Yugo Ando

Subcutaneous adipose tissue–derived stem cells facilitate colonic mucosal recovery from 2,4,6‐trinitrobenzene sulfonic acid (TNBS)–induced colitis in rats

Background: Adipose tissue–derived stem cells (ADSCs) can be easily obtained from subcutaneous adipose tissue, and ADSCs can be demonstrated to display multilineage developmental plasticity. In this study, using TNBS‐induced colitis rats, we show the feasibility of repairing injured intestinal mucosa with adipose tissue–derived stem cells. Methods: The subcutaneous adipose tissue of F344 rats was obtained and digested by collagenase. The digested tissue was cultured in DMEM containing 10% FBS for 1 month. ADSCs were confirmed to differentiate under appropriate conditions into various lineages of cells, including bone, neural cells, adipocytes, and epithelial cells. HGF, VEGF, TGF‐&bgr;, and adiponectin in the culture supernatants of ADSCs were determined by ELISA. ADSCs (107 cells) were injected into the submucosa of the colon to examine their capacity to repair intestinal mucosa injured by TNBS. Results: In the experimental colitis model, the injection of ADSCs facilitated colonic mucosal repair and reduced the infiltration of inflammatory cells. High levels of HGF, VEGF, and adiponectin were detected in the culture supernatants of ADSCs. Moreover, injected ADSCs distributed to several layers of the colon, and some of them differentiated into mesodermal lineage cells. Conclusions: ADSCs can accelerate the regeneration of injured regions in experimental colitis. HGF, VEGF, and adiponectin might be responsible for the regeneration of injured regions in the colon.

Successful immunotherapy of autoimmune cholangitis by adoptive transfer of forkhead box protein 3(+) regulatory T cells.

Treatment of primary biliary cirrhosis (PBC) has lagged behind that of other autoimmune diseases. In this study we have addressed the potential utility of immunotherapy using regulatory T cells (Treg) to treat murine autoimmune cholangitis. In particular, we have taken advantage of our ability to produce portal inflammation and bile duct cell loss by transfer of CD8+ T cells from the dominant negative form of transforming growth factor beta receptor type II (dnTGF‐βRII) mice to recombination‐activating gene (Rag)1–/– recipients. We then used this robust established adoptive transfer system and co‐transferred CD8+ T cells from dnTGF‐βRII mice with either C57BL/6 or dnTGF‐βRII forkhead box protein 3 (FoxP3+) T cells. Recipient mice were monitored for histology, including portal inflammation and intralobular biliary cell damage, and also included a study of the phenotypical changes in recipient lymphoid populations and local and systemic cytokine production. Importantly, we report herein that adoptive transfer of Treg from C57BL/6 but not dnTGF‐βRII mice significantly reduced the pathology of autoimmune cholangitis, including decreased portal inflammation and bile duct damage as well as down‐regulation of the secondary inflammatory response. Further, to define the mechanism of action that explains the differential ability of C57BL/6 Treg versus dnTGF‐βRII Treg on the ability to down‐regulate autoimmune cholangitis, we noted significant differential expression of glycoprotein A repetitions predominant (GARP), CD73, CD101 and CD103 and a functionally significant increase in interleukin (IL)‐10 in Treg from C57BL/6 compared to dnTGF‐βRII mice. Our data reflect the therapeutic potential of wild‐type CD4+ FoxP3+ Treg in reducing the excessive T cell responses of autoimmune cholangitis, which has significance for the potential immunotherapy of PBC.

The Wistar Bonn Kobori rat, a unique animal model for autoimmune pancreatitis with extrapancreatic exocrinopathy

The male Wistar Bonn/Kobori (WBN/Kob) rat is known to be a unique animal model for chronic pancreatitis with widely distributed fibrosis and degeneration of parenchyma because of the infiltration of lymphocytes. In this report, we show that female (but not male) rats develop dacryoadenitis at 3 months of age, and that both male and female WBN/Kob rats develop sialoadenitis, thyroiditis, sclerotic cholangitis and tubulointerstitial nephritis over 18 months of age. The infiltration of CD8+ cells and the deposits of tissue‐specific IgG2b were observed in the injured pancreas and lachrymal glands. Furthermore, the number of regulatory T cells (defined as CD4+ Forkhead box P3+ cells) decreased in the periphery of both male and female WBN/Kob rats, suggesting that the onset of these diseases is attributable, at least, to the failure in the maintenance of peripheral immune tolerance. These features show clearly that WBN/Kob rats are a useful animal model for autoimmune pancreatitis and Sjøgren‐like syndrome or multi‐focal fibrosclerosis in humans. We also show that these autoimmune diseases can be prevented by a newly devised strategy of bone marrow transplantation (BMT) in which bone marrow cells are injected directly into the bone marrow cavity: intrabone marrow–BMT.

