised multi-centre controlled study[END_REF] which were significantly higher than those achieved with conventional medical therapy. In corticosteroid-dependent UC, rates of remission with GMAA were as high as 85%. [START_REF] Hanai | Adsorptive granulocyte and monocyte apheresis versus prednisolone in patients with corticosteroiddependent moderately severe ulcerative colitis[END_REF] There was also evidence for corticosteroid-sparing effects with GMAA, with significantly lower cumulative doses of corticosteroid, [START_REF] Hanai | Adsorptive granulocyte and monocyte apheresis versus prednisolone in patients with corticosteroiddependent moderately severe ulcerative colitis[END_REF] and significantly higher rates of corticosteroid-free remission in patients allocated to GMAA. [START_REF] Hanai | Intensive granulocyte and monocyte adsorption versus intravenous prednisolone in patients with severe ulcerative colitis: an unblinded randomised multi-centre controlled study[END_REF] In addition, adverse event rates were significantly higher in patients assigned to conventional medical therapy. [START_REF] Hanai | Intensive granulocyte and monocyte adsorption versus intravenous prednisolone in patients with severe ulcerative colitis: an unblinded randomised multi-centre controlled study[END_REF] Compared with a sham apheresis procedure in patients with moderately to severely active UC, [START_REF] Sands | A randomized, double-blind, sham-controlled study of granulocyte/monocyte apheresis for active ulcerative colitis[END_REF] GMAA did not achieve significantly higher rates of clinical or endoscopic remission, and adverse events rates were comparable.

However, when only individuals with evidence of mucosal erosions or ulceration at baseline histological examination were considered in the analysis, significantly higher remission rates were observed with GMAA. In terms of "intensive" GMAA regimens versus "conventional" regimens, available data were conflicting. Three trials suggested that "intensive" regimens were associated with generally higher rates of remission, [START_REF] Sakuraba | An open-label prospective randomized multicenter study shows very rapid remission of ulcerative colitis by intensive granulocyte and monocyte adsorptive apheresis as compared with routine weekly treatment[END_REF][START_REF] Sakuraba | A pilot open-labeled prospective randomized study between weekly and intensive treatment of granulocyte and monocyte adsorption apheresis for active ulcerative colitis[END_REF][START_REF] Ricart | Evaluation of 5 versus 10 granulocyteaphaeresis sessions in steroid-dependent ulcerative colitis: a pilot, prospective, multicenter, randomized study[END_REF] and time to achievement of remission was significantly shorter in two RCTs recruiting patients with mildly to moderately active UC. [START_REF] Sakuraba | An open-label prospective randomized multicenter study shows very rapid remission of ulcerative colitis by intensive granulocyte and monocyte adsorptive apheresis as compared with routine weekly treatment[END_REF][START_REF] Sakuraba | A pilot open-labeled prospective randomized study between weekly and intensive treatment of granulocyte and monocyte adsorption apheresis for active ulcerative colitis[END_REF] However, the largest RCT to date demonstrated almost identical remission rates with "intensive"

and "conventional" regimens in corticosteroid-dependent patients with moderately to severely active disease, and no statistically significant difference in the time to remission. [START_REF] Dignass | Clinical trial: five or ten cycles of granulocyte-monocyte apheresis show equivalent efficacy and safety in ulcerative colitis[END_REF] The effect of GMAA on quality of life was not studied in many of the eligible RCTs but, where reported, there appeared to be no significant benefit of GMAA over conventional medical therapy, sham apheresis procedure, or between the various "intensive" and "conventional" regimens.

Strengths of our study include our rigorous methodology. We described our search strategy, eligibility criteria, and data extraction processes in detail. In addition, independent data extraction was undertaken by two reviewers, and discrepancies were checked and resolved. Finally, the majority of the trials we identified used a validated definition of remission as a clinical endpoint, rather than response to therapy, which we believe is a more clinically useful outcome to the physician who is considering GMAA as a treatment option in a patient with chronic active or corticosteroiddependent UC who is unsuitable for, or refuses, surgery.

Limitations of the present study, as with any systematic review and metaanalysis, arise from the quality of the trials included. Only two of the ten eligible and included RCTs were at low risk of bias, [START_REF] Sands | A randomized, double-blind, sham-controlled study of granulocyte/monocyte apheresis for active ulcerative colitis[END_REF] and most trials did not report either the method used to generate the randomisation list, or whether allocation to treatment was concealed. In addition, only three trials reported that investigators were blinded to the assigned therapy. [START_REF] Hanai | Intensive granulocyte and monocyte adsorption versus intravenous prednisolone in patients with severe ulcerative colitis: an unblinded randomised multi-centre controlled study[END_REF][START_REF] Sands | A randomized, double-blind, sham-controlled study of granulocyte/monocyte apheresis for active ulcerative colitis[END_REF] It has been well-described in the systematic review literature that RCTs that do not adhere to such reporting of their methodology, and trials that are not blinded, tend to overestimate the treatment effect associated with the active intervention. [START_REF] Juni | Assessing the quality of controlled clinical trials[END_REF] Only six trials reported data using a true intention-to-treat analysis. 24- 26, 28, 31 Again, this may have led to an overestimation of the success rates with active therapy. A final limitation is the fact that six of the ten RCTs were conducted in Japan, meaning that their results may not be generalisable to a Western population, There has been one previous systematic review to our knowledge, to date, that has examined the efficacy of GMAA in UC. [START_REF] Habermalz | Clinical effectiveness of selective granulocyte, monocyte adsorptive apheresis with the Adacolumn device in ulcerative colitis[END_REF] In contrast to the current study, the authors felt it appropriate to perform a meta-analysis. They reported a statistically significant benefit of GMAA over control therapy, defined using either response to therapy or clinical remission, with a number needed to treat of 5, and concluded that homogeneous evidence from seven RCTs uniformly indicated that GMAA was more successful than drug therapy in achieving response and remission. There are some limitations of this meta-analysis that we have discussed elsewhere, [START_REF] Thanaraj | Is the benefit of granulocyte monocyte adsorptive apheresis in ulcerative colitis overstated?[END_REF] including pooling of data from RCTs regardless of the control intervention, and the inclusion of a pseudo-randomised study in the analysis, [START_REF] Bresci | Treatment of patients with acute ulcerative colitis:conventional corticosteroid therapy (MP) versus granulocytapheresis (GMA): a pilot study[END_REF] a type of study design that is liable to selection bias. In addition, the largest RCT that they identified showed no significant effect of GMAA on remission or response. [START_REF] Sands | A randomized, double-blind, sham-controlled study of granulocyte/monocyte apheresis for active ulcerative colitis[END_REF] There was also statistically significant heterogeneity between studies, [START_REF] Higgins | Measuring inconsistency in meta-analyses[END_REF] perhaps due to underlying differences in the GMAA regimens used in the various trials.

Data from the current study demonstrate some evidence for a benefit of GMAA over conventional medical therapy, in patients with either active or corticosteroid-dependent UC, in terms of clinical remission, corticosteroid-sparing effects, and reduced adverse events rates, but no trial to date making this comparison has been conducted in a Western population. The only study to compare GMAA with a sham apheresis procedure, conducted in both Western and Japanese patients, failed to demonstrate any significant increase in remission rates with GMAA. The authors speculated that their patient population may have differed from those in previous trials in terms of disease activity, and that GMAA may only be effective in patients with severe histological activity, an assertion supported by their post hoc analysis discussed in detail earlier. There seems to be little to choose between various GMAA regimens in terms of clinical remission rates, though time to remission may be shorter with a more "intensive" regimen. Adverse events were no higher with GMAA in most studies, and serious adverse events were rare, suggesting it is a safe treatment option.

