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Mesenchymal stromal cells (MSCs) are known to have immunosuppressive ability and have been used in clinical treatment of acute graft-versus-host disease, one of severe complications of the hematopoietic stem cell transplantation. However, MSCs are activated to suppress the immune system only after encountering an inflammatory stimulation. Thus, it will be ideal if MSCs are primed to be activated and ready to suppress the immune reaction before being administered. Triptolide (TPL) is a diterpene triepoxide purified from a Chinese herb-Tripterygium wilfordii Hook.f. It has been shown to possess anti-inflammatory and immunosuppressive properties in vitro. In this study, we aimed to use TPL to prime umbilical cord-derived MSCs (TPL-primed UC-MSCs) to enter a stronger immunosuppressive status. UC-MSCs were primed with TPL, which was washed out thoroughly, and the TPL-primed UC-MSCs were resuspended in fresh medium. Although TPL inhibited the proliferation of UC-MSCs, 0.01 μM TPL for 24 h was tolerable. The surface markers of TPL-primed UC-MSCs were identical to those of non-primed UC-MSCs. TPL-primed UC-MSCs exhibited stronger anti-proliferative effect for activated CD4+ and CD8+ T cells in the allogeneic mixed lymphocyte reaction assay than the non-primed UC-MSCs. TPL-primed UC-MSCs promoted the expression of IDO-1 in the presence of IFN-γ, but TPL alone was not sufficient. Furthermore, TPL-primed UC-MSCs showed increased expression of PD-L1. Conclusively, upregulation of IDO-1 in the presence of IFN-γ and induction of PD-L1 enhances the immunosuppressive potency of TPL-primed UC-MSCs on the proliferation of activated T cells. Thus, TPL- primed MSCs may provide a novel immunosuppressive cell therapy.

We herein describe a case of Sweet's syndrome (SS) in a patient being treated for pulmonary toxoplasmosis complicated with myelodysplastic syndrome (MDS). The patient's SS developed after the pulmonary toxoplasmosis improved following treatment. We searched his cytokine profiles comprehensively using a bead-based immunoassay. The results showed no elevation of interleukin (IL)-2, interferon (IFN)-γ or IL-17A, and IL-6 was only observed to have increased at the onset of SS, suggesting that the pulmonary toxoplasmosis had been well controlled and that chronic inflammation may have been the cause of SS. Pulmonary toxoplasmosis is an extremely rare occurrence. The cytokine profile can help to clarify the pathological condition of SS and MDS complicated with severely invasive infectious diseases.

Recent studies have reported that mesenchymal stromal cells (MSCs) migrate to areas of inflammation and suppress adverse immune reactions. Bone marrow (BM)-derived MSCs have been successfully used in patients with acute graft versus host disease (GVHD), but the harvesting of BM carries certain risks for the donor. To circumvent these, we obtained MSCs from Wharton's jelly (WJ) derived from umbilical cord and investigated their potential for immunosuppression. In a mixed lymphocyte reaction (MLR), responder T cell proliferation triggered by allogeneic dendritic cells was inhibited efficiently by WJ-MSCs derived from the same donor of responder cells or those from a third party donor. These inhibitory effects were reversed in a dose-dependent manner in the presence of 1-methyl-DL-tryptophan, an inhibitor of the soluble factor indoleamine 2, 3-dioxygenase (IDO). Immunosuppression by WJ-MSCs was also attenuated by blocking cell-cell contact between WJ-MSCs and responder T cells using a Transwell chamber. Moreover, IDO gene expression was induced in both WJ- and BM-MSCs by inflammatory cytokine IFN-γ, but HLA-DR was expressed in BM-MSCs and not in WJ-MSCs upon stimulation by a relatively low concentration of IFN-γ. These results indicate that WJ-MSCs exert their immunosuppressive effects by cell-cell contact with activated T cells and in part through IDO, and suggest the need for cells rather than soluble factors secreted from MSCs to achieve immunosuppressive therapy in severe cases of GVHD.

