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Abstract Aim The National Clinical Database (NCD) of Japan is a nationwide data entry system for surgery, and it marked its 10th anniversary in 2020. The aim was to present the 2020 annual report of gastroenterological surgery of the NCD. Methods The data of the surgical procedures stipulated by the training curriculum for board‐certified surgeons of the Japanese Society of Gastroenterological Surgery in the NCD from 2011 to 2020 were summarized. Results In total, 5 622 845 cases, including 593 088 cases in 2020, were extracted from the NCD. The total number of gastroenterological surgeries increased gradually in these 10 years, except for the year 2020 due to the COVID‐19 pandemic. The annual number of surgeries of each organ, except the pancreas and liver, decreased by 0.4%–13.1% in 2020 compared to 2019. The surgical patients were consistently aging, with more than 20% of all gastroenterological surgeries in 2020 involving patients aged 80 years or older. The participation of board‐certified surgeons increased for each organ (75.9%–95.7% in 2020). The rates of endoscopic surgery also increased constantly. Although the incidences of postoperative complications of each organ increased by 0.7%–7.9% in these 10 years, postoperative mortality rates decreased by 0.2%–1.5%. Conclusions We present here the short‐term outcomes of each gastroenterological operative procedure in 2020. This review of the 10‐years of NCD data of gastroenterological surgery revealed a consistent increase of the number of surgeries (except for in 2020), especially endoscopic procedures, and aging of the Japanese population. The good safety of Japanese gastroenterological surgeries was also indicated.

In developing a formula for manual use in clinical settings, simplicity is as important as accuracy. Whole-liver (WL) mass is often estimated using demographic and anthropometric information to calculate the standard liver volume or recommended graft volume in liver transplantation. Multiple formulas for estimating WL mass have been reported, including those with multiple independent variables. However, it is unknown whether multivariable models lead to clinically meaningful improvements in accuracy over univariable models. Our goal was to quantitatively define clinically meaningful improvements in accuracy, which justifies an additional independent variable, and to identify an estimation formula for WL graft weight that best balances accuracy and simplicity given the criterion. From the Japanese Liver Transplantation Society registry, which contains data on all liver transplant cases in Japan, 129 WL donor-graft pairs were extracted. Among the candidate models, those with the smallest cross-validation (CV) root-mean-square error (RMSE) were selected, penalizing model complexity by requiring more complex models to yield a ≥5% decrease in CV RMSE. The winning model by voting with random subsets was fitted to the entire dataset to obtain the final formula. External validity was assessed using CV. A simple univariable linear regression formula using body weight (BW) was obtained as follows: WL graft weight [g] = 14.8 × BW [kg] + 439.2. The CV RMSE (g) and coefficient of determination (R2) were 195.2 and 0.548, respectively. In summary, in the development of a simple formula for manually estimating WL weight using demographic and anthropometric variables, a clinically acceptable trade-off between accuracy and simplicity was quantitatively defined, and the best model was selected using this criterion. A univariable linear model using BW achieved a clinically optimal balance between simplicity and accuracy, while one using body surface area performed similarly.

Abstract Background We aimed to present the 2019 annual report of the gastroenterological section of the National Clinical Database (NCD). Methods We reviewed 609,589 cases recorded in 2019 and 5,029,764 cases registered from 2011 to 2019 for the 115 selected gastroenterological surgical procedures. Results The main features of gastroenterological surgery in Japan were similar to those described in the 2018 annual report, namely, that 1) operative numbers gradually increased in all procedures, except gastrectomy and hepatectomy, which decreased in these years; 2) in all eight major gastroenterological surgeries, the age distribution tended toward older patients; 3) the morbidity of esophagectomy, hepatectomy, and pancreaticoduodenectomy increased, but mortality was minimized in all procedures; 4) all eight major gastroenterological procedures have increasingly been performed under laparoscopy; and 5) board‐certified surgeons were increasingly involved. These trends in recent years were more prominent in 2019. Conclusions Thanks to the continuous cooperation and dedication of the surgeons, medical staff, and surgical clinical reviewers who registered the clinical data into the NCD, it is possible to understand the comprehensive landscape of surgery in Japan and to disclose new evidence in this field. The Japanese Society of Gastroenterological Surgery will continue to promote the value of this database and encourage the use of feedback and clinical studies using the NCD, now and in the future. Generating further approaches to surgical quality improvement are important directions for future research.

