Effectiveness of Bortezomib in a Patient With Acute Rejection Associated With an Elevation of Donor-Specific HLA Antibodies After Small-Bowel Transplantation: Case Report.

BACKGROUND: A significant association between donor-specific antibody (DSA) and graft rejection has recently been documented. However, confirmed strategy has not been established for DSA-associated rejection after intestinal transplantation (ITx). CASE REPORT: A 20-year-old male patient with chronic intestinal obstruction caused by hypoganglionosis of the entire intestine underwent cadaveric donor ITx with grafting performed on 232 cm of the small intestine, cecum, and a part of the ascending colon. On post-operative day (POD) 14, a histological evaluation showed an acute rejection of indeterminate grade. The patient had severe acute rejection on POD 16, which prompted us to administer bolus steroids and polyclonal anti-thymocyte antibody, along with baseline maintenance immunosuppression. The histopathological findings of the graft indicated typical acute cellular rejection, although C4d was positive. We then detected donor-specific HLA antibody. The patient initially responded well to the therapy and showed decreased histological rejection signs. However, the refractory low-grade rejection persisted in the graft. During this period, the patient showed increased levels of DSA, and we speculated that the persistent rejection was associated with DSA; thus, bortezomib was administered at this stage as a salvage therapy. This rejection was thereafter successfully controlled without severe adverse effect. Twenty-three months after ITx, the patient is currently alive with complete enteral autonomy. CONCLUSIONS: A case of acute graft rejection followed by a marked elevation of DSA is presented. In this particular case, a modified treatment protocol using bortezomib in addition to the typical immunosuppressive agents was effective.

Establishment of educational program for multiorgan procurement from deceased donors.

INTRODUCTION: Multiorgan procurement is not an easy procedure and requires special technique and training. Since sufficient donors are not available for on-site training in Japan, establishment of the educational program for multiorgan procurement is mandatory. MATERIALS AND METHODS: Development of e-learning and simulation using pigs are our main goals. E-learning contains three dimensional computer graphic (3DCG) animations of the multiorgan procurement, explanation of both donor criteria and procurement procedure, and self-assessment examination. To clarify the donor criteria, the risk factors to 3-month survival of the recipients were analyzed in 138 adult cases of liver transplantation. The 3DCG animation for liver procurement was developed, which was used in the lecture prior to the simulation on August 10, 2013. The results of the examination after this lecture (exam 2013) were compared with the results after the lecture without using animation in 2012 (exam 2012). The simulation was performed by 97 trainees divided into 9 teams, and the surveys were conducted. RESULTS: The risk factors for early outcome of the recipients were cold ischemia time (≥ 10 hours), Model for End-stage Liver Disease score (≥ 20), and donor age (≥ 55 years). Results of examination showed that overall percentage of the correct answers was significantly higher in exam 2013 than in exam 2012 (48.3% vs 32.7%; P = .0001). The survey after the simulation of multiorgan procurement revealed that most trainees thought that the simulation was useful and should be continued. CONCLUSION: The novel educational program could allow young surgeons to make precise assessments and perform the exact procedure in the multiorgan procurement.

Interleukin-32 production associated with biliary innate immunity and proinflammatory cytokines contributes to the pathogenesis of cholangitis in biliary atresia.