Long-term donor-specific tolerance in rat cardiac allografts by intrabone marrow injection of donor bone marrow cells.

Background. Donor-specific central tolerance in cardiac allograft can be induced by hematopoietic chimerism via conventional intravenous bone marrow transplantation (IV-BMT). However, there are problems with IV-BMT, such as the risk of graft failure and of the toxicity from conditioning regimens. Methods. A new method for heart transplantation is presented. This method consists of administration of fludarabine phosphate (50 mg/kg) and fractionated low-dose irradiation (3.5 Gy×2 or 4.0 Gy×2), followed by intrabone marrow injection of whole bone marrow cells (IBM-BMT) plus heterotopic heart transplantation. Results. Cardiac allografts with IBM-BMT were accepted and survived long-term (>10 months) showing neither acute rejection nor chronic rejection including cardiac allograft vasculopathy by such conditioning regimens. In contrast, cardiac allografts with conventional IV-BMT were rejected within 1 month after the treatment with irradiation of 3.5 Gy×2 or within 3 months after the treatment with irradiation of 4.0 Gy×2. Macrochimerism (>70%) was favorably established and stably maintained by IBM-BMT but not IV-BMT. Low levels of transient mixed chimerism (<7%) were induced by IV-BMT with fludarabine plus 4.0 Gy×2, but the chimerism was lost within 1 month after the treatment. Conclusions. These findings indicate that IBM-BMT is a feasible strategy for the induction of persistent donor-specific tolerance, enables the use of reduced radiation doses as conditioning regimens, and obviates the need for immunosuppressants.

Extensive Studies on Perfusion Method Plus Intra‐Bone Marrow‐Bone Marrow Transplantation Using Cynomolgus Monkeys

The collection of bone marrow cells (BMCs) using a perfusion method has been advantageous not only because of the low contamination of BMCs with T cells from the peripheral blood but also the enrichment of stromal cells, which support hemopoiesis. Before the application of this new method to humans, its safety needed to be confirmed using cynomolgus monkeys. We therefore performed the perfusion method on more than 100 cynomolgus monkeys using the long bones (such as the humerus and femur) and also the iliac bones (for human application); in the more than 150 trials to date, there have been no accidental deaths. Furthermore, the technical safety of a new method for the intra‐bone marrow (IBM) injection of BMCs (termed IBM‐bone marrow transplantation) has also been confirmed using 30 monkeys.

Amelioration of 2,4,6-trinitrobenzene sulfonic acid-induced colitis in mice by immunoregulatory dendritic cells

BackgroundDendritic cells (DCs) are widely distributed throughout the lymphoid and nonlymphoid tissues, and are important initiators of acquired immunity. They also serve as regulators by inducing self-tolerance. However, it has not been thoroughly clarified whether DCs are somehow involved in the regulation or treatment of inflammatory bowel diseases.MethodsWe established an ileitis model by transmurally injecting 2,4,6-trinitrobenzene sulfonic acid (TNBS) into the lumen of the ileocolonic junction. The kinetic movement of DCs at the inflammatory sites was analyzed histologically and by flow cytometry, and DCs obtained from the small intestine were analyzed in order to determine the expression of paired immunoglobulin-like receptor-A/B (PIR-A/B) by flow cytometry and quantitative RT-PCR. Furthermore, the regulatory role of DCs was directly determined by a transfer experiment using TNBS-induced colitis model mice.ResultsWe observed three DC subsets (PIR-A/Bhigh, PIR-A/Bmed, and PIR-A/B− DCs) in the conventional DCs (cDCs) from day 3, and the number of PIR-A/Bmed cDCs increased from the time the inflammatory responses ceased (day 7). PIR-A/Bmed cDCs actually migrated to the inflamed colon, and ameliorated the colitis induced by TNBS when transferred to colitis-induced recipients. The colitis was greatly exacerbated when mice had been treated with the indoleamine-pyrrole 2,3-dioxygenase (IDO) inhibitor 1-methyltryptophan (1-mT) at the time PIR-A/Bmed cDCs were transferred, indicating that the therapeutic ability of PIR-A/Bmed cDCs is partially dependent on IDO.ConclusionThe PIR-A/Bmed cDCs, which increase in number during the final stages of inflammation, can be used to treat colitis via an IDO-dependent mechanism.

