Nobuyuki Sano

E‐cadherin, α‐catenin and β‐catenin in biliary atresia: Correlation with apoptosis and cell cycle

Biliary atresia (BA) is the most common cause of obstructive jaundice in infancy. Although the etiology of BA remains unknown, the ductal plate malformation has been considered to play an important role in the development of BA. Cell–cell adhesion has long been recognized as one of the most important processes in organogenesis. E‐cadherin is involved in cell–cell adhesion, together with the catenins. Abnormalities of E‐cadherin and associated catenins have not been examined in detail in the liver with BA. We therefore examined immunolocalization of E‐cadherin and α‐ and β‐catenins in the BA liver (n= 45) and compared the findings with those in non‐BA (n= 11) and fetal liver (n= 21). We semiquantitatively evaluated the findings using H score, which were generated according to the percentage of immunopositive cells and their immunointensity. We also examined mRNA localization of E‐cadherin using mRNA in situ hybridization. We then studied the correlation of E‐cadherin immunoreactivity with apoptotic cells, and cyclin‐dependent kinase inhibitor p27Kip1 immunolocalization of bile duct cells in BA liver (n= 10) and fetal liver (n= 10). In fetal liver, H score of E‐cadherin, but not of α‐ and β‐catenins, was significantly lower in the remodeling stage than in the ductal plate (P= 0.0034) and remodeled stages (P= 0.0024). In addition, the H score of E‐cadherin, but not α‐ and β‐catenin, in bile duct cells was significantly lower in BA liver than in non‐BA liver (P= 0.0132). E‐cadherin mRNA hybridization signals were relatively conserved in bile duct cells of BA liver, but decreased in remodeling ductal plate cells of fetal liver. An inverse correlation was detected between the H score of E‐cadherin and the TUNEL labeling index (LI) in both fetal and BA liver. In contrast, a positive correlation was detected between the H score of E‐cadherin and p27 LI in both fetal and BA liver. These findings suggest that impaired expression of E‐cadherin in bile ducts may play an important role in the biological features of BA, possibly associated with cell cycle and apoptosis.

High trough levels of oral FK506 induced by loss of small intestine

Abstract: To establish a safe and effective usage of oral tacrolimus (FK506) in small bowel transplantation (SBTx) recipients, trough levels and area under the curve (AUC) values of FK506 were assessed using swine models of SBTx and short bowel. Thirty‐eight Landrace male piglets were divided into four groups as follows: Group 1, controls (n=13); Group 2, a one‐third small bowel model (n=5); Group 3, a short bowel model (n=10); and Group 4, a one‐third small bowel allograft model (n=10; five donors and five recipients). Piglets of Groups 1 and 3 were further divided into two sub‐groups, according to the route of drug administration: Groups 1a (n=10) and 3a (n=7) received FK506 orally, and Groups 1b (n=3) and 3b (n=3) received FK506 intravenously. Oral or intravenous FK506 was started on post‐operative day 3 and continued until day 7 in each group. On day 7, trough levels and AUC values were measured. In Groups 1a, 2, 3a and 4, trough levels of FK506 were 2.1±1.2 (p<0.01 vs. Group 2, 3a or 4), 11.2±2.1, 23.3±4.8 (p<0.05 vs. Group 2 or 4) and 14.6±3.0 ng/mL, and AUC values were 101±90 (p<0.01 vs. Group 3a or 4), 319&±155, 808±200, and 531±113 ng.h/mL, respectively. Both trough levels and AUC values were lowest in Group 1a and highest in Group 3a. Between Groups 1b and 3b, there was no significant difference in the blood levels of intravenous FK506. The shorter the functioning residual small intestine was, the higher the trough level of oral FK506 was, while the presence or absence of small intestine did not affect blood levels of intravenous FK506. These results suggest that oral FK506 is metabolized in the functioning small intestine during its absorption. Therefore, events which cause intestinal malfunction, such as graft rejection in SBTx, inflammation and loss of small intestine, may adversely raise the trough level of oral FK506.

The functional roles of porcine CD80 molecule and its ability to stimulate and regulate human anti-pig cellular response.

