Junichi Fukui

Prevention of graft-versus-host disease by intra-bone marrow injection of donor T cells.

We have recently found that intra‐bone marrow‐bone marrow transplantation (IBM‐BMT) can be used to prevent graft‐versus‐host disease (GvHD), even when intensive donor lymphocyte infusion (DLI) is carried out. In the present study, in conjunction with IBM‐BMT, allogeneic splenic T cells as DLI were also injected into the bone marrow cavity of lethally irradiated (8.5 Gy) recipients. The extent of GvHD was compared with that of recipients that had received allogeneic IBM‐BMT plus i.v. injection of allogeneic T cells (intravenous DLI [IV‐DLI]). GvHD in recipients treated with allogeneic IBM‐BMT plus IBM‐DLI was far milder than in those treated with allogeneic IBM‐BMT plus IV‐DLI. This was confirmed macroscopically and histopathologically. The frequency of regulatory T cells (Tregs) detected as CD4+CD25+ and CD4+Foxp3+ cells was significantly higher in recipients treated with IBM‐BMT plus IBM‐DLI than in those treated with IBM‐BMT plus IV‐DLI. Donor‐derived helper T (Th) cells polarized to Th2 type in recipients treated with IBM‐BMT plus IBM‐DLI, whereas Th1 cells were dominant in recipients treated with IBM‐BMT plus IV‐DLI. Furthermore, the production of transforming growth factor‐β and hepatocyte growth factor from bone marrow stromal cells was enhanced after IBM‐DLI. Thus, IBM‐BMT plus IBM‐DLI seem to preferentially induce Tregs and Th2, resulting in the prevention of GvHD.

Induction of Senile Osteoporosis in Normal Mice by Intra‐Bone Marrow‐Bone Marrow Transplantation from Osteoporosis‐Prone Mice

A P6 substrain of the senescence accelerated mouse (SAMP6) spontaneously develops osteoporosis early in life. These mice show the clinical signs of osteoporosis, such as elevated levels of urinary deoxypyridinoline (Dpd), decreased bone mineral density (BMD), and a significant loss of trabecular and cortical bone thickness at 12 months of age. Here, we describe the transfer of osteoporosis to a normal strain by the injection of bone marrow cells from SAMP6 donors directly into the bone marrow cavity (intra‐bone marrow‐bone marrow transplantation [IBM‐BMT]). More than 1 month after IBM‐BMT, hematolymphoid cells were completely reconstituted by donor‐derived cells, and bone marrow stromal cells that could differentiate into osteocytes were also found to be of donor origin. In addition, the recipient C57BL/6 mouse showed the features of osteoporosis in the trabecular bone. Decreases in BMD and increases in urinary Dpd were also observed. When the message levels of cytokines (interleukin [IL]‐11, IL‐6, receptor activator of NF‐κB ligand [RANKL], osteoprotegerin, macrophage–colony‐stimulating factor, and insulin‐like growth factor‐1) were examined by reverse transcription‐polymerase chain reaction (RT‐PCR) and real‐time RT‐PCR analysis, IL‐6 and IL‐11 were reduced to a level similar to that in SAMP6 mice, whereas that of RANKL was increased. These findings indicate that not only the hemopoietic system but also the bone marrow microenvironment are reconstituted as a result of IBM‐BMT, and suggest that the development of senile osteoporosis might be attributable to “stem cell disorders.”

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.

An Innovative Approach to Bone Marrow Collection and Transplantation in a Patient with β-Thalassemia Major: Marrow Collection Using a Perfusion Method Followed by Intra-Bone Marrow Injection of Collected Bone Marrow Cells

Using small animals (mice and rats) and monkeys, we have found that the combination of bone marrow collection using the perfusion method (PM) and intra-bone marrow-bone marrow transplantation (IBM-BMT) of the collected cells is safe and effective in treating various intractable diseases. Based on these findings, we attempted to apply this method to humans. We report here the first case of a patient (6 years old) with β-thalassemia major who underwent allogeneic BMT using this new PM + IBM-BMT method. The white blood cell counts of the patient gradually increased to more than 1500/µL by day 47 and continued to increase, reaching the highest level (8600/µL) on day +55. Fluorescence in situ hybridization data on day +33 showed that 98% of the peripheral blood cells were from the donor. Notably, there were no symptoms of graft-versus-host disease (GvHD). However, on day +56, the patient regrettably died of asphyxia resulting from sticky sputum. There was no evidence of infection (in the lung or liver) or GvHD (in the skin) by necropsy. We hope that this case report will help make our new strategies more readily available for the treatment of patients with various intractable diseases.

