Refractory Crohn's Disease Patients Research Articles

The reparative immunologic consequences of stem cell transplantation as a cellular therapy for refractory Crohn's disease.

Treatment strategies for Crohn's disease (CD) suppress diverse inflammatory pathways but many patients remain refractory to treatment. Autologous hematopoietic stem cell transplantation (SCT) has emerged as a therapy for medically refractory CD. SCT was developed to rescue cancer patients from myelosuppressive chemotherapy but its use for CD and other immune diseases necessitates reimagining SCT as a cellular therapy that restores appropriately responsive immune cell populations from hematopoietic progenitors in the stem cell autograft (i.e. immune "reset"). Here we present a paradigm to understand SCT as a cellular therapy for immune diseases and reveal how SCT re-establishes cellular immunity utilizing high-dimensional cellular phenotyping and functional studies of the stem cell grafts. Immunophenotyping using CyTOF, single cell RNA sequencing (scRNA-seq) and T cell receptor (TCR) sequencing was performed on peripheral blood and intestinal tissue samples from refractory CD patients who underwent SCT. The stem cell graft from these patients was analyzed using flow cytometry and functionally interrogated using a murine model for engraftment. Our study revealed a remodeling of intestinal macrophages capable of supporting mucosal healing that was independently validated using multimodal studies of immune reconstitution events including CyTOF and scRNA-seq. Functional interrogation of hematopoietic stem cells (HSCs) using a xenograft model demonstrated that HSCs shape the timing of immune reconstitution, the selected reconstitution of specific cell lineages and potentially the clinical efficacy of SCT. These studies indicate that SCT serves as a myeloid-directed cellular therapy re-establishing homeostatic intestinal macrophages that support intestinal healing and suggest refractory CD evolves from impairment of restorative functions in myeloid cells. Furthermore, we report heterogeneity among HSCs from CD patients which may drive SCT outcomes and suggests an unrecognized impact of CD pathophysiology on HSC in the marrow niche.

Cyclophosphamide-free Mobilisation Increases Safety While Preserving the Efficacy of Autologous Haematopoietic Stem Cell Transplantation in Refractory Crohn's Disease Patients.

Autologous haematopoietic stem cell transplantation [AHSCT] is a therapeutic option for refractory Crohn's disease [CD]. However, high adverse event rates related to chemotherapy toxicity and immunosuppression limit its applicability. This study aims to evaluate AHSCT's safety and efficacy using a cyclophosphamide [Cy]-free mobilisation regimen. A prospective, observational study included 14 refractory CD patients undergoing AHSCT between June 2017 and October 2022. The protocol involved outpatient mobilisation with G-CSF 12-16 μg/kg/daily for 5 days, and optional Plerixafor 240 μg/d [1-2 doses] if the CD34 + cell count target was unmet. Standard conditioning with Cy and anti-thymocyte globulin was administered. Clinical, endoscopic, and radiological assessments were conducted at baseline and during follow-up. All patients achieved successful outpatient mobilisation [seven patients needed Plerixafor] and underwent transplantation. Median follow-up was 106 weeks (interquartile range [IQR] 52-348). No mobilisation-related serious adverse events [SAEs] or CD worsening occurred. Clinical and endoscopic remission rates were 71% and 41.7% at 26 weeks, 64% and 25% at 52 weeks, and 71% and 16.7% at the last follow-up, respectively. The percentage of patients who restarted CD therapy for clinical relapse and/or endoscopic/radiological activity was 14% at 26 weeks, 57% at 52 weeks, and 86% at the last follow-up, respectively. Peripheral blood cell populations and antibody levels post-AHSCT were comparable to Cy-based mobilisation. Cy-free mobilisation is safe and feasible in refractory CD patients undergoing AHSCT. Although relapse occurs in a significant proportion of patients, clinical and endoscopic responses are achieved upon CD-specific therapy reintroduction.

