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Correspondence: Address to Rachele Ciccocioppo, MD, Center for the Study and Cure of Celiac Disease, Department of Internal Medicine, IRCCS San Matteo Hospital Foundation, University of Pavia-Piazzale Golgi, 19-27100 Pavia, Italy.
Cell Factory and Research Laboratory–Department of Pediatrics, Fondazione IRCCS Policlinico San Matteo and Università degli Studi di Pavia, Pavia, Italy
Cell Factory and Research Laboratory–Department of Pediatrics, Fondazione IRCCS Policlinico San Matteo and Università degli Studi di Pavia, Pavia, Italy
Type II refractory celiac disease (RCD), as defined according to the amount of aberrant intraepithelial lymphocytes, is a condition characterized by severe malabsorption syndrome and poor prognosis, with no effective treatment. Based on the regenerative and immunomodulatory properties of mesenchymal stem cells (MSCs), we investigated the feasibility, safety, and efficacy of serial infusions of autologous bone marrow–derived MSCs in a 51-year-old woman with type II RCD. Mesenchymal stem cells were isolated, expanded, and characterized following standard protocols. Monitoring of the patient's malabsorption indexes, mucosal architecture, and percentage of aberrant intraepithelial lymphocytes was scheduled for the time of enrollment, at each infusion, and after 6 months. Determination of mucosal expression of interleukin (IL)-15 and its receptor was also performed. Expansion of MSCs was feasible, and the patient underwent 4 systemic infusions of 2 × 106 MSCs/kg body weight 4 months apart, without adverse effects. During the treatment period, she experienced gradual and durable amelioration of her general condition, with normalization of stool frequency, body mass index, laboratory test results, and mucosal architecture. Remarkably, the expression of IL-15 and its receptor almost completely disappeared. Thus, treatment of RCD with serial MSC infusions seems promising, leading to recovery from the life-threatening condition while blocking the IL-15 pathogenic pathway.
Refractory celiac disease (RCD) is a condition defined by the persistence or recurrence of malabsorption syndrome and intestinal villous atrophy despite a strict gluten-free diet for more than 12 months.
Its hallmark is an accumulation of aberrant intraepithelial lymphocytes (IELs), the amount of which makes it possible to differentiate between type I and type II RCD.
The latter is burdened by a poor prognosis because of severe malnutrition, frequent infections, and a high risk of overt lymphoma, leading to a 5-year survival rate less than 50%.
although the therapeutic use of its blocking agents is still awaited. Therefore, the current treatment is based on the use of nontargeted immunomodulating agents that do not substantially modify the patients' outcome,
No alloantibodies against mesenchymal stromal cells, but presence of anti-fetal calf serum antibodies, after transplantation in allogeneic hematopoietic stem cell recipients.
In addition, they harbor a powerful and multifaceted action on almost all cells involved in the immune response, with the ultimate effect of dampening inflammation and favoring immune tolerance.
2 immune-mediated conditions whose intestinal lesions resemble those of RCD. Following this evidence, we treated a patient with life-threatening malabsorption syndrome due to type II RCD by serial infusions of autologous bone marrow–derived MSCs with gratifying results.
Case Report
In October 2013, a 51-year-old woman with RCD was admitted to the Department of Internal Medicine of the IRCCS Policlinico San Matteo Foundation (Pavia, Italy) because of recurrence of malabsorption syndrome despite having followed a gluten-free diet for 14 years after being diagnosed as having celiac disease. After her initial diagnosis she was treated with budesonide for refractoriness, with good recovery of clinical condition and mucosal architecture lasting five years (Figure 1). At this latest admission, she reported severe diarrhea and fever that caused weight loss, dehydration, and electrolyte imbalance (Table), which placed her in a life-threatening condition.
Figure 1Chronological distribution of the patient's clinical course. The timing of the therapeutic interventions, ie, the diet regimen, corticosteroid therapy, and cellular treatment, together with that of the specific diagnostic procedures, is schematically represented, applying arbitrary distances among them. In detail, regarding the histologic analysis of the duodenal mucosa at the time of diagnosis of refractory celiac disease (RCD) and before mesenchymal stem cell (MSC) infusions (left-hand boxes), the presence of villous atrophy with crypt hyperplasia and heavy inflammatory infiltrate is clearly evident. After 2 MSC infusions (central boxes), a slight improvement in mucosal architecture is seen. At the end of the MSC treatment and after 6 months (right-hand boxes), the recovery of a healthy mucosa with normal architecture is observed. The histologic sections were stained with hematoxylin-eosin (original magnification, ×100). The lower boxes indicate the flow cytometric analyses of intraepithelial lymphocytes at each time point of the MSC treatment, as determined after the cells with strong CD45 expression and low side scatter were selected, and the dot plot cytoplasmic CD3 vs surface CD3, on CD7+ gate, was performed, thus showing the invariable presence of an aberrant T-cell population lacking in surface CD3 but displaying cytoplasmic CD3. AEA = antienterocyte antibody; ATA = anti–tissue transglutaminase antibody; CD = celiac disease; EMA = antiendomysial antibody.