Prevention of graft-versus-host disease by intrabone marrow injection of donor T cells: involvement of bone marrow stromal cells

We have developed a new and effective method for bone marrow transplantation (BMT): bone marrow cells (BMCs) are injected directly into the bone marrow (BM) cavity of recipient mice. The intrabone marrow injection of BMCs (IBM‐BMT) greatly facilitates the engraftment of donor‐derived cells, and IBM‐BMT can attenuate graft‐versus‐host reaction (GVHR), in contrast to conventional intravenous BMT (i.v.‐BMT). Here, we examine the mechanisms underlying the inhibitory effects of IBM‐BMT on GVHR using animal models where GVHR is elicited. Recipient mice (C57BL/6) were irradiated and splenic T cells (as donor lymphocyte infusion: DLI) from major histocompatibility complex‐disparate donors (BALB/c) were injected directly into the BM cavity (IBM‐DLI) or injected intravenously (i.v.‐DLI) along with IBM‐BMT. The BM stromal cells (BMSCs) from these recipients were collected and related cytokines were examined. The recipient mice that had been treated with IBM‐BMT + i.v.‐DLI showed severe graft‐versus‐host disease (GVHD), in contrast to those treated with IBM‐BMT + IBM‐DLI. The suppressive activity of BMSCs in this GVHD model was determined. The cultured BMSCs from the recipients treated with IBM‐BMT + IBM‐DLI suppressed the proliferation of responder T cells remarkably when compared with those from the recipients of IBM‐BMT + i.v.‐DLI in mixed leucocyte reaction. Furthermore, the level of transforming growth factor‐β and hepatocyte growth factor in cultured BMSCs from IBM‐BMT + IBM‐DLI increased significantly when compared with those from the recipients of IBM‐BMT + i.v.‐DLI. Thus, the prevention of GVHD observed in the recipients of IBM‐BMT + IBM‐DLI was attributable to the increased production of immunosuppressive cytokines from BMSCs after interaction with host reactive T cells (in DLI).

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.

Objective and Quantitative Assessment of Postoperative Pain in Digestive Surgery

Background: Pain is associated with subjective factors, making it difficult to assess. The PainVision™ system has been developed to quantitatively assess pain and compare postoperative pain intensity. We investigated the utility of PainVision in assessing postoperative pain in digestive surgery patients.

Staple-Line Reinforcement of the Duodenal Stump With Intracorporeal Lembert's Sutures in Laparoscopic Distal Gastrectomy With Roux-en-Y Reconstruction for Gastric Cancer.

Purpose: We report a duodenal stump reinforcement procedure in laparoscopic distal gastrectomy with Roux-en-Y reconstruction. Methods: We retrospectively reviewed the data of 223 patients who underwent laparoscopic distal gastrectomy with Roux-en-Y reconstruction for gastric cancer. We compared 2 groups: group NR (not reinforced, n=102, June 2009 to December 2011) when we did not perform reinforcement of the duodenal stump, and group R (reinforced, n=121, January 2012 to July 2014) when we did the reinforcement. The duodenum was divided with an endoscopic linear stapler. In group R, the duodenal staple line was reinforced by hand-sewn Lembert’s sutures. Results: There were no significant differences between group NR and R in patients’ characteristics. Duodenal stump leakage occurred in 2 patients in group NR (2.0%). By contrast, in R group, no patients had duodenal stump leakage or fistula. Conclusions: Duodenal stump leakage can be avoided by using reinforcement with Lembert’s sutures.

Evaluation of the effects of postoperative oral nutrition support on body weight in gastric cancer patients by using an elemental diet: A randomized study.

110 Background: Postoperative weight loss causes deterioration in the patient’s quality of life and influences long-term prognosis in gastric cancer patients who have undergone gastrectomy. Moreover, recent retrospective studies indicated postoperative weight loss as a risk factor for premature interruption of S-1 adjuvant chemotherapy. We conducted a prospective randomized controlled study to examine whether the early institution of nutritional support comprising an oral elemental diet (ED) prepared for post-gastrectomy patients with depressed digestive/absorptive function would help prevent postoperative weight loss. Methods: After surgery, patients were randomly assigned to the ED or control groups. The groups were divided according to the surgical methods used (total/distal gastrectomy), clinical stage (≤Ia/>Ia), and patients’ body mass index ( 18.5). In patients assigned to the ED group (Elental, 300 kcal) was provided in addition to the regular diet from the day starting soft rice diet or equi...