FROM RESISTANCE TO RESPONSE: 52 WEEKS OF SUBCUTANEOUS INFLIXIMAB IN REFRACTORY CROHN'S DISEASE PATIENTS AFTER THE MULTIPLE TREATMENT FAILURES

Abstract BACKGROUND Infliximab (IFX), an anti-TNFα monoclonal antibody, has been utilized since the late 1990s as an effective therapeutic agent for the treatment of Crohn's disease (CD). Since 2020, subcutaneous IFX (IFX-SC) has been available for treating CD. This study aimed to conduct a 52-week observation of IFX-SC treatment in refractory CD patients who had failed on at least two previous lines of biologic therapy patients. MATERIALS AND METHODS The study included 32 patients diagnosed with CD who had failed on 2 to 7 previous lines of biological therapy. Observed cohort was monitored at weeks W0, W4, W14, W30, and W52, recording Harvey-Bradshaw Index (HBI) , C-reactive protein (CRP) and fecal calprotectin (FC) concentrations, trough infliximab (TL-IFX) and anti-infliximab (anti-IFX) serum levels. Endoscopic, ultrasonographic and magnetic resonance disease scores (SES-CD, IUS and MaRIA) were determined at baseline and W52. RESULTS 52-week persistence on IFX-SC treatment was recorded in 18 out of 32 patients (56.3%). A significant decrease of HBI (P <0 .0001), CRP (P =0 .0035), FC (P =0.0012), SES-CD (P =0.013), IUS (P =0.0034) and MaRIA (P =0.0261) was observed. TL IFX increased significantly (.0073), and median TL-IFX levels reached 19.75 μg/mL at W52. During the therapy, 8 out of 16 initially anti-IFX positive individuals seroconverted to negative anti-IFX (50 %); no new sensitization to IFX was recorded in the cohort. None of the patients treated with IFX-SC in W52 needed concomitant immune-modifying treatment. Baseline clinical, laboratory, and imaging markers did not demonstrate any predictive value for IFX-SC treatment failure, except for baseline high levels of anti-IFX (P = <0.0001). CONCLUSION In the cohort of refractory CD patients, over half achieved one-year treatment persistence on IFX-SC, displaying a highly favorable trend in all disease activity markers. The IFX-SC treatment appears to be effective and safe; however, further research is needed to identify potential predictors of therapeutic responsiveness. Acknowledgement: This work was supported by the IBD-COMFORT Foundation

Subcutaneous Infliximab in Refractory Crohn's Disease Patients: A Possible Biobetter?

A subcutaneous formulation of infliximab (IFX-SC) approved to treat patients with inflammatory bowel disease may offer improved efficacy versus intravenous infliximab. Patients with refractory Crohn's disease (CD, n = 32) previously treated unsuccessfully with at least 2 biologics were treated with IFX-SC and followed from baseline at Week 0 (W0) to Week 30 (W30). The study's primary endpoint was the treatment's persistence at W30, while secondary goals included the analysis of serum infliximab trough levels (TL IFX), dynamics of anti-IFX antibodies (ATIs), and clinical, serum and fecal markers of CD activity during IFX-SC treatment. Midterm treatment persistence with the continuation of treatment after W30 was 53%. TL IFX median values showed rapid, significant upward dynamics and exceeded 15.5 μg/mL at W30, whereas median ATI levels significantly declined. Among ATI-negative patients at W0 (n = 15), only one showed IFX immunogenicity with newly developed ATIs at W30. Among ATI-positive patients at W0, ATI seroconversion from ATI-positive to ATI-negative status was observed in 10 of 17 patients (58.8%). Patients who had continued IFX-SC treatment at W30 showed significant decreases in C-reactive protein (P = .0341), fecal calprotectin (P = .0002), and Harvey-Bradshaw index (P = .0029) since W0. Patients with refractory CD previously treated with at least 2 biologics exhibited clinically relevant improvement with IFX-SC, which showed less immunogenic potential than IFX-IV and highly stable TL IFX.

Parenteral Methotrexate Is Efficient in the Treatment of Azathioprine Refractory Crohn's Disease.

There is limited data in the literature analyzing the efficacy of methotrexate in Crohn's disease used after thiopurine analogs. We aimed in our study to show the efficacy of methotrexate in Crohn's disease patients who failed to respond to thiopurine treatment. The study included 29 azathioprine refractory patients with Crohn's disease. Intramuscular methotrexate (25 mg/week) in the induction of remission and intramuscular methotrexate (15 mg/week) in 29 CD patients with a median follow-up time of 13 months was performed. In 15 (51.7%) patients, methotrexate was used in combination with anti-Tumour necrosis factor (TNF) (combination group), while it was used in 14 (48.3%) patients in monotherapy (monotherapy group). The mean Harvey-Bradshaw index score significantly decreased in the follow-up period (Wk0 = 7.6, last visit = 4.5, P < .001). Remission and response rates at week 12 were 75.9% and 79.3%, respectively. Maintenance of remission (77.8% vs 37.5%, respectively, P = .1) and response rates (77.8% vs 50%, respectively, P = .3) due to last visit examination were numerically higher in combination group but they were not statistically significant. The cumulative probability of remission maintenance in patients with methotrexate therapy was 72.7%, 33.1%, and 22.0% at 1, 2 ,and 4 years after starting methotrexate, respectively. Our results show that parenteral use of methotrexate is efficacious in inducing and maintaining remission as a step-up agent in azathioprine refractory Crohn's disease patients.