B = basophils; Cl = serum chloride; E = eosinophils; Ig = immunoglobulin; K = serum potassium; L = lymphocytes; MSC = mesenchymal stem cell; M = monocytes; N = neutrophils; Na = serum sodium.
SI conversion factors: To convert hemoglobin values to g/L, multiply by 10; albumin values to g/L, multiply by 10; IgG values to g/L, multiply by 0.01; IgA and IgM values to mg/L, multiply by 10.
a B = basophils; Cl = serum chloride; E = eosinophils; Ig = immunoglobulin; K = serum potassium; L = lymphocytes; MSC = mesenchymal stem cell; M = monocytes; N = neutrophils; Na = serum sodium.
b SI conversion factors: To convert hemoglobin values to g/L, multiply by 10; albumin values to g/L, multiply by 10; IgG values to g/L, multiply by 0.01; IgA and IgM values to mg/L, multiply by 10.
c Reference range, 19-22 (calculated as weight in kilograms divided by height in meters squared).
d Reference range, D-xylose test result greater than 30 mg/dL.
A course of total parenteral nutrition with combined antibiotic and antifungal drug therapy was undertaken after the isolation of Staphylococcus epidermidis in the blood culture, Escherichia coli in the bronchoalveolar lavage fluid, and Candida albicans in the stool. Meanwhile, the search for antiendomysial, anti–tissue transglutaminase, and antienterocyte antibodies proved negative, and the dietary interview confirmed that she was following a strict gluten-free diet. The genomic analyses revealed the presence of 2 heterodimers of susceptibility to celiac disease (HLA-DQA1*0201,*0501 and DQB1*0201,*0202), and the upper endoscopy showed the reduction of duodenal folds with a scalloped profile, mirrored by complete villous atrophy at histologic examination (Figure 1). On immunohistochemical analysis, a polymorphic infiltrate rich in CD3+, CD5+, CD4+, CD20+, and CD138+ cells in the lamina propria was evident, but with rare CD8+ and CD127+ and no CD56+ elements, and the number of IELs was greater than 40 per 100 epithelial cells. Their flow cytometric characterization
displayed the presence of an aberrant subset as defined by the surface phenotype CD3−CD4−CD8−CD7+CD103+ and cytoplasmic CD3+, with values higher than 90% (Figure 1), that was paralleled by the monoclonal rearrangement of the β- and γ-chains of the T-cell receptor.
The patient was thus diagnosed as having type II RCD. The presence of overt lymphoma or other malignancies was ruled out through a whole-body imaging study, an 18F-fluorodeoxyglucose positron emission tomography scan, and double-balloon enteroscopy that showed some ulcerations surrounded by atrophic mucosa. Because her clinical condition did not improve despite eradication of the infections, a course of systemic corticosteroids (intravenous methylprednisolone 1 mg/kg per day) was undertaken for 4 weeks (Figure 1). Nonetheless, the persistence of severe diarrhea placed the patient in such a critical condition that she was unable to undergo bone marrow transplant. Intravenous infusions of autologous bone marrow–derived MSCs were, thus, proposed as compassionate therapy. The protocol was approved by the local ethics committee (IRCCS Policlinico San Matteo Foundation, Pavia, Italy), and the patient gave written informed consent.
Methods
MSC Preparation
Fifty milliliters of bone marrow blood was harvested, and MSCs were isolated and expanded ex vivo according to Good Manufacturing Practice protocols, as previously reported.
Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute.
and clinical use (Supplemental Figure, available online at http://www.mayoclinicproceedings.org). For therapeutic purposes, MSC aliquots were cryopreserved until use, when they were resuspended in a solution of sodium chloride and human albumin 20% (3/1 vol/vol) in a sterile syringe at a concentration of 2 × 106 cells/mL, and infusions of 2.0 × 106 MSCs/kg body weight were scheduled at baseline and at months 4, 8, and 12 (Figure 1).
IEL Characterization
After isolation from mucosal specimens by means of chemical treatment with EDTA disodium salt dihydrate and DL-dithiothreitol (both from Sigma-Aldrich Corp) and centrifugation using a Percoll density gradient (25%-40%-75%; GE Healthcare), the IEL samples were stained with the following fluorochrome-conjugated monoclonal antibodies: anti-CD3, anti-CD4, anti-CD7, anti-CD8, anti-CD103, and APC-Alexa Fluor 750–conjugated anti-CD45 (all from Becton Dickinson-PharMingen) according to the manufacturer's instructions, to undergo multiparameter flow cytometric analysis (Navios, Kaluza software; Beckman Coulter). For permeabilization, the Fix&Perm kit (An Der Grub Bio Research GmbH) was used.