Background: Mesenchymal stromal cells (MSC) are known to have the immunosuppressive ability and have been applied in clinic to treat acute graft-versus-host disease (GVHD), as one of severe complications after hematopoietic stem cells transplantation (HSCT) in Japan. However, MSC are activated to suppress the immune system only upon the stimulation of inflammatory cytokines and the clinical results of MSC therapies for acute GVHD are varied. It is ideal that MSC are primed to be activated and ready to suppress the immunity (=priming) before administration in vivo. Triptolide (TPL) is a diterpene triepoxide purified from a Chinese herb - Tripterygium Wilfordii Hook F (TWHF). It has been shown to possess anti-inflammatory and immunosuppressive properties in vitro. In this study, we aim to use TPL as the activator for umbilical cord-derived MSC (UC-MSC) to entry stronger immunosuppressive status. Methods: The proliferation of UC-MSC with TPL at the indicated concentrations for different time of 24, 48, 72, and 96 hours. Cell counting kit-8(CCK-8) was added in the culture medium to detect cell toxicity and the absorbance was measured using microplate reader. Flow cytometry was used to identify the MSC surface markers expression. TPL-primed UC-MSC were once replaced with fresh medium and co-culture with mixed lymphocyte reaction (MLR) consisted with mononuclear cells (MNCs) stained with CFSE and irradiated allogenic dendritic cell line (PMDC05) in RPMI 1640 medium supplemented with 10 % FBS (complete medium). IDO-1, SOD1, and TGF-β gene expression in TPL-primed UC-MSC and UC-MSC induced by 10 ng/ml IFN-γ and/or 15 ng/ml TNF-α were evaluated by RT-PCR. PDL1 and PDL2 expression in TPL-primed UC-MSC and UC-MSC in response to IFN-γ and/or TNF-α were checked by Flowjo. Results: Exposure of TPL for UC-MSC for 72hour at the concentration above 0.1 μM resulted in the cell damage significantly. Therefore, we added TPL in UC-MSC at 0.01μM of TPL for up to 48 hours, then washed thourouphly for the following culture for experiments. To evaluate the influence of TPL on the surface markers of UC-MSC, we cultured UC-MSC for 4 hours in complete medium following culture with 0.01μM of TPL for 20 hours (TPL-primed UC-MSC). TPL-primed UC-MSC revealed positive for CD105, CD73, and CD90, negative for CD45, CD34, CD14 or CD11b, CD79α or CD19 and HLA-DR surface molecules as same as the non-primed UC-MSC. In MLR suppression by UC-MSC, the TPL-primed UC-MSC activity revealed stronger anti-proliferative effect on the CD4+ and CD8+ T cells activated by allogeneic DC than those of non-primed UC-MSC in MLR. Furthermore, the TPL-primed UC-MSC promoted the expression of IDO-1, SOD1 and TGF-β in response to IFN-γ+/-TNF-α by RT-PCR and enhanced the expression of PD-L1 by FACS analysis. Discussion:In this study, we found the TPL-primed UC-MSC showed stronger antiproliferative potency on CD4+ and CD8+ T cells compared with non-primed UC-MSC. TPL-primed UC-MSC promoted the expression of IDO-1, SOD1 and TGF-β stimulated by IFN-γ+/-TNF-α, although TPL alone did not induce these factors. Furthermore, we found that the PD1 ligand (PD-L1) was induced in TPL-primed UC-MSC, likely IFN-γ enhanced the PD-L1 expression, evaluated by flowcytometry. These results suggested that TPL-primed UC-MSC seemed more sensitive to be activated as the immunosuppressant. Here, we firstly report the new function of TPL to induce the upregulation of immunosuppressive effect, although the mechanisms of TPL inhibition to MSC need to be explore. Conclusively, TPL-primed UC-MSC might be applied for the immunosuppressive inducer of MSC. Figure Disclosures He: SASAGAWA Medical Scholarship: Research Funding; IMSUT Joint Research Project: Research Funding. Nagamura:AMED: Research Funding. Tojo:AMED: Research Funding; Torii Pharmaceutical: Research Funding. Nagamura-Inoue:AMED: Research Funding.