Biliary atresia (BA) is thought to be associated with infections by viruses such as Reoviridae and is characterized histologically by fibrosclerosing cholangitis with proinflammatory cytokine-mediated inflammation. Interleukin (IL)-32 affects the continuous inflammation by increasing the production of proinflammatory cytokines. In this study, the role of IL-32 in the cholangitis of BA was examined. Immunohistochemistry for IL-32 and caspase 1 was performed using 21 samples of extrahepatic bile ducts resected from BA patients. Moreover, using cultured human biliary epithelial cells (BECs), the expression of IL-32 and its induction on stimulation with a Toll-like receptor [(TLR)-3 ligand (poly(I:C)] and proinflammatory cytokines was examined. BECs composing extrahepatic bile ducts showing cholangitis expressed IL-32 in BA, but not in controls. Caspase 1 was expressed constantly on BECs of both BA and control subjects. Furthermore, poly(I:C) and proinflammatory cytokines [(IL-1ß, interferon (IFN)-γ and tumour necrosis factor (TNF)-α] induced IL-32 expression strongly in cultured BECs, accompanying the constant expression of TLR-3 and caspase 1. Our results imply that the expression of IL-32 in BECs was found in the damaged bile ducts of BA and induced by biliary innate immunity via TLR-3 and proinflammatory cytokines. These findings suggest that IL-32 is involved initially in the pathogenic mechanisms of cholangitis in BA and also plays an important role in the amplification and continuance of periductal inflammatory reactions. It is therefore tempting to speculate that inhibitors of IL-32 could be useful for attenuating cholangitis in BA.

Prenatal diagnosis and management of congenital subglottic stenosis associated with congenital esophageal atresia type C.

Most patients with congenital esophageal atresia (EA) have congenital tracheobronchial abnormalities, which may cause respiratory distress, be difficult to treat and have a poor prognosis. The authors report a neonate with EA and congenital subglottic stenosis (SGS) who exhibited severe respiratory distress immediately after birth. After emergency endotracheal intubation with a narrow endotracheal tube, the authors performed total correction of EA and anterior cricoid split (ACS) on day 1 of age. The postoperative course was uneventful. Some reports have stated that it is difficult to make a prenatal diagnosis when SGS is associated with EA and tracheoesophageal fistula (TEF). The anterior cricoid split technique may be suitable for managing moderate SGS even in neonates with EA. Partial resection of the hypertrophic cricoid cartilage is considered effective in preventing restenosis.

Revised value of the eighth-order contribution to the electron g-2.

The contribution to the eighth-order anomalous magnetic moment (g-2) of the electron from a set of diagrams without closed lepton loops is recalculated using a new FORTRAN code generated by an automatic code generator. Comparing the contributions of individual diagrams of old and new calculations, we find an inconsistency in the old treatment of IR subtraction terms in two diagrams. Correcting this error leads to the revised value -1.9144(35)(alpha/pi)4 for the eighth-order term. This theoretical change induces the shift of the inverse of the fine structure constant by -6.411 80(73)x10(-7).

New determination of the fine structure constant from the electron value and QED.

Quantum electrodynamics (QED) predicts a relationship between the dimensionless magnetic moment of the electron (g) and the fine structure constant (alpha). A new measurement of g using a one-electron quantum cyclotron, together with a QED calculation involving 891 eighth-order Feynman diagrams, determine alpha(-1)=137.035 999 710 (96) [0.70 ppb]. The uncertainties are 10 times smaller than those of nearest rival methods that include atom-recoil measurements. Comparisons of measured and calculated g test QED most stringently, and set a limit on internal electron structure.

The expression of intestinal CYP3A4 in the piglet model.

PURPOSE: In our previous study, the blood levels of tacrolimus were higher in the short bowel and small bowel transplantation models than those in controls. Metabolism by intestinal cytochrome p450 3A4 (CYP3A4) has been reported to influence the blood level of orally administered tacrolimus. We performed immunohistochemistry to examine the localization of intestinal CYP3A4. MATERIALS AND METHODS: Twenty-four piglets were divided into three groups: group 1 were controls (n = 11); group 2, ileal resection of 2/3 proximal small intestine (n = 5); and group 3, total small intestinal resection followed by 1/3 allotransplantation of ileum (n = 8). Tacrolimus was orally administered from postoperative days 3 to 7. On day 7, blood samplings were performed for the trough level and the calculation of area under the concentration time curve (AUC). Intestinal specimens from the jejunum, ileum, colon, and ileal graft were obtained on days 0 and 10 for immunohistochemistry of CYP3A4. RESULTS: The trough and AUC values in group 2 were significantly higher than those in group 1. Furthermore those measurements in group 3 animals were higher than in group 2 (trough levels: 2.5 +/- 1.7, 11.2 +/- 2.1, 16.3 +/- 2.7 ng/mL and AUC values: 126 +/- 90, 319 +/- 155, 546 +/- 117 ng. h/mL). The expression levels of CYP3A4 were, jejunum > ileum > ileal graft > colon. CONCLUSIONS: The intensity and extent of CYP3A4 staining diminished in the ileal graft showing an inverse correlation to the blood concentrations.