Finally, data to support any beneficial effect of GMAA on quality of life are sparse.

F o r P e e r R e v i e w

4-5

Objectives 4 Provide an explicit statement of questions being addressed with reference to participants, interventions, comparisons, outcomes, and study design (PICOS).

5

METHODS

Protocol and registration

5 Indicate if a review protocol exists, if and where it can be accessed (e.g., Web address), and, if available, provide registration information including registration number.

N/A

Eligibility criteria 6 Specify study characteristics (e.g., PICOS, length of follow-up) and report characteristics (e.g., years considered, language, publication status) used as criteria for eligibility, giving rationale.

6

Information sources 7 Describe all information sources (e.g., databases with dates of coverage, contact with study authors to identify additional studies) in the search and date last searched.

6 Search 8 Present full electronic search strategy for at least one database, including any limits used, such that it could be repeated.

6

Study selection 9 State the process for selecting studies (i.e., screening, eligibility, included in systematic review, and, if applicable, included in the meta-analysis).

6-7

Data collection process 10 Describe method of data extraction from reports (e.g., piloted forms, independently, in duplicate) and any processes for obtaining and confirming data from investigators.

7

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Data items 11 List and define all variables for which data were sought (e.g., PICOS, funding sources) and any assumptions and simplifications made.

Risk of bias in individual studies

12 Describe methods used for assessing risk of bias of individual studies (including specification of whether this was done at the study or outcome level), and how this information is to be used in any data synthesis.

7

Summary measures 13 State the principal summary measures (e.g., risk ratio, difference in means).

N/A

Synthesis of results 14 Describe the methods of handling data and combining results of studies, if done, including measures of consistency (e.g., I

2

) for each meta-analysis.

N/A

Page 1 of 2

Section/topic # Checklist item Reported on page #

Risk of bias across studies 15 Specify any assessment of risk of bias that may affect the cumulative evidence (e.g., publication bias, selective reporting within studies).

7

Additional analyses 16 Describe methods of additional analyses (e.g., sensitivity or subgroup analyses, meta-regression), if done, indicating which were pre-specified.

N/A

RESULTS

Study selection 17

Give numbers of studies screened, assessed for eligibility, and included in the review, with reasons for exclusions at each stage, ideally with a flow diagram.

and 26

Study characteristics 18 For each study, present characteristics for which data were extracted (e.g., study size, PICOS, follow-up period) and provide the citations.

27-32

Risk of bias within studies 19 Present data on risk of bias of each study and, if available, any outcome level assessment (see item 12).

27-32

Results of individual studies 20 For all outcomes considered (benefits or harms), present, for each study: (a) simple summary data for each intervention group (b) effect estimates and confidence intervals, ideally with a forest plot.

8-14

Synthesis of results 21 Present results of each meta-analysis done, including confidence intervals and measures of consistency.

N/A

Risk of bias across studies 22 Present results of any assessment of risk of bias across studies (see Item 15).

8, 27-32

Additional analysis 23 Give results of additional analyses, if done (e.g., sensitivity or subgroup analyses, meta-regression [see Item 16]).

N/A We agreed with the editor. We added text to a sentence to the Conclusions section of the Abstract on page 3: "High-quality RCTs comparing GMAA with conventional medical therapy or sham procedure in Western populations, with disease activity confirmed endoscopically, are required."

F o r P e e r R e v i e w

We added text to the reporting of data from the Sands study in the Results section on page 11: "These data suggest that GMAA is of benefit in patients with confirmed endoscopically active disease, and that the treatment effect may have been diluted by the inclusion of patients with much milder disease, or perhaps even functional symptoms."

We altered a sentence in the Discussion section on pages 17 to 18 to read: "The authors speculated that their patient population may have differed from those in previous trials in terms of disease activity, and that GMAA may only be effective in patients with severe histological activity, an assertion supported by their post hoc analysis discussed in detail earlier."

We added text to the final paragraph of the Discussion section on pages 18 and 19, such that it now reads: "In summary, this systematic review has demonstrated some evidence for a beneficial effect of GMAA in active or corticosteroid-dependent UC, We agreed with the reviewer that this was an oversight on our part, and thank them for pointing this out. We reported the absolute numbers responding to therapy or in remission, proportions, and P values reported for these differences in each arm in Table 1 on pages 27 to 32.

The PRISMA statement is required to be submitted online by the journal, which was our reason for providing it. We apologise if the reviewer was confused as to why this had been submitted.

We do not report odds ratios as we did not perform a meta-analysis due to underlying differences in study design.

2.A peer-reviewed meta-analysis have already been published (1). This paper is

imperfect but I think the authors' criticism of it is harsh. Their statistical analysis is sound and their overall conclusion seems fair. While I agree that there is considerable heterogeneity in trial design, most of the trials are intervention vs non-intervention (or sham). The published studies are varied, tend to be small and exactly the scenario to perform a meta-analysis in my view.

We tempered our criticism of the aforementioned published meta-analysis in the Discussion section on page 17 to read: "There are some limitations of this metaanalysis that we have discussed elsewhere, [START_REF] Thanaraj | Is the benefit of granulocyte monocyte adsorptive apheresis in ulcerative colitis overstated?[END_REF] including pooling of data from RCTs regardless of the control intervention, and the inclusion of a pseudo-randomised study in the analysis, [START_REF] Bresci | Treatment of patients with acute ulcerative colitis:conventional corticosteroid therapy (MP) versus granulocytapheresis (GMA): a pilot study[END_REF] a type of study design that is liable to selection bias. In addition, the largest RCT that they identified showed no significant effect of GMAA on remission or response. [START_REF] Sands | A randomized, double-blind, sham-controlled study of granulocyte/monocyte apheresis for active ulcerative colitis[END_REF] There was also statistically significant heterogeneity between studies, [START_REF] Higgins | Measuring inconsistency in meta-analyses[END_REF] perhaps due to subtle differences in the GMAA regimens used in the various trials." We did not reject any studies from the systematic review, but rather reported the findings of each faithfully. We merely pointed out the limitations of the studies as reported in the original publications.

We are confused by the reviewer's comment. Blinding and concealment of allocation are two separate issues. As the reviewer correctly states, it is not possible to blind patients in a non-sham study as to whether or not they received GMAA or conventional medical therapy. However, in non-sham studies it would still be possible to report whether or not treatment allocation (the order in which treatments were assigned to patients) was concealed. If treatment allocation is not concealed in an RCT, the randomisation process can be subverted (advertently or inadvertently) by the physician who is recruiting patients making (conscious or unconscious) decisions as to whether or not to include a patient in the trial on the basis of their disease characteristics, if he / she knows which therapy is due to be assigned to the next recruited patient.

Only two non-sham studies did not report blinding of physicians. We attempted to obtain information in both cases, but received no response from the authors. and reject all "risk-of-bias" studies then they only have the Sands study as admissible for evaluation. Hence their conclusion should be " There is insufficient evidence that GMAA is of any benefit in the treatment of ulcerative colitis" Instead their statement is: "Remission rates in excess of 70% were achieved with GMAA in patients with severely active UC, which were significantly higher than those achieved with conventional medical therapy. In corticosteroid-dependent UC, rates of remission with GMAA were as high as 85%." which is baffling.