Efficacy and safety of simultaneous treatment with two biologic medications in refractory Crohn's disease.

Biologic therapies in patients with Crohn's disease often yield low clinical and endoscopic remission rates. After multiple failed therapies, combining two biologic therapies is possibly the sole medical alternative to recurrent surgery. However, data on this approach are limited. To assess the efficacy and safety of concomitant use of two biologic therapies in the largest cohort to date of refractory Crohn's disease patients. Data were extracted from Crohn's disease patients started on dual biologic therapy at two referral centres. Biologics utilised include infliximab, adalimumab, vedolizumab, ustekinumab, certolizumab and golimumab. The primary outcome was endoscopic improvement (>50% reduction in Simplified Endoscopic Score-Crohn's disease [SES-CD] or explicitly stated). Endoscopic remission (SES-CD<3 or stated), clinical response (Crohn's disease-patient-reported outcome-2 score [PRO2] reduced by 8), clinical remission (PRO2<8), and C-reactive protein (CRP) were also assessed. A total of 22 patients with 24 therapeutic trials of dual biologic therapy were identified. The majority of patients had prior surgical resections (91%), stricturing (59%) or penetrating (36%) phenotype, and perianal fistulas (50%). Median number of prior failed biologics was 4. Endoscopic improvement occurred in 43% of trials and 26% achieved endoscopic remission. Fifty per cent had clinical response and 41% achieved clinical remission. There were significant post-treatment reductions in median SES-CD (14.0 [12.0-17.5] to 6.0 [2.5-8.0], P=0.0005], PRO-2 (24.1 [20.3-27.0] to 13.4 [4.6-21.8], P=0.002] and CRP (17.0 [11.0-24.0] to 9.0 [4.0-14.0], P=0.02). Presence of perianal fistulas decreased from 50% to 33%. Adverse events occurred in 13% of trials. Dual biologic therapy was associated with clinical, biomarker and endoscopic improvements in selected patients with refractory Crohn's disease who failed multiple biologics. Further studies are needed to validate this approach.

P073 Double Balloon Enteroscopy in Refractory Crohn's Disease: Jejunal Findings and Clinical Impact After 10 Years of Follow-Up

BACKGROUND: Crohn's disease (CD) is a chronic inflammatory disease that can affect all segments of the gastrointestinal tract. With various phenotypes and progressive course, it can be complicated with fistulas, abscesses or stenosis. Treatment targets includes clinical remission and mucosal healing in order to avoid complicated outcomes. Double balloon enteroscopy (DBE) allows complete evaluation of the small intestine, providing diagnoses and the possibility of interventions such as biopsies, with low complication rates. Some authors suggest that involvement of the proximal small bowel may indicate worse long-term therapeutic outcomes. This study aims to correlate jejunal endoscopic findings observed in baseline DBE with the activity of CD after 10 years of follow-up. METHODS: Twenty paediatric refractory CD patients (2–17 years old) performed DBE by a single experienced specialist after radiological evaluation for stenosis exclusion between 2007 and 2010. After 10 years, the degree of CD activity was assessed by the Harvey-Bradshaw Index (HBI), C-Reactive Protein (CRP) serum dosage, and current therapy through collection of electronic medical records and personal communication. RESULTS: Sixteen 16/20 patients (80%) completed the 10 years follow up period. Two patients (2/16) changed diagnosis (1- monogenic disease, IL-10 and / or IL10R gene mutation; 1 - ulcerative colitis). Previous findings of DBE of the 14 patients corresponded to: 2 active duodenojejunal ulcers, 4 duodenojejunal scar retractions, 1 retraction and pseudopolyps in the proximal ileum, 6 hyperemia and / or edema and / or granularity of the jejunal mucosa, and 1 without alteration. After 10 years, 6/14 patients have abnormal CRP (>5 mg/L), with 3 of them having HBI between 5 and 7, 2 between 8 and 16 and 1 of 4. All of them presented previous jejunal alterations, 5/6 are currently in use of biologic and 3/6 are steroid dependent. The 2 patients who had active ulcers in baseline DBE remain steroid dependent and with higher rates of HBI activity. Eight patients (8/14) have normal CRP, all presenting HBI lower than 5. Of these, 3 are using combined therapy with immunosuppressant and biologic, 1 with biologic monotherapy, 2 with immunosuppressant, and 2 without medication. CONCLUSION(S): Active jejunal disease, observed through DBE, may indicate a more severe phenotype in CD after long-term follow-up, suggesting a distinct phenotype. Evaluation of the proximal small intestine should be considered in refractory paediatric CD patients or those with diagnostic uncertainty.

