Hironori Kato

Adipogenesis induced by human adipose tissue-derived stem cells.

Adipose tissue-derived stem cells (ASCs), including preadipocytes, may play an important role in de novo adipogenesis and are expected to be a useful external source of cells for adipose tissue engineering. In this study, we examined in vivo adipogenesis up to 24 weeks after implantation, induced by human ASCs that were isolated from adipose tissues and expanded in vitro. ASCs proliferated in vitro in the presence of basic fibroblast growth factor (bFGF), and the number of cells increased by more than 1000-fold at the fourth passage. The ability to differentiate into mature adipocytes was maintained up to the third passage. We incorporated designated numbers of third-passage-expanded cells into a type I collagen scaffold and implanted them into the back of nude mice with or without controlled-release bFGF. After the implantation of 2 x 10(6) ASCs with controlled-release bFGF, the greatest cross-sectional surface area of adipose tissue in the scaffold was 1.19 mm(2) at 12 weeks and 2.14 mm(2) at 24 weeks. About 2 x 10(6) ASCs with controlled-release bFGF was the best condition for total adipogenesis. Immunohistochemical analysis with antihuman vimentin antibody showed that the area of human-origin adipose tissue was maximum in the group with 8 x 10(6) ASCs incorporated in a scaffold at both 12 and 24 weeks. The amount of human-origin adipose tissue increased in all groups with implanted ASCs from 12 to 24 weeks. Only trace of human-origin adipose tissue was observed in other groups implanted ASCs. Our results show that human ASCs not only function as progenitor cells for in vivo adipogenesis, but also induce de novo adipogenesis for long period.

Infectious Complications in Living Related Liver Transplantation

During the last 31 months, 50 children between 3 months and 15 years of age have undergone living related liver transplantation (LRLT) for end-stage liver diseases (39 biliary atresia, 2 Budd-Chiari syndrome, 2 progressive intrahepatic cholestasis, 3 liver cirrhosis, 1 Wilson disease, 1 protoporphyria, 1 tyrosinemia, and 1 fulminant hepatitis). Combined FK-506 and low-dose steroids were routinely used for immunosuppression. There were seven deaths, two of which were related to infection (Candida pneumonia and Epstein-Barr virus [EBV]-associated lymphoproliferative syndrome [LPS]). Five patients had a bacterial infection, all of which were associated with surgical complications. Three patients had Candida infection, all of which were malnourished, had biliary atresia, and had been managed with prolonged antibiotics against obstinate ascending cholangitis. There were 14 symptomatic viral infections (1 herpes simplex virus, 1 herpes zoster virus, 5 cytomegalovirus [CMV], 6 EBV, and 1 EBV-associated LPS). Three of the five CMV infections appeared in patients whose graft was ABO-incompatible, who were managed with prophylactic OKT-3. Most of the viral infections (except 1 EBV-associated LPS) were minor and were treated successfully. The low incidence and successful treatment of CMV infection are related to the high compatibility and low incidence of allograft rejection in LRLT. Bacterial and fungal infections can be decreased by greater refinement of surgical technique and more aggressive preoperative management. Treatment of EBV infection is still an unsolved problem.

Possible association of thioredoxin and p53 in breast cancer

Expression of thioredoxin (TRX), a dithiol-reducing enzyme, and mutations of p53 have been detected in various cancer tissues. We recently reported that TRX-dependent redox regulation plays a crucial role in DNA binding activity of p53. In this study, we investigated the possibility of functional association between TRX and p53 in breast cancer. First, we examined the expression of TRX and mutated p53 in 100 primary breast cancer tissues by immunohistochemistry. Expression of TRX was detected in cases of 84/100 (84%) and expression of p53, which means existence of mutated p53, in cases of 63/100 (63%). TRX positive cases was 89% (56/63) in mutant p53 positive cases. Next, we examined the expression of TRX and p53 in breast cancer cell line MCF-7 cells after CDDP treatment or irradiation. CDDP treatment or irradiation augmented expression of TRX and p53 in MCF-7 cells by western blotting. Immunofluorescence cell analysis by confocal microscopy showed that CDDP treatment induced translocation of TRX into nuclei. These results suggest the possible association of TRX with p53-dependent function including DNA repair in breast cancer.