Again, we were confused by the reviewer's comment. We did not reject any studies from the systematic review, but reported the results of each faithfully in the text (and Table 1) of the article. We agreed with the reviewer. The editor also asked us to address this issue. Please see our response to the editor's comments above. were intolerant of it. All medications for UC remained unchanged during the 12 weeks of the trial, so both trial arms received conventional medical therapy. We therefore left our statement unchanged as it is correct in that, to date, there has been no comparison of GMAA alone with conventional therapy alone in Western UC patients.

Reviewer: 2

Comments for Transmission to the Authors

This is an excellent review article that describes clearly the state or art of the apheresis in UC.

We thank the reviewer for their kind comments. No response required.

In the abstract the statement that meta-analysis was not run could be moved into the results section

The reviewer makes a valid point, which we agreed with. We moved this statement to the Results section of the Abstract as the reviewer suggested. We also moved this statement from the last paragraph of the Methods section of the article itself, on page 7, to the first paragraph of the Results section on page 8. We did not report heterogeneity between results of trials we identified in the article as we did not perform a formal meta-analysis. We presumed the reviewer referred to heterogeneity in the meta-analysis by Habermalz and Sauerland, which we had mentioned in the Discussion section. We agreed with the reviewer that this may have been an explanation for some of this heterogeneity, and added this point to our discussion of the aforementioned meta-analysis on page 17 of the Discussion section:

"There was also statistically significant heterogeneity between studies, [START_REF] Higgins | Measuring inconsistency in meta-analyses[END_REF] perhaps due to underlying differences in the GMAA regimens used in the various trials."

INTRODUCTION

  Ulcerative colitis (UC) is an inflammatory disorder of the gastrointestinal (GI) tract, of unknown aetiology. It causes continuous inflammation affecting the rectum,

  the medical literature was conducted using MEDLINE (1950 to August 2010), EMBASE (1946 to August 2010), and the Cochrane central register of controlled trials. Randomised controlled trials examining the efficacy of GMAA in adult patients (> 90% over 16 years of age) with active or corticosteroid-dependent

  number of individuals achieving clinical remission, defined using a Mayo score of 2 or less, at 12 weeks post-randomisation. Secondary outcome measures included endoscopic remission, and change in quality of life from baseline. Overall, 19 (17.0%) of 112 patients were in clinical remission at 12 weeks with GMAA compared with 6 (10.7%) of 56 receiving the sham apheresis procedure (P = 0.36). The proportion of patients achieving endoscopic remission was very similar in both treatment arms (12% versus 11%, P = 1.0). The smaller trial was of identical design, and recruited 47 UC patients from 11 European and 1 Japanese centre. Clinical remission occurred in 5 (16.1%) of 31 and 3 (18.8%) of 16 in the GMAA and sham apheresis arms respectively. Changes in quality of life scores from baseline

  underlying differences in patient characteristics, genetics, or environment.

3

 3 Describe the rationale for the review in the context of what is already known.

19 From:

 19 Moher D, Liberati A, Tetzlaff J, Altman DG, The PRISMA Group (2009). Preferred Reporting Items for Systematic Reviews and Meta-Analyses: The PRISMA Statement. PLoS Med 6(6): e1000097. doi:10.1371/journal.pmed1000097For more information, visit: www.prisma-statement.org. reviewers' comments. My personal opinion is that more might be made of the point that the Sands study showed a positive effect of apheresis in patients with greater mucosal ulceration and that future studies should not only be sham controlled but should take care to exclude patients who do not have mucosal ulceration.

1 .

 1 both clinical remission rates and corticosteroid-sparing effects, but more data from trials conducted in Western patients with endoscopic evidence of active disease are required before its true place in the management of UC is known." descriptive systematic review article on the therapeutic value of selective granulocyte, monocyte adsorptive apheresis with the Adacolumn device in ulcerative colitis. This review is purely descriptive and no attempt was made to perform a meta-analysis or estimate the size of the effect. It is the authors contention that the available studies are not of sufficient quality to perform a meta-analysis. Without any statistical tool to objectively make an assessment of the size of the effect they can merely summarize each study and let the reader decide on the relative merits of each study. There is a table of all the studies reviewed but it does not contain just about the most useful thing: the actual results! The authors must summarise the results in a table for the readers to make that value judgement. What is the value of reproducing the PRISMA statement? That could be left out and the trial results, OR and P values put in.

3 .

 3 The authors have identified 10 RCT. They remark that concealment was "not stated" in the non-sham studies, and these studies were rejected as having "risk of bias". Non-sham studies can at best be single-blinded. If they are still unclear if the physician is blinded, did the authors attempt to ask the trial authors? (in an email age it would be slothful not to).

4 .

 4 Having rejected these studies, the only admissible study is the Sands study.Here the authors conclusions are confused. If they are as rigorous as they claim

5 .

 5 The Sands study (2) itself may be underestimating the true effect of GMAA since only 63/168 (36%) had histological evidence of mucosal inflammation. If a large number of non-inflammed "colitis" patients were recruited this would dilute the effect of an anti-inflammatory intervention. If the Japanese studies were overstating the case for GMAA, it could be argued that the Sands study is equally flawed in the opposite direction. This is where a meta-analysis would be useful in estimating "true" effect size.

Pg. 17 "Pg 18 "

 1718 If this trial were to be included at all, it should also have been excluded in a sensitivity analysis to assess its impact on the bottom-line result of the metaanalysis." No. You either include the study or exclude it both in the metaanalysis and results, you cannot pick and choose. This part of the text has been removed from the article in line with the reviewer's second major point above. Data from the current study demonstrate some evidence for a benefit of GMAA over conventional medical therapy, in patients with either active or corticosteroid-dependent UC, in terms of clinical remission, corticosteroidsparing effects, and reduced adverse events rates, but no trial to date making this comparison has been conducted in a Western population." This is at best a confusing statement at worse plainly wrong. The Sands study is largely in N American populations and compares intervention vs sham, the latter group had "conventional medical therapy" except that sham therapy was included to make it double-blind.We are again unclear as to what the reviewer means by this comment. The Sands study was not a comparison of GMAA with conventional medical therapy, but a trial of GMAA versus sham procedure in UC patients with active disease who were already receiving conventional medical therapy and who had not responded to it, or

  some heterogeneity within trials because different techniques of apheresis have been used with likely different mechanisms of action?

  The bibliographies of all identified

	Thanaraj et al.	7 of 35
	relevant studies were used to perform a recursive search of the literature. Articles
	were assessed independently by two investigators using pre-designed eligibility
	forms, according to the eligibility criteria stated previously. Any disagreement
	between investigators was resolved by discussion with a third investigator.
	Data Extraction
	All data were extracted independently by two investigators on to a Microsoft
	r o F Excel spreadsheet (XP professional edition; Microsoft Corp, Redmond, WA, USA) as
	P
	e
	e r
	R
	e
	v i e
	w

dichotomous outcomes (i.e. remission / response or failure of remission / response). In addition, the following clinical data were extracted for each trial, where available: number of centres, country of origin, geographical region, UC characteristics (severity of UC activity, presence of corticosteroid-dependence or resistance), schedule of GMAA used, duration of therapy, primary outcome measure used to define remission or response following therapy, and duration of follow-up.

  The primary outcome of interest was clinical remission at 12 weeks, according to a clinical activity index ≤ 4, with 162 patients providing data for this analysis. Time to remission and endoscopic activity were also reported.