Real-world short-term effectiveness of ustekinumab in 305 patients with Crohn's disease: results from the ENEIDA registry.

There are limited data of ustekinumab administered according to the doses recommended in the UNITI studies. To assess the real-world, short-term effectiveness of ustekinumab in refractory Crohn's disease (CD) METHODS: Multicentre study of CD patients starting ustekinumab after June 2017 at the recommend dose (260, 390 or 520mg based on weight ~6mg/kg IV week 0 and 90mg subcutaneously week 8). Values for Harvey-Bradshaw Index (HBI), C-reactive protein (CRP) and faecal calprotectin (FC) were recorded at baseline and at weeks 8 and 14. Demographic and clinical data, previous treatments, AEs and hospitalisations were documented. Possible predictors of clinical remission were examined. Three hundred and five patients were analysed (≥2 previous anti-TNFα therapies 64% and vedolizumab 29%). At baseline, 217 (72%) had an HBI >4 points. Of these, 101 (47%) and 126 (58%) achieved clinical remission at weeks 8 and 14, respectively. FC levels returned to normal (<250µg/g) in 46% and 54% of the patients at weeks 8 and 14 respectively. CRP returned to normal (<3mg/L) in the 35% and 41% of the patients at week 8 and 14 respectively. AEs were recorded in 38, and 40 patients were hospitalised. Intolerance to the most recent anti-TNF agent and fewer previous anti-TNF agents were associated with clinical remission at week 14. Endoscopic severity was associated with poor response. This is the first study to show the real-world effectiveness and safety of ustekinumab administered according to the recommended induction regimen in a cohort of highly refractory CD patients.

Autologous Hematopoietic Stem Cell Transplantation in Refractory Crohn's Disease. Experience of Tertiary Medical Center in Brazil

Background Crohn's disease (CD) is a chronic inflammatory and relapsing disease that can affect any segment of the gastrointestinal tract. A significant percentage of these patients suffer an aggressive disease course, refractory to conventional clinical therapy . Surgery may be required, however the disease tends to recur after the resection. Aim Describe the potential clinical benefit of autologous hematopoietic stem cell with T-lymphocyte-depletion as a treatment for aggressive and refractory CD patients. Evaluate its efficacy, safety, toxicity and adverse events and primary and secondary clinical outcomes of follow up after 1year and 3 years. Methods We evaluated 6 patients submitted to an autologous HSCT. All patients were mobilized with cyclophosphamide (Cy) and G-CSF at the dose of 10mcg/kg/dia, The collection of HSC was initiated when the CD34+ count in the peripheral blood was greater than 15cels/mm3. The harvested product was enriched with CD34 + cells through a CliniMacs (Miltenyi Biotec) with a target CD34 + cells above 2.0 × 10E6/kg and less than 1 × 10E4Tcells/kg. The conditioning regimen consisted of cyclophosphamide 50mg/kg for 4 consecutive days, thymoglobulin 1.5mg/kg and methylprednisolone 1g/day from d-5 to d-2. Results Five patients refractory CD, between 43y and 19y (median age 38y), were included and follow the autologous HSCT. Corporal index mass 20 Kg/m2, median course of illness 14y, all negative for X-linked inhibitor apoptosis protein (XIAP). Four patients had penetrating disease and two patients had nonstricturing nonpenetrating phenotipe. Five patients had 2 to 3 previous intestinal surgeries. All patients used aminosalicylates steroids, thiopurines, metotrexate and more than one immunobiological therapies (IFX, ADA, VDZ). All six patients completed the mobilization, conditioning and transplantation phases, during a median time of hospitalization of 35 days. The median of CD34+ cels infused was 7.7 × 10e6/kg. All patients achieved engraftment, with a sustained median neutrophil count of 0.5 × 109/L at a median of 11 days and platelet grafting was 10 days. All patients had life-threatening complications as infectious (KPC, Staphylococcus aureus and septic by E. coli), febrile neutropenia and non-infectious complications as anemia mucositis and red cell transfusion was required. After 1y HSCT, all patients achieved the 1ary and 2nd outcomes: clinical and endoscopic remission (fig) and were steroid and immunosuppressive free remission (CDAI Conclusion HSCT is a complex treatment strategy for severe CD associated with mortality and serious adverse events. Due to its high complexity, risk of infections and death the balance between risks and benefits becomes significantly important in this therapy.