The colon displays an absorptive capacity of tacrolimus.

PURPOSE: In our previous study, blood levels of orally administered tacrolimus were significantly higher in short bowel piglets than in control animals. It has been reported that the blood levels of tacrolimus are influenced by the metabolic activity of intestinal CYP3A4. If tacrolimus may be absorbed in the colon, direct administration of drug into this organ might be useful to augment bioavailability since the expression of CYP3A4 is low at this site. In the present study we evaluated the absorptive capacity of tacrolimus in the colon. MATERIALS AND METHODS: Piglets were divided into four groups: groups 1 and 2 were controls (n = 11 and 3, respectively); group 3 underwent resection of the entire small intestine (n = 8); and group 4 had a catheter placed in the cecum (n = 4). In groups 1 and 3, tacrolimus was administered orally; in group 2 it was given intravenously; and in group 4 it was injected into cecum through the catheter from postoperative days 3 to 7. On day 7, blood samples were obtained for drug measurements to calculate the area under the concentration time curve (AUC) values. RESULTS: The trough level and AUC values of tacrolimus in group 4 as well as in group 3 were significantly higher than those in group 1. In Group 4 animals showed a 60-minute time to peak concentration. CONCLUSIONS: Tacrolimus is absorbed by the colon. Since the blood levels were not influenced by the metabolic activity in the graft bowel, direct administration of tacrolimus into the colon might be useful in small intestinal transplantation.

Revised alpha4 term of lepton g-2 from the Feynman diagrams containing an internal light-by-light scattering subdiagram.

The alpha(4) contribution to the lepton g-2 from a gauge-invariant set of 18 Feynman diagrams containing a light-by-light scattering subdiagram internally has been reevaluated by a method independent of the previous approach. Comparison of two methods revealed a program error in the first version. Correcting this error, the contributions of these 18 diagrams become -0.990 72(10)(alpha/pi)(4) and -4.432 43(58)(alpha/pi)(4) for the electron and muon g-2, respectively. The correction is not large enough to affect the comparison between theory and experiment for the muon g-2, but it does alter the inferred value for the fine structure constant alpha(-1) by 6 ppb.

Clinical significance of c-kit expression in biliary atresia.

The aim of the present study was to examine the clinical significance of c-kit expression in biliary atresia (BA) using formalin-fixed, paraffin-embedded sections from 21 patients with BA. Patients were divided into group I (n = 8) with good liver function; group II (n = 8) with moderate liver dysfunction; and group III (n = 5) with severe liver dysfunction. Choledochal cysts (CDC, n = 5) and normal liver samples (NL, n = 4) served as controls. The results were analyzed and compared among the groups. Most c-kit+ cells were present in the portal tracts, and their numbers in BA were significantly higher than in the controls (11.12 +/- 1.64 vs 2.15 +/- 0.15 [mean +/- standard error], P = 0.02, BA vs CDC; 11.12 +/- 1.64 vs 1.66 +/- 0.52, P = 0.03, BA vs NL). Clinical correlation revealed a significantly higher number of c-kit+ cells in group III versus group I (18.10 +/- 3.62 vs 8.86 +/- 2.51, P = 0.02). These results suggest that c-kit overexpression is associated with an adverse clinical outcome in BA.

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

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.

Cytokeratin subtypes in biliary atresia: immunohistochemical study.