IL-15 and IL-15 Receptor Expression
To assess the involvement of the IL-15 pathway, Western blot analysis was applied on cryopreserved mucosal specimens by using the monoclonal antibodies to human IL-15 (MAB2471, 1:500 dilution; R&D Systems) and, after stripping, to IL-15 receptor α (IL-15Rα) (MAB147, 1:500 dilution; R&D Systems). The blot was finally stripped and incubated with a rabbit anti-human polyclonal anti–β-actin antibody (1:5000 dilution, Abcam) as internal control. The membrane development was performed using the ECL Prime kit, and films were digitalized by the VersaDoc imaging system (Bio-Rad Laboratories). The densitometry was assessed as band intensity and band area by Quantity One software (Bio-Rad Laboratories). As controls, 4 active (3 women and 1 man; age, 47-63 years) and 4 treated (2 women and 2 men; age, 45-56 years) celiac patients and 4 healthy controls (3 women and 1 man; age, 32-44 years) were recruited.
Results
During the 12-month treatment period, the patient experienced progressive improvement in her clinical condition, with normalization of stool frequency and almost all laboratory test values within a month, together with a gradual increase in body weight (Table), despite the fact that parenteral nutrition and systemic corticosteroid use had been discontinued 2 weeks after the first MSC infusion (Figure 1). There was also substantial improvement in the mucosal findings at endoscopy after 2 MSC infusions, accompanied by partial recovery of the architecture at histologic examination; and at the end of treatment, a full macroscopic and microscopic recovery was achieved, and it was still present 6 months after the last infusion (Figure 1), when only a few scars were found at wireless capsule endoscopy. In contrast, the persistence of both the aberrant IEL population, with values always exceeding 90% (Figure 1), and the monoclonal rearrangement of the β- and γ-chains of the T-cell receptor were still evident. Remarkably, no adverse events were recorded either immediately after each infusion or throughout the observation period. Finally, high IL-15 and IL-15Rα expression values at the mucosal level were observed in this patient before MSC treatment (values of band area/intensity in 2013: 0.86/0.89 arbitrary units (AU) and 1.4/1.25 AU, respectively), resembling those found in active celiac disease (mean values: 0.85/0.94 AU and 1.61/1.38 AU, respectively); while after treatment, they almost entirely disappeared (values after 2 MSC infusions [2014]: 0.09/0.56 AU and 0.54/1.21 AU; values at the end of MSC treatment [2015]: 0.12/0.62 AU and 0.73/1.19 AU, respectively), reaching levels similar to those of healthy controls (mean values: 0.11/0.62 AU and 0.22/1.27 AU, respectively). In patients with treated celiac disease, detectable levels of IL-15 and IL-15Rα were evident (mean values: 0.3/0.68 AU and 0.71/1.25 AU, respectively) (Figure 2).
Figure 2Interleukin (IL)-15/IL-15 receptor α (IL-15Rα) mucosal levels. A, Immunoblotting using the monoclonal anti–IL-15, anti–IL-15Rα, and anti–β-actin antibodies on mucosal samples from representative cases of healthy controls (HCs), patients with treated celiac disease (TCD), patients with untreated celiac disease (UCD), and the present patient with refractory celiac disease (RCD) throughout the treatment period. The protein levels of IL-15 (B) and IL-15Rα (C) were measured by scanning densitometry as band area (black bars) and band intensity (gray bars), expressed as mean ± SD arbitrary units (AUs), and normalized toward β-actin levels.
Despite a growing understanding of its pathogenesis, type II RCD remains a challenging condition because no standardized treatment is available and patient outcome is burdened by a high mortality rate due to the frequent progression into enteropathy-associated T-cell lymphoma.
The few cases in which recovery from malnutrition with improvement of mucosal architecture has been observed were those treated with hematopoietic stem cell transplant,
However, in view of the aggressiveness of bone marrow transplant, alternative and safer strategies are eagerly awaited. Thanks to their peculiar biological properties, MSCs represent an attractive and safe tool for cellular treatment in immune-mediated conditions.
prompted us to consider the use of MSCs as a new therapeutic strategy in type II RCD. Accordingly, we treated a patient with serial intravenous infusions of autologous bone marrow–derived MSCs, scheduled 4 months apart, for 12 months. The treatment was feasible, safe, and effective, with normalization of clinical findings within a month, thus successfully overcoming the critical stage, and the recovery of mucosal architecture and absorptive capacity was slower and more progressive throughout the treatment period. The need for serial infusions instead of a single injection arose from previous evidence of loss of efficacy in a case of adult autoimmune enteropathy.