	DISCUSSION
	Remission rates in the two arms were almost identical (43.9% (36 of 82 patients) with This systematic review has collected all available published RCT evidence
	"intensive" versus 40.0% (32 of 80 patients) with "conventional"), and there was no examining the effects of GMAA in both active and corticosteroid-dependent or
	statistically significant difference observed in terms of time to remission (39.9 days resistant UC. Compared with conventional medical therapy, in the form of
	versus 46.8 days) or in endoscopic activity. Overall, 6% of the "intensive" therapy corticosteroids, GMAA appeared superior in several respects. Remission rates in
	arm and 9% of the "conventional" arm experienced a serious adverse event. excess of 70% were achieved with GMAA in patients with severely active UC,
	F o r F o r
	P P
	e e
	e r e r
	R R
	e e
	v i e v i e
	w w

Flow Diagram of Assessment of Studies Identified in the Systematic Review.

  Taken together these data tell us that, rather than designing future RCTs to examine the potential benefit of one GMAA regimen over another, what is actually

	F o r P e e r conducted in Western patients with endoscopic evidence of active disease are required before its true place in the management of UC is known. STATEMENTS OF INTEREST Authors' Declaration of Personal Interests: Sangeetha Thanaraj: none declared. P. John Hamlin: has participated in an advisory board for Otsuka Pharmaceuticals. Alexander C. Ford: none declared. Declaration of Funding Interests: None. F o r P e e r F o r P e e r F o r P e e r F o r P e e r F o r P e e r F o r P e e r F o r P e e r Articles identified in literature search (n = 1729) Articles retrieved for evaluation (n = 16) Excluded (title and abstract revealed Figure 1. Excluded (n = 7) because: not appropriate) (n = 1713)
			•	Not a randomised controlled
	R e v i e R e v i e R e v i e R e v i e R e v i e R e v i e R e v i e R e e i v	• • • •	trial = 3 Not the comparison of interest = 1 Pseudo-randomised trial = 1 Dual publication = 1 Not all patients had UC = 1
	Articles of GMAA in active UC eligible for inclusion (n = 9)	w w w w w w w w

required are convincing high-quality data that GMAA is effective in Western patients with UC. Trials comparing GMAA with conventional medical therapy in corticosteroid-dependent patients are needed to demonstrate that the results from Japanese trials are reproducible. In addition, further rigorous RCTs, of similar design to that of Sands et al.,

[START_REF] Sands | A randomized, double-blind, sham-controlled study of granulocyte/monocyte apheresis for active ulcerative colitis[END_REF]

should compare GMAA with sham apheresis in patients with histological evidence of active disease. One potential niche for GMAA is in patients with chronic subacute UC refractory to, or intolerant of, 5-ASAs or immunomodulators, who are corticosteroid-dependent, but who do not have acute severe disease, and who are therefore unsuitable for either infliximab or ciclosporin.

The only other treatment option at present for this type of patient is surgery, which may not be suitable, or desirable, for all individuals. Therefore studies that compare quality of life in patients in this situation who receive GMAA or panproctocolectomy, followed by either end ileostomy or IPAA, would also be of great interest to both patients and physicians.

In summary, this systematic review has demonstrated some evidence for a beneficial effect of GMAA in active or corticosteroid-dependent UC, in terms of both clinical remission rates and corticosteroid-sparing effects, but more data from trials

Table 1 . Characteristics of Randomized Controlled Trials of Granulocyte / Monocyte Adsorptive Apheresis in Active or Corticosteroid- dependent UC.

 1


N Blanco

E K Gamstedt
email: gamstedt@kth.se

J Costa

tests in beam-type composite specimens

Keywords: A. Polymer-matrix composites (PMCs), B. Delamination, B. Fracture toughness, A. Adhesive joints, Mechanical hinge

During the experimental study of composite delaminations external loads are usually applied by means of steel or aluminium parts bonded to the surface of beam-type specimens. The bonded joints between the metallic parts and the composite specimen might fail, especially when the tests are carried out under extreme temperatures or fatigue conditions. In addition, the point of application of the external load does not coincide with the neutral axis of the specimen beam, inducing non-linear effects that can lead, for example, to incorrect estimations of fracture toughness. In this paper, the relative importance of the non-linear effects in delamination tests is evaluated and the corresponding correction factors discussed. Next, the design of an improved mechanical hinge that avoids non-linear effects, eliminates bonded joints and can be adapted to different specimen thicknesses is introduced.

Beam-type specimens are widely used to experimentally study the static and fatigue growth of composite delaminations under laboratory conditions, as well as to determine the peel and shear resistance of adhesives. These specimens are generally tested in mode I using the double cantilever beam test (DCB), in mode II using the end-notched flexure test (ENF) or the end load split test (ELS) and in mixed-mode I/II using the mixed-mode bending test (MMB) or the mixed-mode end load split test (MMELS). Except for the ENF test, which basically consists of a three-point bending test, the other tests mentioned need a loading system to apply the force. Figure 1 shows a schema of the two systems most commonly used: end blocks and piano hinges [START_REF] Astm D | Standard test method for mode I interlaminar fracture toughness of unidirectional fibre-reinforced polymer matrix composites[END_REF][START_REF] Astm D | Standard test method for mixed mode I-mode II interlaminar fracture toughness of unidirectional fibre reinforced polymer matrix composites[END_REF]. The figure also shows the reduction of the lever arm when the loaded beam deflects under a load applied with eccentricity.

The two loading systems, end blocks and piano hinges, have the advantage of being conceptually very simple and easy to attach to the beams of the specimen. However, their use can introduce non-linear effects during the tests that can lead to error in the data reduction of the experimental results. An additional disadvantage is that they must be adhesively bonded to the specimen. Even though a surface treatment is applied before bonding, the bond strength between the metallic part and the specimen can be low, especially for thermoplastic-matrix composites, eventually resulting in a premature failure of the joint (mainly in fatigue or high temperature tests). Moreover, misalign- ments between the specimen and the load system can appear.

Brandt [START_REF] Brandt | New load introduction concept for improved and simplified delamination beam testing[END_REF] introduced a mechanical hinge to overcome these problems. The hinge consisted of two parts: the fastener box, which is mechanically attached to the beam of the specimen, and the grip plate, which is mounted on the test machine. The fastener box is a metallic block with a slot where the beam of the specimen is fitted (the geometry of the specimen has to be modified, as shown in Figure 2). Even though this hinge overcomes the problems associated with the use of end blocks and piano hinges (the rotation centre of the hinge is very close to the neutral axis of the beam and no adhesive joints are employed), it cannot be precisely adapted to different specimen thicknesses and its manufacture is expensive.

In this work, an improved and easy to manufacture hinge has been designed for delamination and adhesive tests using beam-type composite specimens. The proposed design is mechanically fixed to the specimen to avoid premature failures due to poor bonding and can be easily aligned to the specimen. Moreover, the hinge can be adapted to different specimens with different thicknesses and, most importantly, it avoids non-linear effects.

The article starts with a brief quantitative study of the non-linear effects induced by piano hinges and end blocks on common composite specimens for different delamination tests. Then, the developed hinge is presented with a thorough description of its mechanical design and performance.

2 Non-linear effects in beam-type delamination specimens

The use of end blocks or piano hinges during delamination and adhesive tests generates a series of structural non-linear effects caused by the distance between the rotation centre of the loading system and the neutral axis of the beam (distance l 1 in Figure 1). When the beam of the specimen deflects, the lever arm between the load application point and the delamination front is additionally reduced: a ′′ instead of a ′ in Figure 1(c). In addition, an end block stiffens the end portion of the specimen, including part of the zone between the load line and the delamination front (distance l 2 in Figure 1(a)).