Efficacy of a concomitant elemental diet to reduce the loss of response to adalimumab in patients with intractable Crohn's disease.

Secondary loss of response to adalimumab (ADA-LOR) commonly occurs in patients with Crohn's disease (CD) treated with adalimumab (ADA). We evaluated the efficacy of concomitant elemental diet (ED) therapy to reduce ADA-LOR in adult CD patients. Patients were divided into either an ED (≥900kcal/day) or a non-ED group (<900kcal/day). Cumulative non-ADA-LOR rates were compared between groups. The effects of ED intake to reduce ADA-LOR were also assessed in antitumor necrosis factor-alpha (TNF-α)-naïve and infliximab (IFX)-intolerant or refractory CD patients. Serum ADA and TNF-α levels were measured. We enrolled 117 CD patients into the ED (n=25) or non-ED (n=92) groups. Although the cumulative non-ADA-LOR rate was higher in the ED group than in the non-ED group, ED intake was not an independent reducing factor for ADA-LOR (adjusted hazard ratio=0.725; 95% confidence interval: 0.448-1.180; P=0.196) in all patients. ED intake was significantly more effective in reducing ADA-LOR in IFX-intolerant or refractory patients than in anti-TNF-α-naïve patients in a dose-related manner (P for interaction <0.20). Serum ADA levels did not differ between the groups. Serum TNF-α levels were significantly lower in the ED group than in the non-ED group at week 28 (P=0.044) and week 52 (P=0.043). Concomitant ED therapy reduced ADA-LOR in IFX-intolerant or refractory patients in a dose-related manner. Reductions in the TNF-α levels by concomitant ED intake may contribute to reduce ADA-LOR in CD patients.

Long-term efficacy and safety of ustekinumab in 122 refractory Crohn's disease patients: a multicentre experience.

Long-term outcome of ustekinumab in Crohn's disease (CD) has not been evaluated. To evaluate the long-term efficacy and safety of ustekinumab and identify the predictive factors of ustekinumab failure-free persistence in a cohort of anti-TNF refractory CD patients. We performed a retrospective multicentre cohort study including all consecutive CD patients who began subcutaneous ustekinumab and presented a clinical response (defined as a significant improvement of CD-related clinical symptoms assessed by the patient's physician leading to continued ustekinumab) during the first year of treatment. Primary outcome was treatment failure defined as withdrawal of treatment due to loss of response, intolerance or need for surgery. Eighty-eight of the 122 (72%) CD patients beginning ustekinumab from March 2011 to December 2014, responded to ustekinumab and were followed up until November 2016. Median time on ustekinumab was 26.6 (13.4-34.4) months. Forty-seven patients (54%) continued ustekinumab with a clinical response and 38 (43%) stopped treatment (32 for failure, five for remission and one for pregnancy). Endoscopic response was observed in 82% of patients with endoscopic evaluation and mucosal healing in 39%. Ustekinumab failure-free persistence rates were 78% at 12months, 66% at 24months and 55% at 36months. No predictive factor of ustekinumab failure-free persistence was identified. One severe adverse event was observed (anal adenocarcinoma). In this cohort of refractory CD patients receiving long-term ustekinumab therapy, more than 50% of patients continued ustekinumab treatment with no loss of response, intolerance or surgery and with a good safety profile.

Association of Ustekinumab Concentrations Using Anser® UST Mobility Shift Assay and Clinical Response in Crohnʼs Disease

