Ilse Molendijk

Pretreatment with interferon-γ enhances the therapeutic activity of mesenchymal stromal cells in animal models of colitis

Mesenchymal stromal cells (MSCs) are currently under investigation for the treatment of inflammatory disorders, including Crohns disease. MSCs are pluripotent cells with immunosuppressive properties. Recent data suggest that resting MSCs do not have significant immunomodulatory activity, but that the immunosuppressive function of MSCs has to be elicited by interferon‐γ (IFN‐γ). In this article, we assessed the effects of IFN‐γ prestimulation of MSCs (IMSCs) on their immunosuppressive properties in vitro and in vivo. To this end, we pretreated MSCs with IFN‐γ and assessed their therapeutic effects in dextran sodium sulfate (DSS)‐ and trinitrobenzene sulfonate (TNBS)‐induced colitis in mice. We found that mice treated with IMSCs (but not MSCs) showed a significantly attenuated development of DSS‐induced colitis. Furthermore, IMSCs alleviated symptoms of TNBS‐induced colitis. IMSC‐treated mice displayed an increase in body weight, lower colitis scores, and better survival rates compared with untreated mice. In addition, serum amyloid A protein levels and local proinflammatory cytokine levels in colonic tissues were significantly suppressed after administration of IMSC. We also observed that IMSCs showed greater migration potential than unstimulated MSCs to sites within the inflamed intestine. In conclusion, we show that prestimulation of MSCs with IFN‐γ enhances their capacity to inhibit Th1 inflammatory responses, resulting in diminished mucosal damage in experimental colitis. These data demonstrate that IFN‐γ activation of MSCs increases their immunosuppresive capacities and importantly, their therapeutic efficacy in vivo. STEM CELLS 2011;29:1549–1558

Disappointing durable remission rates in complex Crohn's disease fistula

Background:Despite potent drugs and surgical techniques, the treatment of perianal fistulizing Crohns disease (CD) remains challenging. We assessed treatment strategies for perianal fistulizing CD and their effect on remission, response, and relapse. Methods:Patients with perianal fistulizing CD visiting the Erasmus MC between January 1, 1980 and January 1, 2000 were identified. Demographics, fistula characteristics, and received treatments aimed at the outcome of these strategies were noted. Results:In total, 232 patients were identified (98 male; 42.2%). Median follow-up was 10.0 years (range, 0.5–37.5 yr). Complex fistulas were present in 78.0%. Medical treatment (antibiotics, steroids, immunosuppressants, and anti-tumor necrosis factor) commenced in 79.7% of the patients and in 53.2%, surgery (colectomy, fistulectomy, stoma, and rectum amputation) was performed. Simple fistulas healed more often than complex fistulas (88.2% versus 64.6%; P < 0.001). Rectum involvement was not associated with a lower remission rate, and anti-tumor necrosis factor therapy did not increase complete fistula healing rates in simple and complex fistula. Initially, healed fistulas recurred in 26.7% in case of simple fistulas and in 41.9% in case of complex fistulas (P = 0.051). Only 37.0% of the complex fistulas were in remission at the end of follow-up compared with 66.7% of the simple fistulas (P < 0.001). Conclusions:Only the minority of CD complex perianal fistulas were in remission after conventional treatment strategies after a median follow-up of 10 years. Simple fistulas were more likely to heal than complex fistulas, and less of these healed fistulas relapsed. However, more than 3 quarters of the patients had complex perianal fistulas.

Mesenchymal stromal cell function is not affected by drugs used in the treatment of inflammatory bowel disease

BACKGROUND AND AIMS Mesenchymal stromal cells (MSC) have both multilineage differentiation capacity and immunosuppressive properties. Promising results with MSC administration have been obtained in experimental colitis. Clinical application of MSC for the treatment of inflammatory bowel disease (IBD) is currently under investigation in phase I-III trials in patients with past or concurrent immunomodulating therapy. However, little is known about MSC interactions with these immunosuppressive drugs. To address this issue we studied the combined effect of MSC and IBD drugs in in vitro functionality assays. METHODS The effects of azathioprine, methotrexate, 6-mercaptopurine and anti-tumor necrosis factor (TNF)-α on MSC phenotype, survival, differentiation capacity and immunosuppressive capacity were studied. RESULTS MSC exposed to physiologically relevant concentrations of IBD drugs displayed a normal morphology and fulfilled phenotypic and functional criteria for MSC. Differentiation into adipocyte and osteocyte lineages was not affected and cells exhibited normal survival after exposure to the various drugs. MSC suppression of peripheral blood mononuclear cell (PBMC) proliferation in vitro was not hampered by IBD drugs. In fact, in the presence of 6-mercaptopurine and anti-TNF-α antibodies, the inhibitory effect of this drug alone was enhanced, suggesting an additive effect of pharmacotherapy and MSC treatment. CONCLUSIONS This study demonstrates that, in vitro, MSC phenotype and function are not affected by therapeutic concentrations of drugs commonly used in the treatment of IBD. These findings are important for the potential clinical use of MSC in combination with immunomodulating drugs and anti-TNF-α therapy.