Abstract Background and Aim In 2015, the Japanese Society of Gastroenterological Surgery (JSGS) initiated data verification in the gastroenterological section of the National Clinical Database (NCD) and reported high accuracy of data entry. Remote audits were introduced for data validation on a trial basis in 2016 and formally accepted into use in 2017‐2018. The aim of this study was to audit the data quality of the NCD gastroenterological section for 2016‐2018 and to confirm the high accuracy of data in remote audits. Methods Each year, 45‐46 hospitals were selected for audit. Twenty cases were randomly selected in each hospital, and the accuracy of patient demographic and surgical outcome data (46 items) was compared with the corresponding medical records obtained by visiting the hospital (site‐visit audit) or by mailing data from the hospital to the JSGS office (remote audit). Results A total of 136 hospitals were included, of which 88 (64.7%) had a remote audit, and 124 936 items were evaluated with an overall data accuracy of 98.1%. There was no significant difference in terms of data accuracy between site‐visit audit and remote audit. Accuracy, sensitivity, and specificity of mortality were 99.7%, 89.7%, 100% for site‐visit audits and 99.8%, 97.3%, 100% for remote audits, respectively. Mean time spent on data verification per case of remote audits was shorter than that of site‐visit audits (10.0 minutes vs 13.7 minutes, P

Abstract The National Clinical Database (NCD) of Japan grew rapidly, harvesting over 11 million cases of data between 2011 and 2018 from more than 5000 facilities. This is the Report of the NCD based upon gastrointestinal surgery information in 4 420 175 cases from 2011 to 2018. More than 70% of all gastrointestinal surgeries were performed at certified institutions, and the percentage of surgeries performed at certified institutions was particularly high for the esophagus (93.8% in 2018), liver (89.4%), pancreas (91.3%), and spleen (86.9%). Also, more than 70% of the surgeries were performed with the participation of the board‐certified surgeon. As the patients have been getting older, the morbidities have been increasing. However, the mortalities have been kept at a low level. The rates of endoscopic surgery have been increasing year by year, especially high in low anterior resection (67.0%) and esophagectomy (61.0%). Nationwide, this database is surely expecting to ensure the quality of board certification system and surgical outcomes in gastroenterological surgery.

Abstract Aim To investigate the effectiveness of the institutional medical structure and of the implemented clinical practice guidelines for improving colon cancer surgical outcomes. Methods We conducted a web‐based questionnaire survey among departments registered at the National Clinical Database in Japan from October 2014 to January 2015 to assess the association between quality indicators (QIs), including structure and process indicators (clinical practice guideline adherence), and the risk‐adjusted odds ratio for operative mortality (AOR) after right hemicolectomy for colorectal cancer during the study period. Results Among the 2064 departments registering at least one colorectal surgery during the study period, we obtained responses from 814 departments (39.4%). Our analysis on data from 22 816 patients with right hemicolectomy demonstrated that three structural QIs (certification of training hospitals by the Japanese Society of Gastroenterological Surgery and the presences of board‐certified gastroenterological and colorectal surgeons) were associated with significantly lower AOR (P