Introduction: Ustekinumab (UST), a monoclonal antibody to the p40 subunit shared by interleukins-12 and -23, is approved for treatment of patients with Crohn's disease (CD). Concentrations of other monoclonal antibodies to tumor necrosis factor alpha (TNFa) have been associated with clinical and endoscopic outcomes in CD. The aim of this study was to measure UST concentrations and antibodies to UST (ATU) in sera from patients with Crohn's disease using a commercial grade, drug tolerant test ANSER®UST. Methods: A homogenous mobility shift assay (HMSA) was developed and validated for measuring UST and ATU concentrations in human serum samples. Serum specimens (N=19) from anti-TNFα refractory CD patients receiving UST maintenance treatment were analyzed (Table 1). Clinical response was defined as >50% reduction in symptoms based on physician global assessment (scores of 0-3: ranging from 0 indicating a lack of response to 3 indicating complete response). Mucosal healing was defined as the absence of ulcers on endoscopy. Serum concentrations of CRP and TNFa were measured using electrochemiluminescent and immunoassays, respectively. Two-sample t-test and Fisher's Exact test were used for the statistical analysis. P-value < 0.05 was considered significant.Table: Table. Patient CharacteristicsResults: Dynamic range for the quantitation of ANSER®UST test was 1.56-25 μg/mL (UST) and 1.56-100 U/ml (ATU), inter- and intra-assay precision was < 10% CV and accuracy was within 20%. A wide range of serum UST concentrations were observed in the analyzed patient cohort (min: 0.78 μg/mL, max: 20.85 μg/mL), but ATU was not detected in any patient. The median drug concentrations for clinical responders were significantly higher than the non-responders (5.8 μg/mL vs 2.2 μg/mL, p=0.0025) while the median CRP concentrations were lower (2.8 μg/mL vs 10.2 μg/mL, p=0.03) (Figure 1). Patients with mucosal healing had numerically higher median UST concentrations (4.6 μg/mL vs 3.9 μg/mL, p=0.16); however, this was not statistically significant.Figure: Serum UST and CRP Concentrations in Clinical Responders and Non-Responders.Conclusion: Using a drug-tolerant ANSER®UST test, serum UST concentration was associated with clinical response in a real world biologic-experienced CD patient population. Though the number of patients was small, this study demonstrated the utility of serum UST monitoring. A larger cohort of patients will be needed to determine the optimal clinical cut-offs of serum UST that may predict UST responsiveness.

DOP053 The fecal microbiome as a tool for monitoring and predicting response outcomes in ustekinumab-treated, anti-TNFα refractory Crohn's disease patients: results from the CERTIFI study

DOP053 The fecal microbiome as a tool for monitoring and predicting response outcomes in ustekinumab-treated, anti-TNFα refractory Crohn's disease patients: results from the CERTIFI study

DOP027 Long-term efficacy and safety of ustekinumab in refractory Crohn's disease patients: a multicenter retrospective experience

DOP027 Long-term efficacy and safety of ustekinumab in refractory Crohn's disease patients: a multicenter retrospective experience

OP030 Identification of disease-relevant bacterial signatures in gnotobiotic IL-10 deficient mice using fecal samples from IBD patients undergoing hematopoietic stem cell transplantation

Background: Imbalanced microbial composition has been linked to the pathogenesis of inflammatory bowel diseases (IBD). Hematopoietic stem cell transplantation (HSCT) proved to be successful in inducing remission in severe, highly refractory Crohn's disease (CD) patients, possibly by erasing immune responses against gut microbes. Gnotobiotic mouse models colonized with human microbiota brought insights into the mechanistic aspects of host-microbe interactions. The aim of this study was to assess the functional role of microbiota signatures associated with different disease states. Methods: High-throughput 16S rRNA gene amplicon sequencing was performed on (n=147) fecal samples collected from (n=13) healthy donors and (n=31) HSCT-treated CD patients. Germ-free (GF) wild-type (WT) and IL10–/– mice (129 Sv/Ev; n=12 mice/group) were colonized with fecal microbiota from CD patients before and after HSCT at different disease states. Selection of CD patients for transfer into GF mice was based on clinical and endoscopic disease activity, including paired patient samples collected under remission or relapse following HSCT. Results: Microbiota profiling showed a significantly reduced microbial diversity in patients compared with healthy controls. Patients in remission showed higher microbial diversity than patients in relapse or at active state of the disease. Patients with fistulating or ileal phenotype had the least diverse ecosystems. High level of inter-individual variation was observed. Despite an incomplete transfer of donor microbiota with a 20–40% loss of species-level taxa, humanized mice reflected the dysbiotic features of their respective human donors, indicated by richness and diversity measures. Histopathological evaluation showed moderate to severe inflammation in colon and cecum of the IL10–/– mice associated with microbiota from patients in relapse. In contrast, IL10–/– mice associated with microbiota from patients in remission remained disease-free. To validate the phenotype transfer, we gavaged the mice three times with donor microbiota during the first week of colonization. Remission-associated mice showed higher species richness but still remained disease-free, while relapse-associated mice developed enhanced inflammation measured at the level of fecal complement C3 concentrations. Endpoint microbial composition remained similar, regardless of the number of inoculations and F1 generation of mice displayed a stable engraftment of human microbiota. Conclusions: Transfer of patient-derived fecal microbiota mimics the disease phenotype in gnotobiotic IL10–/– mice. Bacterial composition, not the number of species is responsible for disease initiation. Humanized mice represent a potential tool for recapitulating disease phenotypes in IBD.