To account for these effects, Williams [START_REF] Williams | The fracture-mechanics of delamination tests[END_REF][START_REF] Williams | Large displacement and end block effects in the DCB interlaminar test in mode-I and mode-II[END_REF] introduced two correcting factors, F and N, in the calculation of the compliance (C corrected = C N ) and energy release rate (G corrected = G F N ). F compensates for the reduction of the lever arm due to the eccentricity of the loading system and the rotation of the specimen, whereas N compensates for the stiffening of the specimen when end blocks are used.

The correcting factor N will not be analyzed in this study because it is not required when using piano hinges and, even with end blocks, the effect is smaller than that of F. According to Williams [START_REF] Williams | The fracture-mechanics of delamination tests[END_REF][START_REF] Williams | Large displacement and end block effects in the DCB interlaminar test in mode-I and mode-II[END_REF] and Hashemi et al. [START_REF] Hashemi | The analysis of interlaminar fracture in uniaxial fiber-polymer composites[END_REF][START_REF] Hashemi | Mechanics and mechanisms of delamination in a poly(ether sulfone) fiber composite[END_REF][START_REF] Hashemi | Mixed-mode fracture in fibre-polymer composite laminates[END_REF],

the expression of F for the ELS, ENF and MMELS (mixed-mode end load split) delamination tests is

F = 1 -θ 1 δ L 2 -θ 2 l 1 δ L 2 (1)
where the parameters θ i were determined by Williams It should be noted that the crack length a is required to determine the value of the parameters θ i ( [START_REF] Williams | The fracture-mechanics of delamination tests[END_REF][START_REF] Hashemi | The analysis of interlaminar fracture in uniaxial fiber-polymer composites[END_REF][START_REF] Hashemi | Mechanics and mechanisms of delamination in a poly(ether sulfone) fiber composite[END_REF][START_REF] Hashemi | Mixed-mode fracture in fibre-polymer composite laminates[END_REF]). However, the exact determination of a can be a significant source of uncertainty due to the difficulties in locating the crack tip (the crack front is not straight and the crack length measured on both edges of the specimen is different). Due to this imprecision in the determination of the crack length, the values of F and N can be imprecise.

In order to determine the importance of these non-linear effects, the influence of F and its variation with the crack length is evaluated for the DCB, ELS and MMELS delamination tests. The ENF test is not considered because a different loading system has to be employed. For this purpose, the mechanical properties of a typical unidirectional carbon/epoxy prepreg have been taken

into account (Table 1). The average thickness of the cured plies has been taken as 0.125 mm. The specimen is considered to have 20 plies per beam (total thickness is 2h = 5 mm), the width is b = 20 mm and the effective length is L = 150 mm. For practical reasons, the range of variation of the crack length a is taken between 10 and 100 mm. All the plies are assumed to be oriented with the fibres in the longitudinal direction of the specimen and the interlaminar crack located in the midplane of the specimen.

ACCEPTED MANUSCRIPT

Table 1 Mechanical properties of a typical unidirectional carbon/epoxy prepreg with a ply thickness of 0.125 mm (*critical energy release rate for a mixed-mode G II /G = 0.5). A similar analysis considering three different carbon-reinforced composites was previously conducted by Williams [START_REF] Williams | The fracture-mechanics of delamination tests[END_REF]. However, in this analysis Williams only considered the DCB specimen and did not take into account the value of δ required for crack propagation for every value of a (see [START_REF] Hashemi | The analysis of interlaminar fracture in uniaxial fiber-polymer composites[END_REF] for further details).

E 11 E 22 = E 22 ν 12 = ν 13 ν 23 G 12 = G 13 G 23 G Ic G * I/IIc G IIc (GPa) (GPa) - - (GPa) (GPa) (J/m 2 ) (J/m 2 ) (J/
The details see [START_REF] Blanco | Mixed-mode delamination growth in carbon-fibre composite laminates under cyclic loading[END_REF][START_REF] Blanco | Variable mixed-mode delamination in composite laminates under fatigue conditions: testing & analysis[END_REF][START_REF] Blanco | Analysis of the mixed-mode end load split delamination test[END_REF]).

According to Figure 3(a), the non-linear effects associated with F can be neglected in the case of the DCB test provided that the distance l 1 is sufficiently small (correction factor less than 5 % for the range of a and l 1 considered). This is in good agreement with the maximum value of l 1 calculated according to the ASTM standard for the DCB test [START_REF] Astm D | Standard test method for mode I interlaminar fracture toughness of unidirectional fibre-reinforced polymer matrix composites[END_REF] and the mechanical and geometrical properties considered. In this case, l 1,max ≈ 18 mm.

In Figure 3(b), it can be observed that the influence of the correction factor F is really important for the ELS test, especially for relatively short cracks.

In fact, when the distance l 1 is equal to 20 mm, the value of F can be as high as 3.5. Unexpectedly, it may be observed that the correction factor does not tend to unity for vanishing values of l 1 and short crack lengths, as it would be expected. In a similar way, the influence of F for the MMELS test (Figure the top and bottom plates inserting a metallic plate with the appropriate thickness t between the top case and the specimen.

The grip is connected to the testing machine and carries the load to the specimen through the fastener box. The design of the grip is very similar to that proposed by Brandt [START_REF] Brandt | New load introduction concept for improved and simplified delamination beam testing[END_REF] and is composed of two parts: the grip plate and the grip arm. Both the grip plate and the grip arm have a pin at one extreme to act as the rotation point for the fastener box and specimen. The grip arm is connected to the grip plate by the two guiding pins while a locking bolt secures the joint between both parts. A schema of the proposed grip is shown in Figure 4(c).

The assembly of the designed hinge is shown in Figure 5(a). The metallic plate with the appropriate thickness t and a generic specimen are also represented in the figure. For reasons of simplicity, an ELS or MMELS specimen is represented, so only one beam of the specimen has to be loaded. Fitting between the bottom case and the specimen is achieved by cutting the extreme part of the unloaded beam, as shown in the figure.

In the case of the ELS or MMELS delamination tests, the designed hinge does not require a special specimen lamination process. The specimens can be laminated in the usual way and fitting them into the hinge only requires cutting the extreme end of the unloaded beam, as shown in Figure 5(a). In fact, the proposed hinge was used by the authors in different MMELS fatigue delamination tests under varying mode mix conditions at room temperature [START_REF] Blanco | Mixed-mode delamination growth in carbon-fibre composite laminates under cyclic loading[END_REF][START_REF] Blanco | Variable mixed-mode delamination in composite laminates under fatigue conditions: testing & analysis[END_REF]. In total, the hinge supported more than 20 million cycles without any slippage between it and the specimen or any other functional problem. Two different specimens with different thicknesses in the loaded beam, 0.65 and 2.6 mm, were employed for the tests. The hinge could be easily adapted to each specimen thickness by using two metallic plates of different thicknesses so that correct alignment between the load application point and the neutral axis was obtained. In all cases, the loading point of the hinge and the neutral axis of the beam were coincident. In total, the hinge has been used for more than 20 million loading cycles at different load-displacement levels without any problem, especially those related to end blocks and piano hinges.

  Fig. 2. Cross-section of the fastener box designed by Brandt [3] without and with the mounted specimen.