Abstract Aim To investigate the frequency and risk factors of perioperative, symptomatic venous thromboembolism (VTE) after gastroenterological surgery. Methods We assessed the frequency of and risk factors for VTE after eight gastroenterological procedures (total 516 217 cases including, gastrectomy, total gastrectomy, hepatectomy, esophagectomy, right hemicolectomy, low anterior resection, pancreaticoduodenectomy, and acute pan‐peritonitis surgery) based on data from the National Clinical Database. Data collected between 2011 and 2013 (382 124 cases) were used as a test set, and data from 2014 (134 093 cases) were used as a validation set. Results The frequency of deep vein thrombosis (DVT) was 0.3% (382 124 cases), and the incidence of pulmonary embolism (PE) was 0.2% (382 124 cases) ranging from 0.1% to 0.7% for DVT and from 0.1% and 0.3% for PE among eight surgeries, respectively. Analyses using pre‐and intra‐operative factors identified the top three risk factors for VTE as esophagectomy, pancreaticoduodenectomy, and hepatectomy. Using pre‐, intra‐, and postoperative factors, the second through fourth risk factors were sepsis, prolonged ventilation >48 hours and readmission within 30 days. The highest risks factor for PE using pre‐, intra‐, and postoperative factors were any cardiac events. Unplanned intubation was the fourth risk factor. Conclusion The risk for DVT and PE differed for each surgical procedure. VTE and PE risk factors changed after considering postoperative factors. It may be necessary to reconsider the prophylaxis depending on whether the complication occurs after surgery, particularly breathing and cardiac complications.

Abstract Aim Gastric cancer is the second leading cause of cancer death worldwide. Surgery is the mainstay treatment for gastric cancer. There are no prediction models that examine the severity of postoperative morbidity. Herein, we constructed prediction models that analyze the risk for postoperative morbidity based on severity. Methods Perioperative data were retrieved from the National Clinical Database in patients who underwent elective gastric cancer resection between 2011 and 2012 in Japan. Severity of postoperative complications was determined by Clavien‐Dindo classification. Patients were randomly divided into two groups, the development set and the validation set. Logistic regression analysis was used to build prediction models. Calibration powers of the models were assessed by a calibration plot in which linearity between the observed and predicted event rates in 10 risk bands was assessed by the Pearson R2 statistic. Results We obtained 154 278 patients for the analysis. Prediction models were constructed for grade ≥2, grade ≥3, grade ≥4, and grade 5 in the development set (n = 77 423). Calibration plots of these models showed significant linearity in the validation set (n = 76 855): R2 = 0.995 for grade ≥2, R2 = 0.997 for grade ≥3, R2 = 0.998 for grade ≥4, and R2 = 0.997 for grade 5 (all: P

Abstract Background The aim of the present study was to develop a risk calculator predictive of postoperative pneumonia in patients undergoing gastroenterological surgery. Methods We analyzed data from 382 124 patients undergoing eight main gastroenterological surgeries between 2011 and 2013 using the National Clinical Database in Japan. A risk model was developed using multivariate logistic regression analysis with patient data from 2011 to 2012 (n = 247 604) and validated using data from 2013 (n = 134 520). Results Pneumonia was observed in 11 105 patients (2.9%). After the input of significant primary disease and surgical procedures, 18 patient characteristics including gender, chronic obstructive pulmonary disease, sepsis, and need for any assistance in the activities of daily living, six laboratory parameters, and two intraoperative factors were used for risk calculation. Area under the receiver‐operating characteristic curve was 0.822 (95% confidence interval, 0.817‐0.826) in the derivation group and 0.826 (0.819‐0.832) in the validation group. Conclusion The risk calculator accurately predicted the occurrence of pneumonia following gastroenterological surgery.

Abstract The Japanese Society of Gastroenterological Surgery (JSGS) and the American College of Surgeons National Surgical Quality Improvement Program (ACS‐NSQIP) have collaboratively developed several clinical projects since 2011 using two nationwide clinical registries with the goal of achieving further improvement of surgical quality in both countries. In this review, the historical viewpoints and the collaboration between JSGS and ACS and their use of nationwide registries [National Clinical Database (NCD) and NSQIP] for research are reviewed. We have carried out a joint project, the 30‐day Mortality Risk Model Study and, currently, we are working on several joint projects such as the Morbidity‐Mortality Study, Japan‐USA Calibration Study, Geriatric Study, and Safety Culture Study as well as Auditing in JSGS/NCD with reference to the NSQIP method. These joint projects will continue to provide us with important information and data to drive improvements in surgical care in both countries. This will also help us to identify any unknown weaknesses in the health‐care systems of the USA and Japan.