Lymphatic Mapping With Fluorescence Navigation Using Indocyanine Green and Axillary Surgery in Patients With Primary Breast Cancer

Abstract:  The indocyanine green fluorescence (ICGf) navigation method provides real‐time lymphatic mapping and sentinel lymph node (SLN) visualization, which enables the removal of SLNs and their associated lymphatic networks. In this study, we investigated the features of the drainage pathways detected with the ICGf navigation system and the order of metastasis in axillary nodes. From April 2008 to February 2010, 145 patients with clinically node‐negative breast cancer underwent SLN surgery with ICGf navigation. The video‐recorded data from 79 patients were used for lymphatic mapping analysis. We analyzed 145 patients with clinically node‐negative breast cancer who underwent SLN surgery with the ICGf navigation system. Fluorescence‐positive SLNs were identified in 144 (99%) of 145 patients. Both single and multiple routes to the axilla were identified in 47% of cases using video‐recorded lymphatic mapping data. An internal mammary route was detected in 6% of the cases. Skip metastasis to the second or third SLNs was observed in 6 of the 28 node‐positive patients. We also examined the strategy of axillary surgery using the ICGf navigation system. We found that, based on the features of nodal involvement, 4‐node resection could provide precise information on the nodal status. The ICGf navigation system may provide a different lymphatic mapping result than computed tomography lymphography in clinically node‐negative breast cancer patients. Furthermore, it enables the identification of lymph nodes that do not accumulate indocyanine green or dye adjacent to the SLNs in the sequence of drainage. Knowledge of the order of nodal metastasis as revealed by the ICGf system may help to personalize the surgical treatment of axilla in SLN‐positive cases, although additional studies are required.

The reversal of recurrence hazard rate between ER positive and negative breast cancer patients with axillary lymph node dissection (pathological stage I-III) 3 years after surgery

BackgroundsPrognostic factors are defined as biological or clinical measurement associated with overall survival and/or disease-free survival. Previous studies have shown that patients with estrogen receptor (ER) positive cancers have a better prognosis than patients whose cancers do not have these receptors.MethodsThis study investigated the assessment of variables in defining prognosis of 742 breast cancer women with pathological stage (pTNM) I-III diagnosed between 1980 and 2005 at the Kyoto University Hospital in Japan, by age, clinical stage (cTNM), pTNM, the numbers of positive lymph nodes (pN), and ER status.ResultsMultivariate analysis demonstrated that pTNM and ER status were the independent prognostic factors for overall survival, and that pTNM and pN were the independent prognostic factors for disease-free survival. For the 0- to 2-year interval, the hazard of recurrence was higher for the ER-negative patients than the ER-positive patients, and beyond 3 years the hazard was higher for ER-positive patients.ConclusionThe present study confirmed the previous reports which showed favorable prognosis of the patients with lesser pTNM or positive ER status. A reversal of recurrence hazard rate between ER positive and negative breast cancer patients beyond 3 years after operation was detected. The fact may indicate the importance of long term adjuvant hormone therapy for ER positive cancer patients.

Living related liver transplantation across ABO blood groups with FK506 and OKT3

In living related liver transplantation (LRLT), the use of graft livers across ABO blood groups is unavoidable since the organ donor is usually one of the recipients parents. This report presents our initial experiences with LRLT, focusing on ABO-incompatible cases. From June 1990 to May 1992, we successfully performed a series of 34 LRLT on children (15 males and 19 females) ranging in age from 7 months to 15 years. Overall recipient survival rates were 90% (25/28) in elective LRLT and 50% (3/6) in emergency LRLT. These cases were classified into three groups: ABO blood group-identical (n=21), compatible (n=10), and incompatible (n=3). The immunosuppressive regimen consisted of FK506 and low-dose steroids in the first two groups. In the incompatible cases, exchange transfusion was performed to decrease anti-A and/or-B antibody titers before LRLT, and prophylactic OKT3 was added to FK506 and steroids after LRLT. No significant difference in recipient and graft survifal was observed among the groups. In the identical group, no rejection episodes have been observed thus far. Rejection occurred in two out of the ten compatible cases. Among the incompatible cases, one recipient had mild rejection and was treated. The remaining two recipients have had no rejection episodes thus far. Although all three recipients had cytomegalovitus (CMV) infection, they were successfully treated with gancyclovir, and no lethal infection has developed in any of these cases. The present results suggest that graft livers from living related donors across ABO blood groups can function well with FK506, low-dose steroids, and prophylactic OKT3 without causing lethal complications.

What is the most appropriate scan timing for intraoperative detection of malignancy using 18F-FDG-sensitive gamma probe? Preliminary phantom and preoperative patient study.