Cyclophosphamide Pulse Therapy in Severe Refractory Crohn's Disease: A Retrospective Multicenter Case Series

Background and Aims: In Crohn's disease (CD) patients still remain refractory to current regimens, including biologicals. Previous data from small single-center studies indicated cyclophosphamide pulse therapy (CPT) to be effective for induction of remission at least in steroid-refractory cases. The aim of the present study was to study the efficacy and safety of CPT in mainly tumor necrosis factor (TNF)-refractory complicated CD patients. Methods: Patients with refractory CD undergoing CPT were identified in 13 centers of the German IBD Study Group and retrospectively registered. In total, 41 patients (12 male, 29 female, median age 36 years, range 18-72 years) were included for analysis. Seventy-eight percent of these had previously been treated with thiopurines and 90% had previously received anti-TNF antibodies. Former steroid treatment was found throughout the cohort. Results: Patients received a median number of 5 (1-13) pulses every 28 (13-54) days in a period of 120 (12-411) days. A median dose of 766 (600-1,200) mg and a median cumulative dose of 4,500 (750-9,750) mg was given. A clinical response (reduction in the Harvey-Bradshaw Index [HBI] ≥2 points) was found in 68% of the patients and clinical remission (HBI <5 points) in 32%. Steroids could be reduced from 31 to 12 mg per day over all patients. Side effects were recorded in 71% (n = 29) of the patients. Three patients terminated CPT due to side effects. No patient died. Conclusion: Our data point to CPT as a therapeutic alternative for induction of remission in patients with severe refractory courses of CD including TNF antagonists. CPT might serve as bridging for maintenance treatment.

Transferrin Level Before Treatment and Genetic Polymorphism in HFE Gene as Predictive Markers for Response to Adalimumab in Crohn's Disease Patients.

Tumor necrosis factor α inhibitors (anti-TNF) have improved treatment of several complex diseases, including Crohn's disease (CD). However, the effect varies and approximately one-third of the patients do not respond. Since blood parameters as well as genetic factors have shown a great potential to predict response during treatment, the aim of the study was to evaluate response to anti-TNF treatment with adalimumab (ADA) between genes HFE and TF and haematological parameters in Slovenian refractory CD patients. Single nucleotide polymorphisms (SNPs) rs1799852 in gene TF and rs2071303 in gene HFE were genotyped in 68 refractory CD patients for which response has been measured using inflammatory bowel disease questionnaire (IBDQ) index. Haematological parameters and IBDQ index were determined before therapy and after 4, 12, 20 and 30weeks. We found novel strong association between SNP rs2071303 in gene HFE and response to ADA treatment, particularly patients with G allele comparing to A allele had better response after 20weeks (p=0.008). Further, we found strong association between transferrin level at baseline and treatment response after 12, 20 and 30weeks, where average transferrin level before therapy was lower in responders (2.38g/L) compared to non-responders (2.89g/L, p=0.005). Association was found between transferrin level in week 30 and SNP rs1799852 (p=0.023), and between MCHC level before treatment and SNP rs2071303 (p=0.007). Our results suggest that SNP in gene HFE as well as haematological markers serve as promising prognostic markers of response to anti-TNF treatment in CD patients.

Efficacy and safety of thalidomide in the treatment of 29 refractory Crohn's disease patients