Activation of granulocytes by direct interaction with dendritic cells

Granulocytes from human peripheral blood were co‐cultured with conventional dendritic cells (cDC) or plasmacytoid DCs (pDC) to examine the effects of DCs on the activation or function of granulocytes. After co‐culture of granulocytes with DCs, expression of the activation markers of granulocytes (CD63 and CD64) was up‐regulated, and increased expression of CD50, the activation marker and ligand for CD209 (DC‐SIGN) was also observed. The interaction of granulocytes with DCs was visualized as the cluster where DCs, especially cDCs, were surrounded by granulocytes to form a ‘rosette’. After co‐culture of granulocytes with cDCs, the secretion of elastase from granulocytes was enhanced significantly when examined cytohistochemically and by enzyme‐linked immunosorbent assay. An increase in myeloperoxidase (another activation index of granulocytes) was also observed after co‐culture with DCs. These findings suggest the functional and phenotypical activation of granulocytes by interaction with DCs. Furthermore, we examined the involvement of adhesion molecules in the granulocyte–DC interaction, and found that CD209 participates to some extent in this interaction.

Downregulation of MicroRNA-21 in Colonic CD3+ T Cells in UC Remission.

Background:In active inflammatory bowel disease (IBD), microRNA expression profiling consistently features disease-specific signatures, and microRNA-21 (miR-21) has been shown to be upregulated in the inflamed colon of patients with active ulcerative colitis (UC). However, the cellular sources of miR-21 expression in IBD tissues have not yet been identified. We sought to determine the expression levels of miR-21 and one of its downstream target genes, programmed cell death 4 (PDCD4), in CD3+ T cells isolated from the colonic mucosa of patients with active IBD, inactive IBD, and non-IBD controls. Methods:Colonic biopsies were treated with collagenase V. CD3+ T cells were isolated using MACS CD3 positive selection. Total RNA was converted to cDNA. Real-time PCR reactions were performed with PCR primers for miR-21, SNORD95, PDCD4, and GAPDH. Results:The expression of miR-21 was statistically significantly downregulated in CD3+ T cells from patients with UC in remission as compared to active disease (P = 0.0193). miR-21 negatively regulates PDCD4 expression. As predicted, the mRNA level of PCDC4 in CD3+ T cells was upregulated in UC and Crohns disease in remission as compared to active disease (UC active versus UC remission: P = 0.0008, Crohns disease active versus Crohns disease remission: P = 0.0215) and in patients with UC in remission as compared to healthy controls (P = 0.0226). Conclusions:Although miR-21 expression is downregulated, PDCD4 is upregulated in CD3+ T cells during the remission phase of UC. Our results indicate that miR-21 and related pathways in colonic T cells may play a role in limiting pathogenic T-cell responses and may constitute future target candidates to induce remission in UC.

Tubular adenomas with clear cell change in the colorectum: A case with four lesions and a review of the literature