Abstract Background The Japanese National Clinical Database (NCD) is a large‐scale, nationwide, web‐based data entry system that is linked to the surgical board certification system and covers almost all surgical cases carried out in Japan. Aim To evaluate outcomes according to the gastroenterological section of the NCD. Methods The 115 surgical procedures stipulated by the “Training Curriculum for Board‐Certified Surgeons in Gastroenterology” were registered from 2011 to 2017. The number of surgeries, preoperative comorbidities, and short‐term outcomes were compared between registration periods. Results In total, 3 818 414 cases have been registered. More than 70% of all surgeries were carried out at certified institutions. The annual number of cases has been increasing year after year, and the aged population has also been increasing. Although the rates of preoperative comorbidities and postoperative complications have been increasing, the postoperative mortality rate has remained relatively low; in 2017, the 30‐day mortality rate was 1.0% among those who underwent esophagectomy, 0.7% among those who underwent distal gastrectomy, 1.1% among those who underwent total gastrectomy, 1.3% among those who underwent right hemicolectomy, 0.5% among those who underwent low anterior resection, 1.3% among those who underwent hepatectomy, and 1.3% among those who underwent pancreaticoduodenectomy. The annual rate of endoscopic surgery dramatically increased over 7 years between 2011 and 2017, especially for low anterior resection (29.5%‐62.6%) and esophagectomy (31.0%‐56.1%). Conclusion This database is expected to ensure the quality of the board‐certification system and surgical outcomes in gastroenterological surgery.

Crizotinib, the first clinically available inhibitor of anaplastic lymphoma kinase (ALK) gene rearrangement, is generally well tolerated. In contrast, neutropenia induced by crizotinib is a commonly reported grade 3 or 4 adverse event. In such cases, interruption and dose reduction of crizotinib might be necessary for some patients with severe neutropenia. However, information concerning clinical experience and management of severe neutropenia is currently limited. In this report, the successful management of crizotinib-induced neutropenia by dose reduction of crizotinib in a patient with ALK-positive non-small cell lung cancer is described.

To assess the use of laparoscopic surgeries (LS) and the association between its performance and hospitals' preference for LS over open surgeries.LS is increasingly used in many abdominal surgeries, albeit both with and without solid guideline recommendations. To date, the hospitals' preference (LS vs. open surgeries) and its association with in-hospital outcomes has not been evaluated.We enrolled patients undergoing 8 types of gastrointestinal surgeries in 2011-2013 in the Japanese National Clinical Database. We assessed the use of LS and the occurrences of surgery-related morbidity and mortality during the study period. Further, for 4 typical LS procedures, we assessed the hospitals' preference for LS by modeling the propensity to perform LS (over open surgeries) from patient-level factors, and estimating each institution's observed/expected (O/E) ratio for LS use. Institutions with O/E>2 were defined as LS-dominant. Using hierarchical logistic regression models, we assessed the association between LS preference and in-hospital outcomes.Among 1,377,118 patients undergoing gastrointestinal procedures in 2,336 participating hospitals, use of LS increased in all 8 procedures (35.1% to 44.7% for distal gastrectomy (DG), and 27.5% to 43.2% for right hemi colectomy (RHC)). Those operated at LS-dominant hospitals were at an increased risk of operative death (OR 1.83 [95%CI, 1.37-2.45] for DG, 1.79 [95%CI, 1.43-2.25] for RHC) compared to standard O/E level hospitals (0.5≤O/E