Immunomodulatory Effects of Mesenchymal Stromal Cells in Crohn’s Disease

The ability of mesenchymal stromal cells (MSCs) to suppress immune responses combined with their potential to actively participate in tissue repair provides a strong rationale for the use of MSCs as a new treatment option in diseases characterized by inflammation and severe tissue damage, such as Crohns disease (CD) and perianal fistulas. Multiple studies have shown that MSCs suppress a range of immune cells, such as dendritic cells (DC), naïve and effector T cells, and natural killer (NK) cells. Recently published papers attribute the immunosuppressive capacity of MSCs to soluble factors produced by MSCs, such as prostaglandin E2 (PGE2), inducible nitric oxide synthase (iNOS), and indoleamine 2,3-dioxygenase (IDO). Promising results are obtained from phase I and II clinical trials with autologous and allogeneic MSCs as treatment for refractory CD and perianal fistulas; however the question remains: what are the molecular mechanisms underlying the immunomodulating properties of MSCs? This paper highlights the present knowledge on the immunosuppressive effects of MSCs and its complexity in relation to CD and perianal fistulas.

Improving the outcome of fistulising Crohn's disease

Fistulas are a frequent manifestation of Crohns disease (CD) and can result in considerable morbidity. Approximately 35% of all patients with CD will experience one fistula episode during their disease course of which 54% is perianal. The major symptoms of patients with perianal fistulas are constant anal pain, the formation of painful swellings around the anus and continuous discharge of pus and/or blood from the external fistula opening. The exact aetiology of perianal fistulas in CD patients remains unclear, but it is thought that a penetrating ulcer in the rectal mucosa caused by active CD forms an abnormal passage between the epithelial lining of the rectum and the perianal skin. Genetic, microbiological and immunological factors seem to play important roles in this process. Although the incidence of perianal fistulas in patients with CD is quite high, an effective treatment is not yet discovered. In this review all available medical and surgical therapies are discussed and new treatment options and research targets will be highlighted.

Intraluminal injection of mesenchymal stromal cells in spheroids attenuates experimental colitis

BACKGROUND AND AIMS In recent years, mesenchymal stromal cells [MSCs] emerged as a promising therapeutic option for various diseases, due to their immunomodulatory properties. We previously observed that intraperitoneally injected MSCs in experimental colitis form spherical shaped aggregates. Therefore, we aggregated MSCs in vitro into spheroids and injected them intraluminally in mice with established colitis, to investigate whether these MSC spheroids could alleviate the colitis. METHODS We injected 0.5 x 10(6) MSCs in spheroids, 2.0 x 10(6) MSCs in spheroids, or phosphate-buffered saline [PBS] as a treatment control, via an enema in mice with established dextran sulphate sodium [DSS]-induced colitis. Body weight was measured daily and disease activity score was determined at sacrifice. Endoscopy was performed to evaluate mucosal healing. After sacrifice, both systemic and local inflammatory responses were evaluated. RESULTS Intraluminally injected MSC spheroids alleviated DSS-induced colitis, resulting in significantly less body weight loss and lower disease activity score at sacrifice when a high dose of MSC spheroids was administered. However, the percentage of mucosal lesions in the distal colon and endoscopy scores were not significantly lower after treatment with 2.0 x 10(6) MSCs in spheroids compared with PBS-treated mice. Systemic inflammation marker serum amyloid A [SAA] was significantly reduced after treatment with 2.0 x 10(6) MSCs in spheroids. In addition, local cytokine levels of IFN-ɣ, TNF-α, IL-6, and IL-17a, as well as numbers of macrophages and neutrophils, showed a clear decrease-though not always significant-after intraluminal injection of the MSC spheroids. CONCLUSION Intraluminally injected MSC spheroids at least partially attenuate experimental colitis, with fewer phagocytes and proinflammmatory cytokines, when a high dose of MSCs in spheroids was administered.

Endoscopic Administration of Mesenchymal Stromal Cells Reduces Inflammation in Experimental Colitis

Background Mesenchymal stromal cells (MSCs) are a potential therapeutic modality in inflammatory bowel diseases (IBDs) because of their immunomodulatory and regenerative properties. However, when injected systemically, only a small portion of the cells, if any, reach the inflamed colon. In this study, we assessed whether endoscopic injections of MSCs into the intestinal wall of the inflamed colon affect the course of experimental colitis. Furthermore, we investigated if injection of aggregated MSCs in spheroids could enhance their therapeutic ability. Methods Expression levels of in vivo MSC aggregates and in vitro MSC spheroids were compared with monolayer cultured MSCs for both anti-inflammatory and pro-regenerative factors. Subsequently, MSCs and MSC spheroids were injected endoscopically in mice with established dextran sulfate sodium (DSS)-induced colitis. Results Endoscopically injected MSCs and MSC spheroids both alleviated DSS-induced colitis. Furthermore, both in vivo and in vitro MSC spheroids showed increased expression of factors important for immunomodulation and tissue repair, compared with monolayer cultured MSCs. Despite differential expression of these factors, MSC spheroids showed similar clinical efficacy in vivo as single-cell suspension MSCs. Analysis of serum samples and colon homogenates showed that local MSC therapy resulted in increased levels of interferon-γ, indoleamine 2,3-dixoygenase, and interleukin-10. Conclusions Endoscopic injections of MSCs and MSC spheroids in the inflamed colon attenuate DSS-induced colitis. Our data show that endoscopic injection can be a feasible and effective novel application route for MSC therapy in patients with luminal IBD.