[ 4 ]

 4 and Hashemi et al.[START_REF] Hashemi | The analysis of interlaminar fracture in uniaxial fiber-polymer composites[END_REF][START_REF] Hashemi | Mechanics and mechanisms of delamination in a poly(ether sulfone) fiber composite[END_REF][START_REF] Hashemi | Mixed-mode fracture in fibre-polymer composite laminates[END_REF]. To the knowledge of the authors, no specific expressions have been formulated for the MMB test to determine these parameters. For the DCB test, the length of the interlaminar crack a, must be used instead of the length of the specimen,[START_REF] Astm D | Standard test method for mode I interlaminar fracture toughness of unidirectional fibre-reinforced polymer matrix composites[END_REF][START_REF] Astm D | Standard test method for mixed mode I-mode II interlaminar fracture toughness of unidirectional fibre reinforced polymer matrix composites[END_REF][START_REF] Williams | The fracture-mechanics of delamination tests[END_REF][START_REF] Williams | Large displacement and end block effects in the DCB interlaminar test in mode-I and mode-II[END_REF][START_REF] Davies | Protocols for interlaminar fracture testing of composites[END_REF][START_REF] Robinson | Interlaminar fracture toughness. Mechanical Testing of Advanced Fibre Composites[END_REF].

  variation of F with the crack length for the DCB, ELS and MMELS tests for different values of l 1 is shown in Figure 3. In this figure, F has been calculated considering the displacement δ that corresponds to the critical value of the energy release rate of the material. Thus, for every value of a, the δ required for crack propagation is determined using G Ic for the DCB test and G IIc for the ELS test. In the case of the MMELS test, δ is calculated for every crack length according to the critical energy release rate of the material under mixed-mode I/II, G I/IIc . Actually, for this test, the mixed-mode ratio G II /G (mode II energy release rate over total energy release rate) varies from G II /G = 0.335 when a = 10 mm to G II /G = 0.416 when a = 100 mm (for further

3Fig. 4 .

 4 Fig. 4. Schema of the (a) top case, (b) bottom case and (c) grip of the designed load hinge.

Fig. 5 .

 5 Fig. 5. (a) View of the assembly of the hinge and the specimen (an ELS or MMELS delamination specimen is considered for simplicity) and (b) designed load hinge during a MMELS test where the load application point is centred at the neutral axis of the beam.

Figure 5 (

 5 b) shows the hinge used during the MMELS tests and how the external load is applied to the neutral axis of the loaded beam of the specimen.The adaptation of the designed hinge to the DCB and MMB tests can be ACCEPTED MANUSCRIPT problematic, as in the case of using the hinge designed by Brandt. Two bottom cases have to be fixed to the specimen and two top cases and grips must be employed. The first problem appears when the two bottom cases are fixed in the delamination plane between the beams of the specimen without excessively opening the interlaminar crack. The second problem in adapting this type of hinge to DCB or MMB tests is the interference between two sets of top cases and grips.There are three alternatives to adapting the designed hinge to the DCB or MMB test and to overcoming the problem of fixing two bottom cases to the specimen. The first possibility is to machine the specimens or use a specific process for their manufacturing in order to obtain a geometry similar to the one proposed by Brandt (see Figure2and[START_REF] Brandt | New load introduction concept for improved and simplified delamination beam testing[END_REF] for details). A second possibility is to carefully force two bottom cases between the beams of the specimen and then fix them to two upper cases. The advantage of this alternative is that the geometry of the specimen does not have to be modified as in the previous case. The disadvantage of this method is that an initial displacement of the beams is introduced and forces an initial opening, without measured load, of the interlaminar crack. This alternative has been used by the authors in different static DCB and MMB tests for the characterisation of interlaminar crack resistance of carbon fibre-reinforced polymer laminates with very good results and values of G similar to those obtained with piano hinges[START_REF] Vicens | Ensayo de doble viga en voladizo para la propagación de grietas interlaminares en modo I en materiales compuestos: estudio experimental comparativo en tres laboratorios[END_REF][START_REF] Vicens | New mechanical fixtures for load introduction in DCB tests: design and performance[END_REF].The third option for adapting the designed hinge to the DCB and MMB tests is based on the use of the usual specimen geometry but only the top cases of the proposed hinge are bonded at each beam of the specimen. Although this alternative includes the disadvantage of an adhesive joint between specimen and hinge, the load is still applied to the neutral axis of the beam. Nonetheless, to avoid any interference between the two sets of top cases and the grips when using the hinge for the DCB or MMB test, one of the sets has to be manufactured with an increased distance between the shaft holes and shafts of the top case and the grip, respectively (see Figures4(a) and 4(c)). The increased distance must be sufficient to avoid interference when superposing the two top cases and grips.The designed hinge has been used by the authors in a multitude of different static and fatigue delamination tests, including DCB, MMB and MMELS tests, involving beam-type specimens with different thicknesses[START_REF] Blanco | Mixed-mode delamination growth in carbon-fibre composite laminates under cyclic loading[END_REF][START_REF] Blanco | Variable mixed-mode delamination in composite laminates under fatigue conditions: testing & analysis[END_REF][START_REF] Vicens | Ensayo de doble viga en voladizo para la propagación de grietas interlaminares en modo I en materiales compuestos: estudio experimental comparativo en tres laboratorios[END_REF][START_REF] Vicens | New mechanical fixtures for load introduction in DCB tests: design and performance[END_REF].

4 ConclusionsA

 4 Figure 1a

  Figure 1c

(a) DCB (b) ELS

ACCEPTED MANUSCRIPT

cannot be neglected, requiring the use of different correction factors (which in turn exhibit some inconsistencies for short crack lengths). Consequently, the hinge presented in this work allows calculation of the energy release rate components without taking into account correcting factors that complicate the calculations.

The hinge is manufactured with top and bottom cases. A metallic plate can be inserted between the top case and the specimen, so the hinge can fit a wide range of specimen thicknesses while keeping the load application point centred at the neutral axis of the beam. Some indications on how to adapt the proposed hinge for DCB and MMB delamination and adhesive tests, in which two sets of hinges are required, have been given.

Acknowledgments

The authors would like to acknowledge the manufacturing skills of Bertil Dolk and Bengt Möllerberg, workshop technicians at KTH Solid Mechanics (Royal Institute of Technology, Stockholm). The authors would also like to acknowledge financial support from the Spanish Government under research project MAT2006-14159-C02-01.

Shafts

Guiding pins

Locking bolt

Grip arm

Grip plate


Andrea Terenzi

Carla Vedova

Giovanni Lelli

Jovan Mijovic

Luigi Torre

Luca Valentini

Josè M Kenny

Chemorheological Behavior of Double-Walled Carbon Nanotube Epoxy Nanocomposites

Keywords: A. Nano composites, B. Thermal properties, D. Differential scanning calorimetry (DSC), D. Rheology

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Introduction

Carbon nanotubes (CNTs) are promising fillers for composite materials to improve mechanical behavior and electrical and thermal transport [START_REF] Tans | Room-Temperature Transistor Based On A Single Carbon Nanotube[END_REF]. Across many different applications, CNTs/polymer composites have been extensively studied [START_REF] Xie | Dispersion and alignment of carbon nanotubes in polymer matrix: A review[END_REF][START_REF] Qian | Load transfer and deformation mechanisms in carbon nanotube-polystyrene composites[END_REF][START_REF] Wei | Thermal expansion and diffusion coefficients of carbon nanotube-polymer composites[END_REF]. The key problem for CNTs/polymer composite elaboration is the dispersion, compatibilization, and stabilization of the CNTs in the polymer matrix.