Strategies to reduce the immunogenicity of pancreatic islets and to prevent the activation of proinflammatory events are essential for successful islet engraftment. Pretransplant islet culture presents an opportunity for preconditioning to improve outcomes of islet transplantation. We previously demonstrated that ex vivo mitomycin C (MMC) pretreatment and subsequent culture significantly prolonged graft survival. Fully understanding the biological process of pretreatment could result in the development of a protocol to improve the survival of islet grafts. Microarrays were employed to conduct a comprehensive analysis of genes expressed in untreated or MMC-treated rat islets that were subsequently cultured for 3 d. A bioinformatics software was used to identify biological processes that were most affected by MMC pretreatment, and validation studies, including in vivo and in vitro assay, were performed. The gene expression analysis identified significant downregulation of annotated functions associated with cellular movement and revealed significant downregulation of multiple genes encoding proinflammatory mediators with chemotactic activity. Validation studies revealed significantly decreased levels of interleukin 6 (IL-6), monocyte chemoattractant protein 3 (MCP-3), and matrix metallopeptidase 2 (MMP2) in culture supernatants of MMC-treated islets compared with controls. Moreover, we showed the suppression of leukocyte chemotactic activity of MMC-treated islets in vitro. We also showed that MMC-treated islets secreted lower levels of chemoattractants that synergistically reduced the immunogenic potential of islets. Histological and immunohistochemical analyses of the implant site revealed that infiltration of monocytes, CD3-positive T cells, and B cells was decreased in MMC-treated islets. In conclusion, the ex vivo pretreatment of islets with MMC and subsequent culture can reduce the immunogenic potential and prolong the survival of islet grafts by inducing the suppression of multiple leukocyte chemotactic factors.

The recovery of all of the islets contained in a pancreas is the goal of islet isolation for transplantation. This study reveals an environment that injures the isolated islets during digestion and proposes a new model for optimal islet isolation. Islets were isolated from Wistar rat pancreases by stationary collagenase digestion while the digestion time was varied at 15, 30, 60, and 120 min. The digested pancreas and islets were analyzed histologically and adenosine nucleotides were measured. Overnight cultured islets (40 islets) were cocultured for 30 min with the supernatants obtained from pancreatic collagenase digestion at different digestion periods in order to assess the toxic environment. The peak yields of islets were obtained at 30 min of digestion. The histological study of digested pancreas showed that the exocrine cells lost their cellular integrity at 120 min of digestion, but the islet cells were left intact. Accordingly, the ATP levels of the pancreatic tissue decreased during the digestion period. The coculture experiment demonstrated that the islets cultured with the supernatants from the collagenase digestion showed digestion time-dependent disruption of the cellular integrity of islets in accordance with a rapid decrease of ATP levels in the islets. The addition of serine protease inhibitors into this coculture clearly showed protection of islets, which maintained high ATP levels in association with intact membrane integrity as assessed by AO/PI staining. Morphological deterioration of islets as well as a marked ATP decrease was evident in the entire digested pancreas as well as in islets cocultured in the supernatants from the collagenase digestion. Various factors toxic to the islets can therefore be analyzed in future experiments using this coculture model for obtaining a good yield of viable islets.

Mizoribine (MZ) inhibits the differentiation and proliferation of helper T and B cells after antigen recognition by suppressing the purine biosynthesis pathway and nucleic acid synthesis. MZ has been used in kidney transplantation, but distinct data are unavailable for islet transplantation. The present study investigated the efficacy of MZ for islet xenotransplantation. Immunosuppressive effects of MZ were determined by mixed lymphocyte reaction (MLR) assay in vitro. Toxicities for Wistar rat islets were determined by adenosine triphosphate (ATP) contents of islets during 3-day culture and stimulation index in response to glucose after culture. Immunosuppressive effects in vivo were tested in a Wistar-to-B6 islet xenotransplantation model. MZ was administered continuously for 28 days subcutaneously or intramuscularly. MZ inhibited MLR response by approximately 50% at 0.1 μg/ml. ATP contents decreased with MZ >100 μg/ml, while stimulation index was maintained. Continuous infusion of MZ at 10 mg/kg maintained blood concentrations at 0.13–0.19 μg/ml, while intramuscular injection of MZ at 100 mg/kg/day (peak 520 μg/ml at 1 h postinjection) resulted in below measurable levels (