Recently, umbilical cord (UC) has become attracted source of mesenchymal stromal cells (MSCs) for immunotherapy such as treatment of severe acute graft versus host disease, because of abundant sources and ease of collection without invasive process. In previous reports on bone marrow-derived MSCs, there is diversity of immunosuppressive mechanisms of MSCs, which have not yet fully clarified in UC-MSCs. Here we studied the immunosuppressive properties of UC-MSCs on the activated immune system in vitro. Methods; UC was collected after obtaining informed consent from the mother. UC-MSCs were obtained from frozen-thawed UC tissue with explant method and expanded in culture medium. Mixed lymphocyte reaction (MLR) consisted with mononuclear cells (MNCs) stained with CFSE and allogeneic dendritic cell (DC) line (PMDC05) co-cultured with or without UC-MSCs was carried out, and the proliferation of CD4 and CD8-positive lymphocytes were analyzed by flowcytometry followed by FlowJo software. Migratory activities of UC-MSCs toward activated lymphocytes in MLR were evaluated by counting the migrating cells stained with DAPI through the insert filter under the microscope. The percentage of CD4+CD25+FOXP3+ regulatory T cells (Treg) before and after co-culture with UC-MSCs was analyzed by flowcytometry. Monocyte phenotypes co-cultured with UC-MSCs directly or separately by insert filter were analyzed with anti-CD14, CD206, and CD168 antibodies. Results: The UC-MSCs were positive for CD105, CD73, CD90, and negative for CD45 and HLA-DR. HLA-DR, CD80, CD86, and CD40 were negative even in the high concentration of IFN-γ, while BM-MSCs became positive for HLA-DR. PD-L2 was constitutively expressed in UC-MSC, while PD-L1 was induced upon the addition of IFN-γ. In MLR, responder T cell proliferation triggered by allogeneic DC was inhibited efficiently by 3rd party derived UC-MSCs. Mean+/- SD distribution index of proliferation in CD4- and CD8- positive cells co-cultured with UC-MSCs showed 7.6+/-4.4% and 8.8+/-3.9% compared to those without UC-MSCs (n=4, as different donor derived MSCs). This inhibition was attenuated by the addition of 1-Methyl-dl-tryptophan, Indoleamine 2, 3-dioxygenase (IDO-1) inhibitor, in a dose-dependent manner. The supernatant of MLR showed the increase of IFN-γ and TNF-α, which were also suppressed in MLR co-cultured with UC-MSCs. UC-MSCs migrated toward activated lymphocytes in MLR compared with those of non-activated lymphocytes significantly, which was inhibited by the addition of AG1296, known as platelet-derived growth factor inhibitor; and PPP as insulin-like growth factor (P Discussions and Conclusion: These results demonstrated that UC-MSCs have the diverse immunosuppressive potency through migration toward the activated lymphocytes, suppressing activated T cells proliferation, regulatory T cells induction, and transition of monocyte phenotype. And UC-MSCs are the feasible and abundant source for immunotherapy. Disclosures Nagamura-Inoue: AMED: Research Funding. Nagamura:AMED: Research Funding. Tojo:AMED: Research Funding; Torii Pharmaceutical: Research Funding.

Background: For clinical cord blood (CB) transplantation, CB is processed using a standard hydroxyethyl starch protocol generally within 48 h of collection at room temperature. However, for tissue stem cell research, mononuclear cells (MNCs) were isolated from CB using a Ficoll-Paque density-gradient method. Here we report the effect of storage temperature and time from CB collection to processing on the cord blood mononuclear cells (CB-MNCs) isolated using a density-gradient method. Methods:We processed CB using a Ficoll-Paque density-gradient method to collect the cells in the MNC layer. Cells were analyzed using an automatic blood cell counter, and CD34 cells were counted according to the ISHAGEmethod. Results: The recovery rate of viable MNCs in the CB-MNC layer was inversely related to the time from collection to processing of CB samples. However, recoveries of total nucleated cell and CD34 were not affected by the time from collection to processing. The percentage of neutrophil contamination in the MNC layer increased significantly with increasing time from CB collection to processing (n=100, p