Background. The pig is currently considered to be the most likely candidate for a xenogenic-organ source. Anti-pig human T-cell response via co-stimulatory molecules has been studied with great interest. The soluble form of porcine CD80 has recently been cloned and characterized, but the sequence of the transmembrane form has not been determined. The purpose of this study was to investigate the functional interaction between porcine CD80 and human T cells using the full-length clone of porcine CD80. Materials and Methods. Specific complementary DNA (cDNA) clones encoding porcine CD80 were isolated and sequenced using rapid amplification of cDNA ends-polymerase chain reaction. Polymerase chain reaction-amplified cDNA coding for the open reading frame of the porcine CD80 transmembrane form was subcloned into an expression vector and then transfected into Chinese hamster ovary (CHO) cells. CHO cells transfected with porcine CD80 (CHO-pCD80) were co-cultured with human CD4+ T cells and then interleukin-2 secretion was measured and transferred pCD80 expression in these human T cells was detected by flow cytometry. Results. We cloned and determined the complete nucleotide sequence for the transmembrane form of porcine CD80. Results from our T-cell co-stimulatory assay showed significant interleukin-2 production when co-stimulated with CHO-pCD80. Human naïve CD4+ T cells acquired xenogenic pCD80 molecules in the process of T-cell activation. Conclusions. Findings from this study seem to suggest that pCD80 has the functional ability to regulate human anti-pig cellular response. In addition, genetic manipulation of porcine co-stimulatory molecules offers a potentially new therapeutic strategy to prevent xenogeneic rejection across species.

Clinical significance of 99mTc-DTPA-galactosyl human serum albumin liver scintigraphy in follow-up patients with biliary atresia

PURPOSE Technetium 99m DTPA-galactosyl human serum albumin (GSA) liver scintigraphy was performed in follow-up patients with biliary atresia, and its clinical significance was investigated. METHODS Between 1994 and 2001, GSA liver scintigraphy was performed 153 times in 57 follow-up patients. HH15, LHL15, and H/L15 (HH15/LHL15) were obtained. Patients were divided into 3 groups according to the clinical status (good, n = 17; fair, n = 24; poor, n = 16). The correlation between these parameters and liver function tests was examined. Twenty-six patients of the 57 underwent 3 serial GSA scintigraphies and also were divided into 3 groups (good, n = 13; fair, n = 8; poor, n = 5). (3rd/1st)H/L15 (3rd H/L15/1st H/L15) was obtained and compared. RESULTS H/L15 had a correlation with serum albumin and serum cholinesterase. H/L15 was statistically different among 3 groups (good, 0.97 +/- 0.15; fair, 0.94 +/- 0.09; poor, 1.12 +/- 0.21; P <.05). Although most patients in the good (10 patients; 76.9%) and fair (7 patients; 87.5%) groups showed (3rd/1st)H/L15 of less than 1.1, 3 patients (60%) in the poor group showed (3rd/1st)H/L15 of more than 1.1. (3rd/1st)H/L15 in the poor group was significantly higher than those in good and fair groups (P <.05). CONCLUSIONS Technetium 99m-GSA liver scintigraphy is useful to assess the functional hepatic reserve in follow-up patients with biliary atresia. Serial assessment with GSA scintigraphy can provide the trend of the patients liver condition and can estimate the prognosis of the liver.

Comparison of Β-Adrenergic blocking activity of dichloroisoprenaline (H 56/28, I.C.I. 50172, LB46), methoxamine (MJ 1999) and propranolol in the canine femoral vascular bed

Die blockierende Wirkung intra- arteriell verabreichter,Β-adrenergischer Mittel wurde am femoralen Gefässgebiet des Hundes untersucht. DCI, H 56/28 und LB 46 per se regten den arteriellen Blutstrom mit zunehmender Dosierung an, während Methoxamin in umgekehrter Weise wirkte. Eine hemmende Wirkung auf den durch Isoprenalin angeregten Blutstrom fand sich in folgender Reihenfolge: LB 46≧H 56/28 > Propranolol > MJ 1999=DCI. I.C.I. 50172 und Methoxamin fehlt diese Wirkung.