The etiology of biliary atresia (BA) remains unknown, but ductal-plate malformation and insufficient ductal-plate remodeling have been suggested to play important roles, so it is beneficial to examine the maturation and differentiation of bile ducts in BA. Different epithelial types are characterized by the expression of specific cytokeratin (CK) subtypes. CK can therefore serve as a 'lineage marker' of epithelial cells. CK subtypes have not been previously examined in BA. In this study, we examined the maturation of bile-duct cells in BA (n = 45) using immunohistochemistry of CK subtypes, with mouse monoclonal antibodies to CAM5.2, and CK subtypes 7, 8, 13, 14, 17, 19 and 20. We then compared these findings with pediatric non-BA (n = 11) and fetal (n = 21) liver. We semiquantitatively evaluated the findings using a H score method. In the fetal liver, immunoreactivity for CAM5.2, CK-7, CK-8 and CK-19 was detected in bile-duct cells, and CAM5.2 and CK-8 immunoreactivity was also detected in hepatocytes. The distribution of these CK subtypes was the same in fetal, pediatric non-BA and BA liver. However, CK-7 immunoreactivity was markedly weaker in bile ducts of fetal (H scores: ductal plate 0 +/- 0; remodeling 9.5 +/- 40.3; remodeled 37.3 +/- 60.8) and BA (H score: 200.9 +/- 55.3) liver compared to non-BA liver (H score: 251.1 +/- 33.5). In addition, CK-20 was detected in the bile ducts of the fetal and BA liver, but not in non-BA liver. These findings suggest that the expression patterns of CK subtypes in bile-duct cells in BA are similar to that in developing bile-duct cells, which is indicative of bile-duct cell immaturity.

Survival patterns in biliary atresia and comparison of quality of life of long-term survivors in Japan and England.

BACKGROUND/PURPOSE: Portoenterostomy is an accepted method of achieving bile drainage in biliary atresia, but there is a paucity of data, including formal quality-of-life (QoL) studies, on long-term survivors. This report includes survival analysis and QoL studies from the world's largest series of cases treated in Japan (1951 to 1998). The Japanese QoL results are compared with a matched group of UK patients from King's College Hospital, London. METHODS: One hundred fifteen Japanese surviving portoenterostomy patients were studied and comparison of trends in survival calculated from 6-year period cohorts. Liver function and hematologic status in a group of 30 long-term survivors (14 to 24 years) were compared with 25 patients from England, (14 to 23 years). Twenty-five Japanese and 21 UK patients (SF-36) completed a QoL questionnaire. RESULTS: Median survival times in Japanese patients before 1975 were less than 1 year but increased to 18 years after 1975. Hematologic and liver function test results did not show any significant differences between the Japanese and UK patients. QoL studies in the UK patients showed no significant difference from normative, general population data. Japanese patients underperformed in general health (P = .01), role emotional (P = .05) and role physical (P = .07) but, overall, there was no significant difference between the Japanese and UK patients except for marginal differences in indices of general health and vitality (P = .06 and .04, respectively). CONCLUSIONS: Long-term survival rate in the Japanese patients increased dramatically from 1 year to 17 years after 1975. The QoL of survivors was comparable in Japan and England. The satisfactory comparison with normative population data suggests that we should continue to use portoenterostomy as the primary treatment for biliary atresia. J Pediatr Surg 36:892-897.

CD8+ T cells infiltrating into bile ducts in biliary atresia do not appear to function as cytotoxic T cells: a clinicopathological analysis.

It is speculated that immune mechanisms are involved in bile duct damage in biliary atresia (BA), as in primary biliary cirrhosis (PBC). In BA, however, no reports have described the in situ distribution of cytotoxic T lymphocytes (CTLs) using specific markers, nor has the clinical association been clarified. The present study describes the immunohistochemical distribution of CD8+ T cells and the relevant markers [perforin, granzyme B, FasL (CD95L)] in 47 cases of BA operated upon at days 12-79. The results were compared with those of PBC. In BA, CD8+ T cells infiltrated bile ducts in a way similar to that observed in PBC. However, in sharp contrast to PBC, none of the inflammatory cells infiltrating into the bile ducts in BA expressed cytotoxic markers such as perforin, granzyme B, or Fas ligand (FasL). Clinical follow-up of patients with BA revealed that a greater degree of infiltration of bile ducts by CD8+ T cells is associated with better liver function. Taken together, these data suggest the absence of direct CTL activity against bile ducts in BA in the postnatal period.