The latter referred to a 61-year-old woman with a life-threatening malabsorption syndrome due to corticosteroid-refractory adult autoimmune enteropathy as diagnosed on the basis of positivity of antienterocyte autoantibodies and villous atrophy with inflammatory infiltration of the small bowel mucosa unresponsive to any dietary restrictions, including a gluten-free diet.
She had undergone 2 intravenous infusions of 1.8 × 106 MSCs/kg body weight 2 weeks apart with dramatic amelioration of her clinical conditions and prompt disappearance of the specific autoantibodies and mucosal lesions. However, this improvement was transient, and after a short time all the clinical, serologic, and histologic features of autoimmune enteropathy recurred. A possible explanation for this phenomenon is the trapping of most MSCs in the lungs owing to their large size,
In this regard, the short life span of MSCs when injected in vivo may explain their loss of efficacy and the need for serial infusions, as also observed in patients with Crohn disease, where the best outcome was obtained when serial infusions were performed,
In addition, a direct regenerative action cannot be excluded because MSCs have been shown to electively populate the intestine in the injured area and improve lesions to the same extent as their hematopoietic counterpart
this groundbreaking demonstration of an almost complete disappearance of IL-15 and IL-15Rα expression directly at the mucosal level provides a rationale for the wide use of MSCs in this condition. In this regard, note that MSCs are also capable of inhibiting the proliferation of IL-15–stimulated natural killer cells and their cytotoxicity,
Mesenchymal stem cells inhibit natural killer-cell proliferation, cytotoxicity, and cytokine production: role of indoleamine 2,3-dioxygenase and prostaglandin E2.
Thus, in the face of the persistence of aberrant IELs, MSCs seem to rescue the intestinal epithelium from the autoimmune attack, allowing the regrowth of villi and the normalization of absorptive function with striking results in terms of clinical findings and quality of life of the patient. The possibility that the pathogenic/aberrant clones may take longer to disappear and/or that further mechanisms are involved cannot be currently ruled out. In this regard, note that even after hematopoietic stem cell transplant, the percentage of IELs did not undergo substantial modification, and a few patients still developed a T-cell lymphoma,
thus indicating that aberrant IELs are highly resistant even to the conditioning regimen applied.
Conclusion
The MSCs from this patient with type II RCD display all the biological properties needed for therapeutic use, and their systemic infusion proved safe and successful in aiding the regeneration of small intestinal mucosa while dampening the IL-15 pathway. Scheduling of the infusions seems important to achieve a sustained effect, and the absence of risks and adverse events is another crucial point to bear in mind when choosing among the therapeutic options available in this specific clinical setting. Further investigations and systematic clinical trials are upcoming to definitively establish the usefulness of this therapeutic strategy in RCD, possibly either type I or type II, because no data supporting the a priori exclusion of a subset are available.
Acknowledgments
We thank Costanza Alvisi, MD, who performed all the endoscopic examinations by biopsy sampling; Giuseppina C. Cangemi, MSc, Alessandra Pasini, BSc (Department of Internal Medicine, Fondazione IRCCS Policlinico San Matteo, Pavia, Italy), Roberta Riboni, MSc (Department of Human Pathology, IRCCS Policlinico San Matteo Foundation), and Laura Catenacci, MSc (Cell Factory and Research Laboratory, Department of Pediatrics, Fondazione IRCCS Policlinico San Matteo), who provided technical support; and Susan West, who performed a thorough revision of the English text.
Supplemental material can be found online at http://www.mayoclinicproceedings.org. Supplemental material attached to journal articles has not been edited, and the authors take responsibility for the accuracy of all data.
References
Malamut G.
Cellier C.
Refractory coeliac disease: epidemiology and clinical manifestations.
No alloantibodies against mesenchymal stromal cells, but presence of anti-fetal calf serum antibodies, after transplantation in allogeneic hematopoietic stem cell recipients.
Optimization of in vitro expansion of human multipotent mesenchymal stromal cells for cell-therapy approaches: further insights in the search for a fetal calf serum substitute.
Mesenchymal stem cells inhibit natural killer-cell proliferation, cytotoxicity, and cytokine production: role of indoleamine 2,3-dioxygenase and prostaglandin E2.
Grant Support: This study was funded by a research grant from Fondazione IRCCS Policlinico San Matteo (No. 579, code 08064409) and by a grant from the Ministero dell'Università e Ricerca Scientifica (PRIN 2011, code 2010K34C45_007) (R.C.). These sources of funding were not in any way involved in the study design, collection, analysis and interpretation of data; writing of the manuscript; or the decision to submit the article for publication.
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