To the Editor: Clear cell change, defined by the presence of clear and/or vacuolated cytoplasm without obvious mucin, occurring in colorectal neoplasms has extremely rarely been reported, although various types of metaplasia or differentiation, including squamous morules and Paneth cell proliferation have been documented in colorectal tumors. One series reported a prevalence of clear cell change in colorectal adenomas of 0.086% (3/3486 cases); only 18 cases have been reported in the English-language literature. In this case report, we describe an additional case with multiple colorectal tubular adenomas showing clear cell change, and also discuss the possible pathogenesis of this phenomenon in colorectal adenomas. A 63-year-old Japanese male with a past history of prostatectomy for prostate cancer (Gleason score 3þ 41⁄47, pT2c) 1 year earlier, underwent a colorectal endoscopic examination to investigate a positive fecal occult blood test. He was found to have 25 polyps (2 in the cecum, 9 in the ascending colon, 5 in the transverse colon, 1 in the descending colon, 5 in the sigmoid colon, and 3 in the rectum). Polypectomy was performed for all the lesions. Microscopic examination of two of the polyps in the ascending colon revealed that approximately 50% of one polyp and 10% of the other polyp showed clear cell change, and the remaining portion of each represented a typical tubular adenoma with high-grade atypia (dysplasia) (Fig. 1a). Clear cell change was characterized histopathologically by the presence of clear cytoplasm in tall columnar neoplastic cells. These neoplastic cells had hyperchromatic elongated nuclei without conspicuous nucleoli. Most of the neoplastic cells had clear cytoplasm in both the apical and subnuclear portions of the cell with apically oriented nuclei (Fig. 1b,c); but in some neoplastic cells, the clear cytoplasm was present only in the apical portion with the nuclei located in the base of the cells. These neoplastic cells had clear multi-vacuolated cytoplasm, and the sizes of the vacuoles varied (Fig. 1b,c). Abrupt transition from typical tubular adenoma cells to the clear cells was observed in both polyps (Fig. 1a–c). No adenocarcinomatous component was noted in either polyp. One polyp in the descending colon and one rectal polyp appeared to be typical tubular adenomas with low-grade atypia (dysplasia) showing clear cell change (approximately 10% of cells). Periodic acid-Schiff (PAS) staining with or without diastase digestion demonstrated that most of the neoplastic clear cells did not have material that was stained by PAS, whereas the typical tubular adenoma cells contained substance that were stained by PAS and digested by diastase. Moreover, none of the clear cells contained material that was stained by alcian blue. The results of the immunohistochemical studies are summarized in Table S1 (the polyp from the descending colon was too small to be evaluated). The clear cells were negative for MUC2 expression, although MUC2 was expressed in the conventional tubular adenoma cells (Fig. 1d). MUC5AC and MUC6 were not expressed in either the clear cells or the conventional adenoma cells. CD10 was expressed in the neoplastic clear cells of all polyps (Fig. 1e). In the conventional tubular adenoma component, CD10 was expressed in one polyp in the ascending colon, but not expressed in the two other polyps. Most of the neoplastic clear cells in the three polyps showed positive immunoreactivity for adipophilin (Fig. 1f), although none of the conventional tubular adenoma cells in the three polyps were positive for adipophilin. Ki-67 labeling index (LI) was 35% and 50% (high-grade dysplasia), and 33% (low-grade dysplasia) in the clear cell components (45%, 62%, and 53% in the conventional adenoma components, respectively). Scattered p53-positive cells were observed in both the neoplastic clear cells and conventional tubular adenoma cells in all polyps. Neither SALL4 nor alpha-fetoprotein was expressed in the clear cells. The remaining 12 polyps were conventional tubular adenomas without clear cell change. Moreover, five polyps (1 in the cecum, 2 in the ascending colon, and 2 in the transverse colon) were sessile serrated adenomas/polyps, and four polyps (2 in the ascending colon, 1 in the transverse colon, and 1 in the sigmoid colon) were hyperplastic polyps. In this report, we described a case with four colorectal tubular adenomas showing clear cell change. Table S1 summarizes the clinicopathological features of the previously reported cases of colorectal adenoma with clear cell change as well as the present one (24 lesions from 20 patients, including the recently reported Japanese case). This extremely rare phenomenon is most commonly observed in the left-side colon (13/20 lesions), and rare in the ascending colon (3 lesions). Most of the patients had a single lesion; only one previously reported case had two lesions. The present case was unique, because clear cell

Esophageal Large-Cell Neuroendocrine Carcinoma with Inconsistent Response to Treatment in the Primary and Metastatic Lesions

Esophageal large-cell neuroendocrine carcinoma (NEC) is a rare malignant tumor that is characterized by high-grade malignancy and a poor prognosis. However, the rarity of esophageal NEC has prevented the development of an established treatment, and no reports have described a discrepancy in the effectiveness of cisplatin plus irinotecan between primary and metastatic lesions. A 43-year-old Japanese man was referred to our hospital with refractory epigastralgia. A previous gastrointestinal endoscopy had revealed a 50-mm type 2 tumor in the abdominal esophagus. The pathological findings indicated poorly differentiated squamous cell carcinoma. Contrast-enhanced computed tomography revealed a metastatic liver tumor. One cycle of fluorouracil and cisplatin was not effective, and endoscopy was repeatedly performed. The pathological findings indicated a large-cell malignant tumor with tumor cells that were positive for CD56, synaptophysin, and Ki-67 (> 80%). Based on a diagnosis of esophageal large-cell NEC with a metastatic liver tumor, the patient received cisplatin plus irinotecan biweekly. After 4 months, computed tomography revealed marked shrinkage of the metastatic tumor, but the patient complained of dysphagia. Endoscopy revealed enlargement of the primary tumor, which was then treated using radiotherapy plus fluorouracil and cisplatin. The primary tumor subsequently shrank, and the patient’s symptoms were relieved, but the metastatic tumor grew. Thus, chemoradiotherapy could be an option for managing a primary esophageal large-cell NEC that does not respond to chemotherapy alone. However, the possibility of an inconsistent response to therapy in primary and metastatic lesions should be considered.