Background aims The human umbilical cord (UC) is a rich source of mesenchymal stromal cells (MSCs), which have been reported to have multi-lineage potential. The objectives of this study were to investigate the characteristics and capacity of UC-MSC neurosphere formation and whether this event enhances the propensity of UC-MSCs to undergo neural differentiation. Methods UC-MSCs were collected by the improved explant method. UC-MSCs and neurosphere-forming UC-MSCs (UC-MSC-neurospheres) were induced to undergo neurogenic differentiation, the latter of which were induced by suspension culturing in the presence of epidermal growth factor and basic fibroblast growth factor. The differentiation and migratory capacities of the individual cultures were then compared on the basis of the expression of neural markers, as measured by immunocytochemistry, immunoblotting and quantitative real-time polymerase chain reaction and transwell assays, respectively. Results Both UC-MSCs and UC-MSC-neurospheres were capable of differentiating into neurogenic cells when cultured in neurogenic differentiation medium. However, pre-conditioned UC-MSC-neurospheres exhibited significantly higher expression of neural markers—including microtubule-associated protein ( MAP2 ), MUSASHI1 , glial fibrillary acidic protein ( GFAP ), and NESTIN —compared with those derived from UC-MSCs directly. Moreover, UC-MSC-neurospheres expressed significantly higher levels of the stemness markers NANOG , KLF4 and OCT4 than did UC-MSCs. Migration assays also revealed that both UC-MSCs and UC-MSC-neurospheres actively migrate toward glucose-depleted cells. Conclusions Neurogenic differentiation potential probably is greater in UC-MSC-neurospheres than in UC-MSCs. Thus, UC-MSC-neurospheres may serve as a better source of cells for neurogenic regenerative medicine.

Idiopathic thrombocytopenic purpura associated with renal cell carcinoma is relatively rare. We report the case of a 48-year-old woman with massive renal cell carcinoma, measuring approximately 20 × 14 × 14 cm, who presented with severe thrombocytopenia: platelet count, 2000 cells/μL. After confirming normal bone marrow, she received high-dose dexamethasone and intravenous gamma globulin, which raised the platelet count to normal levels. She then underwent left radical nephrectomy. The pathological examination showed chromophobe renal cell carcinoma. After the resection, the platelet count was maintained within the normal range without any treatment. The current case is the first report of chromophobe renal cell carcinoma causative of severe idiopathic thrombocytopenic purpura.

The measurement of CD34+ cells is becoming important as an assessment of the quality of cord blood (CB) units for transplantation. In the cryopreserved sample, it is demanded accurate method that excludes non-viable cells those are damaged during freezing and thawing. The ProCOUNT method which we have used so far is impossible to distinct non-viable cells from viable cells in counting CD34+ cells.In this study, we compared the ProCOUNT method with the 7-AAD (7-amino actinomycin D) method, which can detect and exclude non-viable cells from CD34+ cell analysis. In fresh CB (n=50), the concentrations of CD34+ cells by the ProCOUNT and 7-AAD methods were 63.1±64.6/μL and 68.3±65.7/μL, respectively. In frozen-thawed CB (n=60), concentrations by the ProCOUNT and the 7-AAD method were 97.9±71.5μ/L and 67.6±48.6/μL, respectively, this difference being significant (p

BACKGROUND: Prolonged periods of marrow hypoplasia have been a problem in cord blood transplantation. DMSO is thought to produce osmotic shock to the progenitors when the thawed cells are infused into the patients. To solve this problem, a 2× dilution method originally developed in the New York Blood Center showed earlier myeloid engraftment,1 although follow-up clinical studies have not performed. STUDY DESIGN AND METHODS: To clarify the influence of the removal of DMSO by this method on the speed of engraftment in unrelated cord blood transplantation, 46 adult patients with cord blood units processed by the Tokyo Cord Blood Bank from September 1998 to March 31, 2002 were studied. Twenty-four patients received 2.6 ± 0.71 × 107 nucleated cells per kg without washing (nonwashed group), while 22 patients were received 2.7 ± 0.52 × 107 nucleated cells per kg after 2× dilution washing (washed group). RESULTS: The cumulative incidence of engraftment was not significantly different between the two groups. Median neutrophil recovery (≥5 × 109/L) in the nonwashed and washed groups was 26 and 25 days, respectively, and the median platelet recovery (≥20 × 109/L) in patients with myeloid engraftment was 44 and 40 days, respectively (NS). On the other hand, the doses of CFCs and CD34+ cells showed the influence on myeloid and platelet recovery. CONCLUSION: A 2× dilution after thawing cord blood did not result in the improvement of myeloid engraftment speed.