Problems during and after pregnancy of former biliary atresia patients treated successfully by the Kasai procedure.

BACKGROUND/PURPOSE: The aim of this study was to investigate the problems and the quality of life during and after pregnancy of the patients who had undergone Kasai operation and to find out a strategy for follow-up during the period of their pregnancy. METHODS: A questionnaire was sent to 134 institutions of the Japanese Biliary Atresia Society with the following questions: (1) Do you have any pregnancy cases in patients who had undergone Kasai operation? (2) Did she have any menstrual problem? (3) Did she have any problem during pregnancy and delivery? (4) Did she have any change in liver function tests after delivery? (5) Did she have any early and long-term problem after delivery? (6) Did the baby have any problem? (7) Was there any special care or comment about the pregnancy of the biliary atresia patients? The responses were analyzed. RESULTS: Fourteen institutions reported 16 cases of pregnancy, 23 cases of delivery, and 2 cases of abortion. The causes of abortion in the 2 cases were attributed to hemorrhagic shock after massive bleeding from esophageal varices and serious atopic dermatitis, respectively. Other problems during pregnancy were abruption of placenta, fetal distress leading to caesarian section, and development of liver dysfunction leading liver transplantation. Problems after delivery included deterioration of liver function in 6 patients (37.5%), attacks of ascending cholangitis in 4 patients (25.0%), and severe fatigue with liver dysfunction from nursing the baby leading to liver transplantation. Only 3 of 16 (18.8%) patients were free of any problems. No abnormality was seen in the babies. CONCLUSIONS: Even if the patients with biliary atresia lead a good postoperative course, unexpected complications can occur when they become pregnant. Close long-term follow-up is required for proper management of pregnancy in biliary atresia patients.

In situ CD14 expression in biliary atresia: comparison between early and late stages.

PURPOSE: The aim of this study was to compare the in situ expression of CD14 between early and late stage of biliary atresia (BA) to determine if a time course of CD14 expression exists in BA. METHODS: Immunohistochemical analysis of membrane-bound CD14 expression was carried out in periodate-lysine-paraformaldehyde (PLP)-fixed frozen sections from 9 early- (obtained during Kasai procedure) and 6 late- (obtained during liver transplantation) stage cases of BA. Normal liver (n = 3) and choledochal cysts (n = 5) served as normal controls and disease controls respectively. RESULTS: In the early stage, 6 patients (66.66%) showed extensive CD14 expression (grade 3 [G(3)], more than 50% positive cells), whereas no CD14-positive cells could be detected in 4 patients (66.66%) in the late stage. In both stages, most of the positive cells were observed in the parenchyma of the hepatic lobules where Kupffer cells and sinusoidal endothelial cells stained positive. Arterial and venous endothelium, bile duct cells, and hepatocytes were negative for CD14. Double immunohistochemistry in the early stage showed a higher colocalization rate of CD14 and CD68 in the sinusoidal locations (33.69 +/- 9.270% [mean +/- SEM]) than in the portal tract (7.6+/-4.64% [mean +/- SEM]; P<.05). Similar pattern of colocalization also was observed in the late stage. In the normal controls no expression of CD14 could be detected, whereas in the disease controls only 1 case showed mild expression (grade 1 [G(1)], 1% to 10% positive cells) and the rest showed no expression of CD14. CONCLUSION: These results suggest that CD14 expression in BA is a dynamic phenomenon having time-related change with overexpression in the early stage and reduced expression in the late stage.

E-cadherin, alpha-catenin and beta-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 alpha- and beta-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 alpha- and beta-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 alpha- and beta-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.