One of the goals of islet transplantation is to transplant viable islets without host immunosuppression. The present study was designed to determine whether pretreatment of islets with mitomycin-C (MMC) followed by culture enhances islet survival in a rat-to-mouse xenogeneic combination. WS(RT1k) rat islets pretreated with various concentrations of MMC (0, 3.2, 10, 32, 100, 320, and 1000 μg/ml) were tested for viability by in vitro insulin secretory capacity and vital staining of islets. The MMC-treated islets (10 μg/ml) cultured for various periods (4, 20, or 40 h, 3 or 7 days) were transplanted into the renal subcapsular space of STZ-induced diabetic C57BL/6 (B6: H-2b) mice. MMC-treated or nontreated islets were subjected to microarray gene analysis and immunohistological study. Evaluation of in vitro insulin secretory capacity and vital staining of islets indicated that MMC at a dose ≤32 μg/ml is nontoxic and preserves islet function. Marked prolongation of graft survival was noted with half of islet grafts surviving indefinitely (>100 days) when 10 μg/ml of MMC-treated islets was transplanted after 40 h or 3 days in culture, but not when they were transplanted within 4 h following treatment or at 7 days following treatment, indicating that there is a critical culture period necessary for successful islet graft survival. Microarray analysis suggested possible genes for this prolongation with TGF-β highly expressed in MMC-treated islets subjected to culture for 3 days. Our results indicate that MMC treatment followed by a critical culture period induces marked prolongation of rat islet xenograft survival in nonimmunosuppressed recipient mice, offering a strategy for islet transplantation without immunosuppression.

The liver is thought to be an immunologically privileged organ in the response to inoculated antigens. We previously demonstrated that it is possible to localize inoculated antigens to the liver alone or only to extrahepatic tissue using orthotopic syngeneic liver transplantation (OSLT). In this study, we analyzed more detailed mechanisms of the anti-alloimmune response in the liver. DA rat spleen cells were systemically injected into WS rats (donor spleen cell inoculation, DSI). In the sensitized liver-grafted (SLG) group, after DSI, liver grafts were retrieved from sensitized WS rats, then transplanted into naive WS rats. In the sensitized liver-removed (SLR) group, after DSI, WS rats were totally hepatectomized and given livers transplanted from naive WS rats. All the rats were challenged with heterotopic heart grafts 10 days after DSI. Mean heart graft survival in the control, DSI, SLG, and SLR groups were 11.6 ± 1.6, 10.7 ±2.4, 4.4 ± 1.0, and 24.6 ± 6.3 days, respectively. Accelerated rejection in the SLG group as well as graft prolongation in the SLR group disappeared when OSLT was performed 2 days after DSI or later. Irradiation of DA splenocytes before inoculation did not alter graft survival in SLG. However, pretreatment with gadolinium chloride prior to DSI attenuated the antidonor response in the SLG group. In conclusion, a vigorous antidonor response occurred in the liver after systemic inoculation of spleen cells. It peaked 1 day after DSI and disappeared rapidly. Kupffer cells seemed to play an important role in this phenomenon.

We have previously shown that mitomycin C (MMC) treatment of donor tissue resulted in significant prolongation of graft survival in allo- and xenotransplantation models. However, the mechanisms involved in this prolongation are not clearly understood. This study aims to shed light on the immune responses to MMC-treated islet xenografting under the kidney capsule. Collagenase-digested WS (RT1 k ) rat islets incubated for 30 min with MMC and subsequently cultured for 20 h were transplanted into the renal subcapsular space of streptozotocin-induced diabetic C57BL/6 (B6;H-2 b ) mice. The grafts were harvested on postgrafting day 7 and sections were prepared and stained by hematoxylin and eosin (H&E). Histological study of the grafts in a group not treated with MMC showed marked cellular infiltration and destruction of islet clusters, whereas that of MMC-treated grafts demonstrated a bleb formation under the kidney capsule, in which islet cell clusters were reorganized, creating a layer of cells fixed to the interior of the bleb. Minimal invasion by inflammatory cells was observed only at the edge of the bleb, and most islet cells were protected from these infiltrating cells. In conclusion, MMC treatment induces remodeling of islet structure and forms a bleb under the kidney capsule, where no inflammatory cell infiltration occurs, suggesting that this site is a kind of immunologically privileged environment for xenografted islets.