In this regard a number of excellent reviews have been published on the functionalization of CNTs [START_REF] Tasis | Chemistry of carbon nanotubes[END_REF][START_REF] Sun | Functionalized carbon nanotubes: Properties and applications[END_REF][START_REF] Hirsch | Functionalization of single-walled carbon nanotubes[END_REF] covering covalent attachment of functional moieties through reaction onto CNTs and noncovalent adsorption or wrapping of various functional molecules. Double-walled carbon nanotubes (DWNTs) [START_REF] Saito | Anomalous potential barrier of double-wall carbon nanotube[END_REF] are considered as the simplest member of the family of multiwalled carbon nanotubes (MWNTs), consisting of two coaxial single-walled carbon nanotubes (SWNTs). It is expected to have unique physical and mechanical properties due to its special double wall structure if compared to that of SWCNTs and MWCNTs [START_REF] Dai | Carbon nanotubes: Synthesis, integration, and properties[END_REF][START_REF] Liu | Electrical transport in doped multiwalled carbon nanotubes[END_REF].

Nanotube-epoxy composites have been widely studied [START_REF] Ajayan | Aligned carbon nanotube arrays formed by cutting a polymer resin-nanotube composite[END_REF][START_REF] Kao | A Raman spectroscopic investigation of heating effects and the deformation behaviour of epoxy/SWNT composites[END_REF][START_REF] Frogley | Polarized resonance Raman spectroscopy of singlewall carbon nanotubes within a polymer under strain[END_REF][START_REF] Puglia | Analysis of the cure reaction of carbon nanotubes/epoxy resin composites through thermal analysis and Raman spectroscopy[END_REF][START_REF] Gojny | Functionalisation effect on the thermo-mechanical behaviour of multi-wall carbon nanotube/epoxy-compo sites[END_REF][START_REF] Wagner | Stress-induced fragmentation of multiwall carbon nanotubes in a polymer matrix[END_REF][START_REF] Cooper | Investigation into the deformation of carbon nanotubes and their composites through the use of Raman spectroscopy[END_REF][START_REF] Nuriel | Measuring fiber-matrix interfacial adhesion by means of a 'drag-out' micromechanical test[END_REF]. Thus, the functionalization of DWNTs represents an open issue for the preparation and manipulation of DWNT based nanocomposites with multifunctional properties. Even if some attempts have been done to functionalize them [START_REF] Datsyuk | Double walled carbon nanotube/polymer composites via in-situ nitroxide mediated polymerisation of amphiphilic block copolymers[END_REF][START_REF] Ayala | Tailoring N-Doped Single and Double Wall Carbon Nanotubes from a Nondiluted Carbon/Nitrogen Feedstock[END_REF] no data exist at our knowledge on how their dispersion affects the rheological properties of DWNT based polymer nanocomposites.

In the present work, we used grafted-N-Methyldiethanolamine on carboxy-functionalized DWNTs to investigate the chemorheology of the amino-functionalized DWNTs during the cure reaction of an epoxy system. A chemorheological model has been also developed for this new type of nanocomposite. The MDEA and MDEA/C-DWNTs systems were studied with the H-NMR technique. 1 H-NMR analysis was performed by using CDCl 3 as solvent and tetramethylsilane as internal reference. A Bruker FT-NMR Avance400 400MHz spectrometer equipped with a BVT3200 controller of temperature and a BBI 5 mm probe for liquid was used. 1 H spectra were recorded by using the following parameters: acquisition time 1s, receiver gain 263, dwell time 124,8 µs, pre-scan delay 6 µs, relaxation delay 1s, frequency of observe channel 400,13 MHz, power level for pulse -3 dB, 90° high power pulse 5.80 µs and a number of scan of 32.

Experimental Details

Isothermal and dynamic tests were performed by means of a differential scanning calorimeter (DSC) Mettler Toledo 822e/400 coupled with an intercooler. The procedure adopted was the following: isothermal tests at six different temperatures (145°C, 150°C, 155°C, 160°C, 165°C and 170°C) for 2 hours, followed by a dynamic post-cure from 0°C to 300°C at 10°C/min to determine the residual reactivity of the polymer matrix. Dynamic tests from -50°C to 350°C at three different heating rates (2°C/min., 5°C/min. and 10°C/min.) were performed as well to calculate the maximum networking enthalpy of the reacting systems.

The rheological tests were performed with a Rheometric Scientific Ares rheometer with parallel plates (∅ 25mm); all tests were done in the same isothermal and dynamic conditions of the calorimetric experiments with a frequency of 1Hz and a strain of 1.5%. In these tests the evolution of the viscosity during the curing reaction both in isothermal and dynamic conditions is monitored. It is well known that during the cure of a thermosetting matrix the viscosity increases from a very low value (liquid like) up to an infinite point (gelation) [START_REF] Janezic | Implicit Runge-Kutta Method for Molecular Dynamics Integration[END_REF].

Such increase, being of many order of magnitude, cannot be monitored using the same precision over the whole field. Therefore in order to be able to accurately evaluate the gelation point of the resin, an high viscosity transducer must be used during the tests, this produces an increase of the scattering of the data in the low viscosity region.

Both calorimetric and rheological data were analysed by using kinetic and chemorheological mathematical models [START_REF] Janezic | Implicit Runge-Kutta Method for Molecular Dynamics Integration[END_REF], with the aim to develop a software tool able to predict the behaviour of the analyzed systems.

Results and Discussion

The H-NMR analysis was used in order to get more detailed information on the emergence of specific interactions between the MDEA and C-DWNTs (Fig. 1). If we compare the spectra of the MDEA system with that of MDEA/C-DWNTs one, it is clear how the presence of C-DWNTs causes the enlargement of the peaks with a consequent fine-structure loss. In particular the NH 2 signal shift from 3.4 ppm for pure MDEA to 3.2 ppm for MDEA with C-DWNTs suggests that the DWNT carboxylic groups react with the MDEA ammine groups leading to the formation of the -NHCO groups.

The SEM images of the composite fracture surfaces are reported in Fig. 2. With respect to the sample obtained by mixing the tubes with the DGEBA (Fig. 2 A displacement of the gel point at lower time and temperature is due to the fact that the functionalized nanotubes interacts with the epoxy matrix via the amino groups immobilized on the tube sidewall, reducing the mobility of chains of the epoxy molecules [START_REF] Valentini | Use of Plasma Fluorinated Single-walled Carbon Nanotubes for the Preparation of Nanocomposites with Epoxy Matrix[END_REF].

The analysis of the calorimetric and rheological data has been performed by means of a chemorheological model, which relates the evolution of the viscosity to both the degree of reaction and the temperature conditions. In particular, a kinetic model is required to predict the rate of reaction and the degree of cure as a function of time and temperature.

6 By using isothermal and dynamic calorimetric tests, it is possible to monitor the evolution of the degree of cure [START_REF] Halley | Chemorheology of thermosets-an overview[END_REF]:

( ) ( ) ( ) , y T K T f t α α α ∂ = = ⋅ ∂ (1)
Where α is the degree of cure, t the time, K(T) is a function of temperature and f(α) is a function of the degree of cure.

The function selected for the analysis in the present work is given by the combination of two auto-catalytic peaks [START_REF] Torre | Cure kinetics of epoxy anhydride nanocomposite systems added with reactive flame retardants[END_REF]. Therefore, Eq. ( 1) can be rewritten as follows:

( ) ( ) ( ) ( ) 1 2 1 2 1 max 2 max n n m m d K T K T dt α α α α α α α = ⋅ ⋅ - + ⋅ ⋅ - (2)
Where α max is the maximum degree of cure, K 1 and K 2 are Arrhenius-like functions of the temperature and m 1 , m 2 , n 1 , n 2 are specific parameters.