Background aims Human umbilical cord (UC) has become a notable source for mesenchymal stromal cells (MSCs) that can migrate to areas of inflammation and damaged tissue and can suppress excess immune reactions and to repair, respectively. Although UC is a solid tissue, there are several advantages, including repeatable uses from the same donor sample when needed and the possibility of future explorations for cells with unknown potential, if we could cryopreserve the UC as a living tissue material. However, because the cryoprotectants in the previous reports included animal- or allogeneic human-derived serum or no serum, the frozen-thawed UC-MSCs were inferior to fresh UC-MSCs in cell proliferation. The objective of this study was to find a suitable cryopreservation method of UC for clinical use. Methods The UC was cut in cross-section and incised longitudinally, immersed in the cryoprotectant and frozen slowly. Later, it was thawed and minced rapidly, and the fragments of UC were cultured by improved explant method. Results The highest yield of cells was obtained from frozen-thawed UC with serum- and xeno-free cryoprotectant, STEM-CELLBANKER, when compared with others. The cells derived from frozen-thawed UC stored in STEM-CELLBANKER expressed the phenotypes of MSCs, retained the immunosuppressive properties in allogeneic mixed lymphocyte reactions and the differentiation potentials (into adipocyte and chondrocytes) comparable to those derived from fresh UC. Conclusions UC can be cryopreserved in serum- and xeno-free cryoprotectant as a living tissue while keeping its growth and functions equivalent to fresh UC. Our method is simple and feasible for clinical use.

Trichophyton verrucosum, a causative agent of bovine dermatophytosis in Japan is considered to transfer easily from infected cattle to other healthy ones. No studies have ever tried to elucidate the distribution of T. verrucosum in breeding environment. In this study, we investigated the distribution of T. verrucosum in infected young calf skin and healthy skin. Hairs or scale samples were collected from 29 lesional skin, and 46 non-lesional skin of the 19 and 34 infected calves, respectively, both varying in age 2 to 6 months old, and 35 hair samples from the 29 healthy ones. They were directly inoculated on medium. The detection rates of T. verrucosum-positive were 58.6% from the lesional parts, 34.8% from the non-lesional parts and 17.1% from the healthy parts. The isolation of T. verrucosum from the calves with no skin lesion due to dermatophytosis implied that this infection might be easily transmitted by the contact with the infected calves. T. verrucosum infection in cattle poses a serious problem in animal husbandry. It is also important for public health to take preventive measures against the infection.

Recently, cord blood transplantation (CBT) for adult is rapidly increasing in number, Especially for the patients over 50 years of age. By the end of 2003, 224 units were shipped for the patients Group I: 50 y.o.21%. We analyzed 152 patients with hematological malignancies including ALL, AML, CML, MDS, malignant lymphoma, myeloma and neuroblastoma reported from CBT center in the world by the end of 2003. Patients and Methods:Group I included 58 patients with 13 standard risk and 45 high risk patients and showed mean±SD of age; 5.3 ±4.1y.o.,BW; 20.5±13.4kg, CB volume at collection; 91.6±27.1ml, NC; 5.5±3.3x107/kg, CFC; 8.6±6.4x104/kg, CD34; 1.5±1.1x105/kg, Group II: 64 cases with 25 standard and 39 high risk patients and age; 30.6±10.3y.o., BW; 51.9±9.5kg, CB volume at collection;116.6±27.7ml, NC;2.6±0.7x107/kg, CFC;5.2±2.6x104/kg and CD34;0.8±0.5x105/kg, Group III: 30 cases with 9 standard and 21 high risk patients and age; 54.1±3.3y.o., BW;55.9±11.0kg, CB vol.at collection;118.8±24.7ml NC;2.4±0.4x107/kg, CFC;4.8±1.9x104/kg and CD34;0.8±0.4x105/kg. The patients who underwent CBT for graft failure (GF) of prior transplant were excluded. Conditioning regimen in Group I demonstrated 54 patients with full regimen and 4 with reduced intensity regimen (RIST); in Group II, 62 patients with full regimen and 2 RIST; and in Group III, 14 cases full regimen and 15 cases RIST. Results: Cumulative myeloid engraftment was seen 67.2% in Group I, 73.4% in Group II and 46.7% in Group III (*Group II vs. Group III: P Conclusion: The application of CBT has been expanded to the elderly patients (>50 y.o.), although the conditioning regimen and the special medical care for the complications in the early pahse after UCBT has remained to be discussed.