In this study, we examined the effect of mitomycin C (MMC) treatment on graft survival and evaluated its efficacy in immunomodulation of islet graft for transplantation. Male WS rats were used as islet donors and streptozotocin-induced diabetic C57BL/6 mice as recipients. The isolated islets were treated with MMC at concentrations of 0, 0.1, 1, 3.2, 10, 32, 100, 320, and 1000 μg/mL for 30 min, and were cultured for 20 h. Then, 300–400 islets were transplanted into the renal subcapsular space of diabetic mice. Significant prolongation of graft survival was obtained when the islets were treated with MMC at a concentration of 10, 32, or 100 μg/mL (MST 23 ± 7.4, 17.5 ± 5.4, 29.6 ± 9.7 days: p < 0.003, p < 0.012, p < 0.001, respectively, vs. 12.3 ± 2.7 days for culturing alone). Islets treated with MMC at a concentration of 320 μg/mL or more failed to restore normoglycemia in the diabetic recipient mice after transplantation. Viability of islets incubated with doses up to 100 μg/mL, assessed under the confocal microscope after propidium iodide and Hoechst 33342 staining, was maintained well comparable to that of freshly isolated islets, while those treated at 320 μg/mL was significantly decreased. Thus, a therapeutic window for MMC efficacy was found at concentrations from 10 μg/mL to 100 μg/mL. This modality is simple and effective and underlying molecular mechanisms need to be determined in the future.

It is controversial whether or not microchimerism (MC) is responsible for the induction and maintenance of donor-specific tolerance. We have shown that intraportal injection (i.p.) of donor splenocytes induces a long-term graft survival of liver and heart in rats. In this study, we examined by polymerase chain reaction (PCR) the status of MC in the liver, spleen, and blood of rat cardiac recipients following i.p. or intravenous injection (i.v.) of donor splenocytes. Male DA (RTl a ) and Wistar (RTl k ) rats were used as donors and recipients, respectively. Heterotopic heart transplantation was performed 10 days after i.p. or i.v. injection of 5 × 10 7 DA spleen cells. DA cardiac allografts were rejected with a mean survival time (MST) of 11.9 ± 1.6 (n = 10) days in nontreated recipients. Injections (i.v.) led to no significant prolongation of graft survival (MST: 11.2 ± 1.9 days, n = 6), while i.p. injection induced a significant prolongation of graft survival (MST: 18.6 ± 1.5 days, n = 5) over the control. Either i.p. or i.v. injection alone resulted in systemic MC in these organs throughout the observation time over 60 days. MC was detected in the spleen, liver, and blood of cardiac recipients 7 days after transplantation and also even after cessation of cardiac heartbeat 21 days after transplantation. This was the case with either i.p. or i.v. group, which showed MC on day 7 after transplantation and persistent MC after cessation of the heartbeat. These data suggest that the presence of MC in the liver, spleen, and the blood of transplant recipients may not be responsible for immunological unresponsiveness to donor antigens.

Recently, we reported that intraportal (IP) injection of donor spleen cells (SPCs) prevented liver allograft rejection. Moreover, we developed a new method using polymerase chain reaction (PCR)-mediated restriction fragment length polymorphism (RFLP) analysis, and demonstrated micro-chimerism (MC) at the DNA level in the spleen 14 days after IP injection. In the present study, the long-term presence of injected allogeneic SPCs was investigated at the cellular level by immunofluorescence staining as well as the DNA level using RFLP analysis. Male ACI (RT1 a rats were used as the donors and Lewis (RT1 1 ) rats as the recipients. After DNA preparation from the lymphoid organs, RT1Bβ domain 1 region was amplified by PCR, and RFLP analysis was performed with PVuII restriction enzyme. In the immunofluorescence staining, the monoclonal antibody, MN4-91-6, was used to detect the injected donor ACI SPCs in a frozen specimen. We did not detect MC in Lewis rats intravenously injected with 5 x 10 7 ACI SPCs on day 14. On the other hand, stable chimerism in the spleen was observed in intraportally injected rats up to 28 days after injection at not only the DNA level but also the cellular level. No chimerism was detected in other organs (including the thymus, lymph nodes, and liver). In conclusion, the long-term presence of injected allogeneic SPCs in the spleen was demonstrated after IP injection but not after IV injection, and this phenomenon may be one of the mechanisms involved in portal venous immunosuppression.