P050 Intraperitoneally injected mesenchymal stromal cells home significantly more often to the intestines in colitis mice compared to healthy controls

was to quantify binding of CZP, IFX, ADA and ETA to FcRn and to measure FcRn-mediated transcytosis of these agents. Methods: A BiacoreTM assay was used to determine the binding of CZP, ADA and IFX to human FcRn. Anti-TNFs were passed over an FcRn-coated chip for 5min at a range of concentrations from 21 670nM to determine the on-binding rate; a buffer at pH 6.0 was used to allow optimum binding. The off-rate was followed for a further 5min by running buffer alone over the chip. MDCK II cells transfected with human FcRn were used to measure FcRn-mediated transcytosis using a pH 5.9 buffer on the apical side and pH 7.2 on the basolateral side. The antiTNFs and the control antibody (P146), which possessed an Fc modified to prevent binding to FcRn, were biotinylated to allow visualization. The amount of each anti-TNF transcytosed across the cell layer over 4hrs was measured by MSD assay. Results: IFX (132nM) and ADA (225nM) had relatively high binding affinity to FcRn while the binding affinity of ETA to FcRn was approximately 5 to 10-fold lower (1500nM). In contrast, CZP did not bind to the FcRn with any measurable affinity. The mean levels of transcytosis seen with IFX and ADA were 249.6 ng/mL and 159.5 ng/mL, respectively (n = 3). Transcytosis of ETA (81.3 ng/mL) was lower than that of ADA and IFX. In contrast, the level of CZP transcytosis (3.2 ng/mL) was significantly lower than that observed with the other anti-TNFs tested. The control antibody P146 also showed low transfer (5.9 ng/mL). Since neither the control antibody nor CZP bind to FcRn, the levels detected are probably due to low level nonspecific leakage across the cell layer. Conclusions: CZP does not have an Fc and thus did not bind FcRn. Moreover, no FcRn-mediated CZP transcytosis was detected. In contrast, ADA and IFX had relatively high binding affinity to FcRn and were actively transcytosed. ETA showed lower binding affinity to FcRn and subsequent transcytosis, compared to IFX/ADA, but FcRn-mediated ETA transport could still be measured. These results explain previously observed active transport of anti-TNFs across the placenta seen in patients treated with IFX and ADA, whereas only low levels were observed with CZP.

DOP087 Only one third of Crohn's disease patients have sustained remission of perianal fistulas

patients were colonoscoped at 18 months. HRQoL was assessed with a general (SF36) and disease-specific (IBDQ) questionnaire pre-operatively and at 6, 12 and 18 months. CRP, CDAI and faecal calprotectin (FC) were measured longitudinally. Results: 174 patients (median age 38, 55% female) were included. HRQoL was poor pre-operatively: median SF36 = 40 (where maximum=100, Australian normal = 70 90) and IBDQ= 120 (maximum=224, average score in Australian Crohn’s disease patients=156). For all patients both SF36 and IBDQ improved significantly at 6 months to 78 and 178 respectively, and this was sustained at 12 months (81 and 183) and 18 months (80 and 182 respectively). Females had lower HRQoL than males post-op at 6 (SF36 p = 0.012; IBDQ p = 0.007) and 12 months (SF36 p = 0.001, IBDQ p = 0.006). Smokers had poorer HRQoL compared to non-smokers at both 12 and 18 months: SF36 at 12 month p = 0.002, and IBDQ at 12 and 18 months (p = 0.046, p = 0.047 respectively). Persistent endoscopic remission, thiopurine or adalimumab therapy and treatment step up were not associated with changes in HRQoL. There was a significant inverse correlation between CDAI and both SF-36 and IBDQ at 6, 12 and 18 months. HRQoL did not correlate with endoscopic remission, CRP or FC. Conclusions: Intestinal resection of all macroscopic Crohn’s disease, with a focus on maintaining remission, is associated with significant and sustained improvement in general and disease-specific HRQoL. The lower HRQoL in female patients and smokers may reflect partly their lower QoL in the healthy and IBD populations, but this requires further investigation. A higher clinical disease activity index, but not direct measures of active disease or type of drug therapy, is associated with a lower HRQoL, suggesting that symptoms reflect subjective personal factors and not active mucosal disease or drug effects.