The final conversion degree reached by the system was found to be a sigmoid function of the cure temperature [START_REF] Lelli | Modelling of the chemo-rheological behaviour of thermosetting polymer nanocomposites[END_REF]:

( ) ( ) ( ) max 0.5 1 1 exp exp T T T α β β = + ⋅ ⋅ -⋅ (3)
where T 0.5 is the absolute temperature (K) that allows the achievement of the half of the maximum networking degree and β is a temperature-independent parameter that influences the slope of the sigmoid function.

The evolution of the degree of cure can be calculated by solving the Eqs. ( 1) and ( 2). As a classical integration is not possible, a specific algorithm based on the fourth-order Runge Kutta method was implemented for the numerical integration.

The model shows a very good fitting in both isothermal and dynamic conditions for all the materials tested. The comparisons among experimental data and model predictions in specific isothermal and dynamic conditions are reported in Figs. 5(a) and 5(b).

In Fig 5(a) it is important to note that at 160°C the pure DGEBA/MDEA system reaches a maximum degree of cure of approximately 0.65 and about 66 min. are necessary to get α=0.5.

At the same temperature conditions, the nanocomposite obtained with MDEA modified C-DWNTs reaches a maximum degree of cure of 0.99 and the time necessary to reach the degree of reaction of 0.5 is about 20 min. Moreover, it is possible to observe that a temperature of 210°C is necessary to make the neat system reach an α max similar to that of the filled system. The kinetic parameters are reported in Tab. 3.

The following equation relates the T g of the system to the degree of cure [START_REF] Havlicek | Theoretical modelling of the rheological behaviour of an epoxy-amine system during curing[END_REF]:

( )

1 1 1 0 q T T T g g g α α α α - = + + ⋅ ⋅ - ∞ (4)
Where T g0 and T g∞ are the glass transitions temperatures for the monomer and the fully cured system and q is a fitting parameter.

Finally, the following equation correlates the viscosity to the degree of cure [START_REF] Castro | Studies of mould filling and curing in the reaction injection moulding process[END_REF][START_REF] Opalicki | Chemorheology and curing kinetics of toughened epoxy matrices for high performance composites[END_REF][START_REF] Mijovic | Modelling of chemorheology thermoset cure by modified WLF equation[END_REF][START_REF] Ivankovic | Curing kinetics and chemorheology of epoxy anhydride systems[END_REF]:

( )

1 2 ( ) ( ) exp ( ) rheo n g g g g g C T T T C T T α α η η α α α - = ⋅ - ⋅ + - - (5)
where α g is the extent of reaction at the gel point, T g (α) is a function describing the dependence of the T g on the networking degree and n rheo is a constant to be determined by regression analysis of the experimental data.

  Carboxy-functionalized double-walled carbon nanotubes (C-DWNTs) were purchased from Nanocyl (purity ≈ 90 vol%, carboxylic group concentration 1.24 at.%; data supplied by Nanocyl). The polymeric matrix of the nanocomposite under investigation was obtained from a diglycidyl ether of bisphenol A-based (DGEBA) epoxy resin (D.E.R.330 provided by Dow) and a solid N-Methyldiethanolamine (MDEA, provided by Sigma-Aldrich). The C-DWNTs were stirred in the MDEA at the melting temperature of the amine (i. e. 100°C for 45 min.). The C-DWNTs grafted with MDEA, MDEA/C-DWNTs, were added to obtain a percentage of 1wt% respect to the DGEBA monomer maintaining a stoichiometric molar ratio (1:2.33) with MDEA. An alternative preparation method consisting of firstly mixing the C-DWNTs with DGEBA and then by adding the MDEA moiety, was investigated. The morphology of the sample fracture surfaces was investigated by field emission scanning electron microscopy (FESEM).

  (a)), one can observe that the dispersion of the C-DWNTs is very good (Figs. 2(b) and 2(c)) with the bundle-like structure observed in the inset of Fig. 2(a) that is completely lost giving to the sample a very good homogeneity on the entire surface. This result, together with the H-NMR analysis reported above, confirms that the presence of a MDEA fraction distributed over C-DWNTs sidewalls is responsible for the obtained good dispersion and the homogeneity of the sample.The results of the calorimetric test in isothermal and dynamic conditions are reported in Figs.

  3(a) and 3(b), respectively. These results show an important catalytic effect of the C-DWNTs on the cure reaction of the DGEBA/MDEA system with a shift of the cure reaction peak toward lower time (in isothermal tests) and lower temperature (in dynamic test). Moreover, it is also possible to note that the cure rate (Fig. 3(b)) for the system obtained with MDEA modified C-DWNT is higher than the conversion speed of the neat epoxy system. The rheological measurements (Figs. 4(a) and 4(b)) confirm what observed in the calorimetric tests. Also in this case, the onset of the cure reaction for the filled system is shifted to lower time and temperatures, respectively. The results obtained from calorimetric and rheological tests are summarized in Tabs. 1 and 2.

  The comparisons among experimental data and chemoreological model (numerically integrated according to the 4 th -order Runge-Kutta method[START_REF] Janezic | Implicit Runge-Kutta Method for Molecular Dynamics Integration[END_REF]) for the DGEBA/MDEA and DGEBA/MDEA/C-DWNTs systems are reported in Figs.6(a) and 6(b), respectively. The parameters of the model are reported in Tab. 3. From these figures it is evident the accordance between the experimental and the model. Moreover, the higher reaction enthalpy together with the T 0.5 and β parameters (Tab. 3) obtained for the nanocomposite make the sigmoid function (Eq. 3) more steeper than that calculated for the DGEBA/MDEA neat system. This could lead to the conclusion that the C-DWNTs play a catalytic role in the networking process. On the other hand, all the remaining kinetic parameters (Tab. 3) are lower in the case of the nanocomposite (i.e. the reaction goes more slowly), probably due to some kind of obstacle played by the tubes against the mobility of polymer chains.

  we reported how N-Methyldiethanolamine modified carboxy-DWNTs are used to obtain a cross-linked epoxy nanocomposite. Calorimetric and rheological tests reveal how the presence of MDEA modified C-DWNTs has a strong influence on the maximum degree of cure and on the gel time of the epoxy system. The use of kinetic and chemoreological models show an excellent agreement with the experimental data being the catalytic effect of the tubes observed also in the variation of specific parameters of these models. Following these indications we retain that the presented synthesis strategy could have implications toward building some novel architectures with nanotubes and polymers. Aknowledgments The authors thank Dr. Federica Meloni for the SEM analysis, Dr. Michele Marini and Prof. Francesco Pilati (Materials and Environmental Engineering Department, University of Modena and Reggio Emilia) for the H-NMR analysis.

Fig. 1 .

 1 Fig. 1. H-NMR spectra of MDEA and MDEA/C-DWNTs systems.

Fig. 2 .Fig. 3 .Fig. 4 .Fig. 5 .Fig. 6 .

 23456 Fig. 2. (a) FESEM image of the DGEBA/MDEA nanocomposite obtained with nanotubes

  dynamic rheological tests of DGEBA/MDEA and DGEBA/MDEA/C-DWNTs systems. Tab. 3. Characteristic kinetic and rheological parameters for DGEBA/MDEA and DGEBA/MDEA/C-DWNTs systems.

Figure 1 Figure 2

 12 Figure 1

Figure 4 Figure 5

 45 Figure 4

Figure 6 Tab. 2

 62 Figure 6

Table Captions

 Captions Tab. 1. Time of maximum reaction rate and maximum reaction peak temperature for isothermal and dynamic tests of DGEBA/MDEA and DGEBA/MDEA/C-DWNTs systems. Tab. 2. Time onsets for isothermal rheological tests and onset and gel temperatures for