Objective To evaluate the efficacy and safety of thalidomide (100 to 200mg per day) in the treatment of adult refractory Crohn's disease (CD). Methods From July 2008 to February 2013, 29 refractory CD patients were enrolled in thalidomide (100 to 200 mg per day)cohort study. The clinical activity was evaluated by simplified CD activity index. Patients in clinical remission underwent colon endoscope examination, and mucosal healing was assessed by simple endoscopic score for Crohn's disease (SES-CD). Adverse reactions (ADR) were also observed. Results Among the 29 CD patients, 23 males and six females, the baseline of 19 patients (65.5%) were in clinical active period and 10 in clinical remission period. Among patients with baseline in clinical active period, three patients did not reach the target dose because of ADR, the left 16 patients were treated with thalidomide for one year and 14 patients achieved clinical remission.The median time of inducing clinical remission was one month. A totle of 24 patients with clinical remission induced by thalidomide and with baseline in clinical remission period were assessed in efficacy evaluation of mucosal healing. Thalidomide was withdrawn in three patients in six months because of ADR and colonoscopy evaluation did not complete, while the other 21 patients received colonoscopy evaluation among whom 33.3%(7/21) achieved mucosal healing after two years of thalidomide treatment. Numbness of the hands, feet or mouth, somnolence and dermatitis were the top three ADR of thalidomide treatment. A total of nine patients withdrew the medication because of ADR (four (13.8%) with numbness of the hands, feet or mouth, four (13.8%) with dermatitis and one (3.4%) with leukopenia). Conclusions Thalidomide 100 to 200 mg per day can induce clinical remission and mucosal healing in refractory CD. However, it has some adverse reactions and close monitoring and follow up are required during treatment. Key words: Thalidomide; Crohn disease; Remission induction; Mucosal healing; Adverse reactions

Cellular immunotherapy of chronic inflammation and autoimmunity using autologous antigen-specific T regulatory (Treg) cells

Among the most innovative immunotherapies, autologous cellular immunotherapy aims at treating patients with their own immune cells that, before reinjection, have been educated ex vivo to fight disease. Antigen-specific regulatory T cells (Ag-Treg) are recognized as a promising tool for therapeutic intervention in patients with chronic inflammation and autoimmunity. Ovasave ® is an autologous ovalbumin-specific Treg (Ova-Treg) product that is in development for the treatment of severe Crohn's disease patients refractory to available treatments. Ovasave ® has shown a good tolerability and dose-related efficacy in a first-in-man open-labeled phase I/IIa study in refractory Crohn's disease patients. In this study, a 75% response rate and a 38% remission rate on the CDAI (Crohn's Disease Activity Index) were observed at the best dose. In addition, clinical response was correlated with a decrease in vitro proliferation of patient's white blood cells in response to ovalbumin, indicating that decreased proliferation to Ag-Treg specific antigen could be used as a biomarker of clinical response. A randomized controlled phase IIb clinical study with Ovasave will start mid-2014 to confirm the efficacy and tolerability of Ovasave ® in refractory's Crohn's disease patients. Ag-Treg manufacturing has been developed to be highly cost-effective and logistically simple: multiple years of treatment doses are manufactured by one processing from one blood sample. Ovasave ® is the lead product from TxCell's ASTrIA platform Ag-Treg products. From this platform, Col-Treg, an autologous collagen-II specific based product is in the final preclinical stage of development for the treatment of patients with autoimmune uveitis, an orphan chronic inflammatory condition of the eye, with a phase I/IIa clinical study planned for 2015. With Ovasave ® and Col-Treg as novel, personalized cellular immunotherapies for chronic autoimmune inflammatory diseases, Ag-Tregs represent a new therapeutic opportunity for patients who today have few or no treatment options.

Fecal microbiota transplantation through mid-gut for refractory Crohn's disease: safety, feasibility, and efficacy trial results.

The gut microbiota plays a pivotal role in the intestinal diseases. Fecal microbiota transplantation (FMT) might be a rescue therapy for refractory inflammatory bowel disease. This study aimed to evaluate the safety, feasibility, and efficacy of FMT through mid-gut for refractory Crohn's disease (CD). We established standardized laboratory protocol and clinical work flow for FMT. Only refractory CD patients with Harvey-Bradshaw Index (HBI) score ≥ 7 were enrolled for this study. All included patients were treated with single FMT through mid-gut and assessed during follow-up. Metagenomics analysis showed a high concordance between feces sample and purified fecal microbiota from same donors. Standardized fecal microbiota preparation and clinical flow significantly simplified the practical aspects of FMT. Totally, 30 patients were qualified for the present analysis. The rate of clinical improvement and remission based on clinical activity at the first month was 86.7% (26/30) and 76.7% (23/30), respectively, which was higher than other assessment points within 15-month follow-up. Patients' body weight increased after FMT, and the lipid profile improved as well. FMT also showed a fast and continuous significant effect in relieving the sustaining abdominal pain associated with sustaining CD. This is a pilot study with the largest sample of patients with refractory CD who underwent single FMT. The results demonstrated that FMT through mid-gut might be a safe, feasible, and efficient rescue therapy for refractory CD.