PurposeTo evaluate the appropriate post-injection timing for hand-held-gamma-ray-detecting probe (GDP) scanning for the intraoperative detection of malignancy after preoperative F-18 FDG (FDG) injection.MethodsPatient study with superficially located cancer was performed on three patients before operation by dual-phase whole-body PET at 2 and 6–7 hr post-injection of FDG (370 MBq), and by probe scanning from the skin at several points at 1, 3, 5, and 7 hr after FDG injection. TNRa (tumor-adjacent-normal ratio) and TNRc (tumor-contralateral-normal ratio) were calculated. Phantom study was also performed to determine basic GDP function.ResultsThe patient study revealed that tumors showed constant TNRa (0.9–1.3) and TNRc (1.1–3.0) by GDP count rate, and that there was no tendency of an increase in TNRa with time. The standard deviations of GDP count rate were lower at 1–3 hr post-injection compared with those of delayed scans. While delayed PET showed an increase or no change in the tumor FDG uptake, the decrease of normal tissue FDG uptake was not adequate to create higher TNRs. The phantom study revealed that LN model showed TNRa of 1.7 or greater by GDP count rate (cps) when background contained no FDG, but that they showed TNRa of 1.3 or less when the background contained 4% of the LN FDG activity per ml.ConclusionThe present study suggests that higher FDG count rate of tumors at 1–3 hr post-injection would be more suitable for the gamma-probe detection compared with lower count rate at 6–7 hr delayed scans with wide standard deviations.

Efficient proliferation and adipose differentiation of human adipose tissue-derived vascular stromal cells transfected with basic fibroblast growth factor gene.

Human vascular stromal (VS) cells obtained from mature adipose tissue were transfected with an adenovirus vector carrying the basic fibroblast growth factor (bFGF) gene. bFGF protein was observed in VS cell nuclei 24 h after transfection and in the cytoplasm and extracellular space 72 h after transfection. Naive VS cells were almost static in vitro and proliferated in a dose-dependent manner on stimulation with recombinant bFGF (rbFGF). However, bFGF-transfected VS cells proliferated spontaneously to the same extent as naive VS cells when stimulated with rbFGF at 100 ng/ml. The former cells started to proliferate on day 3 after transfection and the proliferation pattern was similar to that of the latter cells, although only a slight amount of bFGF protein was detected in the culture medium when the bFGF-transfected cells started to proliferate. The proliferation of bFGF-transfected VS cells was completely inhibited by bFGF neutralizing antibody, which also completely inhibited the proliferation of naive VS cells stimulated with rbFGF. Under conditions favoring differentiation to adipocytes, bFGF-transfected VS cells stopped proliferating and started to accumulate lipid in the cytoplasm. bFGF-transfected VS cells, which spontaneously and efficiently proliferate while preserving their ability to differentiate into adipocytes, may be an adequate cell source for human adipose tissue regeneration.

Soluble CD23 as a sensitive marker for Epstein-Barr virus-related disorders after liver transplantation

Plasma levels of the soluble fragments of Fc epsilon RII/CD23 (sCD23/IgE-binding factor) were measured to assess the level of activation of B lymphocytes associated with Epstein-Barr virus infection in 28 patients who received living-related liver transplantation and were treated with FK506 and steroids. In 6 patients with symptoms of EBV infection (EBV-related disorders), the plasma concentration of sCD23 increased to more than 9.8 ng/ml at the onset of symptoms. In a patient with B cell lymphoma, the plasma levels of sCD23 increased significantly when peripheral lymphadenopathy was noticed, and remained more than 10 ng/ml during the terminal period. In 4 of 6 patients, the increase of plasma levels of sCD23 preceded the increase of anti-EBV capsid antigen IgM. In the other 2 of 6 patients, there was no significant increase of the antibody, despite the integration of EBV DNA in the mononuclear cells in their ascites. The plasma levels of sCD23 of the patients without symptoms of EBV infection did not exceed 7.5 ng/ml. In contrast, the proportion of CD20+/CD23+ B lymphocytes in peripheral blood mononuclear cells was not significantly different in the patients with EBV-related disorders and those with latent asymptomatic EBV infection. Therefore, the plasma level of sCD23 is a sensitive and useful marker of EBV-related polyclonal and/or monoclonal B cell proliferation in transplanted patients with immunosuppression.

Changes in portal vein hemodynamics after hepatic portoenterostomy in biliary atresia

Portal vein (PV) shrinkage sometimes eliminates the possibility of liver transplantation in biliary atresia patients after hepatic portoenterostomy. To determine the factors leading to PV shrinkage, we performed a serial sonographic study of the portal venous system in 21 children. Cross-sectional PV area and mean portal venous velocity (PVV) were reduced in patients with refractory cholangitis and those with gastroesophageal varices and cholangitis. Although the reduction in cross-sectional PV area was greater in patients with four-time laparotomy than single laparotomy, the mean PVV was not reduced by repeated laparotomy. Patients with varices were lower in age, weight, mean PVV, cross-sectional PV area for age, and had higher serum total bilirubin levels. In conclusion, refractory cholangitis is a significant factor in shrinking the PV. With active bile drainage, varices spontaneously regress, the PV increases in both caliber and total length per unit hepatic volume, and PVV normalizes. It is suggested that pulsed Doppler PV sonography can help to determine the optimal time for liver transplantation referral in biliary atresia patients with progressive cirrhosis.