Yuichi Iwaki

CADAVERIC SMALL BOWEL AND SMALL BOWEL–LIVER TRANSPLANTATION IN HUMANS

Five patients had complete cadaveric small bowel transplants under FK506 immunosuppression, one as an isolated graft and the other 4 in continuity with a liver. Three were children and two were adults. The five patients are living 2-13 months posttransplantation with complete alimentation by the intestine. The typical postoperative course was stormy, with sluggish resumption of gastrointestinal function. The patient with small intestinal transplantation alone had the most difficult course of the five, including two severe rejections, bacterial and fungal translocation with bacteremia, renal failure with the rejections, and permanent consignment to renal dialysis. The first four patients (studies on the fifth were incomplete) had replacement of the lymphoreticular cells in the graft lamina propria by their own lymphoreticular cells. Although the surgical and after-care of these patients was difficult, the eventual uniform success suggests that intestinal transplantation has moved toward becoming a practical clinical service.

Multiscreen Serum Analysis Of Highly Sensitized Renal Dialysis Patients For Antibodies Toward Public And Private Class I Hla Determinants: Implications For Computer-predicted Acceptable And Unacceptable Donor Mismatches In Kidney Transplantation

A multiscreen serum analysis program has been developed that permits a determination of antibody specificity for the vast majority of highly sensitized patients awaiting transplantation. This program is based on a 2 x 2 table analysis of correlations between serum reactivity with an HLA-typed cell panel and incorporates two modifications. One implements the concept of public HLA determinants based on the serologic crossreactivity among class I HLA antigens. The other modification derives from the premise that most highly sensitized patients maintain the same PRA and antibody profiles over many months and even years. Monthly screening results for patients with persistent PRA values can therefore be combined for analysis. For 132 of 150 highly sensitized patients with >50% PRA, this multiscreen serum analysis program yielded information about antibody specificity toward public and private class I HLA determinants. The vast majority of patients (108 of 112) with PRA values between 50 and 89% showed antibody specificity generally toward one, two, or three public markers and/or the more common private HLA-A, B antigens. For 24 of 38 patients with >90% PRA, it was possible to define one or few HLA-specific antibodies. The primary objective of the multiscreen program was to develop an algorithm about computer-predicted acceptable and unacceptable donor HLA-A, B antigens for patients with preformed antibodies. A retrospective analysis of kidney transplants into 89 highly sensitized patients has demonstrated that allografts with unacceptable HLA.A-A, B mismatches had significantly lower actuarial survival rates than those with acceptable mismatches (P = 0.01). This was shown for both groups of 32 primary transplants (44% vs. 67% after 1 year) and 60 retransplants (50% vs. 68%). Also, serum creatinine levels were significantly higher in patients with unacceptable class I mismatches (3.0 vs. 8.4 mg% [P = 0.007] after 2 weeks; 3.9 vs. 9.1 mg% [P = 0.014] after 4 weeks). Histopathologic analysis of allograft tissue specimens from 47 transplant recipients revealed a significantly higher incidence of humoral rejection (P = 0.02), but not cellular rejection, in the unacceptable mismatch group. These results suggest that the multiscreen program can establish which donor HLA-A.B mismatches must be avoided in kidney transplantation for most highly sensitized patients. For 18 of 150 high PRA renal dialysis patients, the multiscreen program could not define HLA-specific antibody. Most patients had >90% PRA, and many of their sera appeared to contain IgM type nonspecific lymphocytotoxins that could be inactivated by dithioerythreitol (DTE). Preliminary studies have shown that this treatment enabled the detection of HLA-specific antibodies upon subsequent screening on many occasions. These data suggest that non-HLA specific reactivity revealed by multiscreen analysis can often be removed by DTE treatment. Multiscreen analysis offers an attractive approach to regional organ-sharing programs for highly sensitized renal transplant candidates. It enables the development of an efficient strategy for donor selection based on the computer assignment of acceptable HLA-A, B mismatches for each patient.

Platinum Nanoflowers on Scratched Silicon by Galvanic Displacement for an Effective SALDI Substrate

We report a new and facile method for synthesizing 3D platinum nanoflowers (Pt Nfs) on a scratched silicon substrate by electroless galvanic displacement and discuss the applications of the Pt Nfs in surface-assisted laser desorption/ionization-mass spectrometry (SALDI-MS). Surface scratching of n-type silicon is essential to induce Pt Nf growth on a silicon substrate (to obtain a Pt Nf silicon hybrid plate) by the galvanic displacement reaction. The Pt Nf silicon hybrid plate showed excellent SALDI activity in terms of the efficient generation of protonated molecular ions in the absence of a citrate buffer. We propose that the acidity of the Si-OH moieties on silicon increases because of the electron-withdrawing nature of the Pt Nfs; hence, proton transfer from the Si-OH groups to the analyte molecules is enhanced, and finally, thermal desorption of the analyte ions from the surface occurs. Signal enhancement was observed for protonated molecular ions produced from a titania nanotube array (TNA) substrate on which Pt nanoparticles had been photochemically deposited. Moreover, surface modification of the Pt Nf silicon hybrid plate by perfluorodecyltrichlorosilane (FDTS) (to obtain an FDTS-Pt Nf silicon hybrid plate) was found to facilitate soft SALDI of labile compounds. More interestingly, the FDTS-Pt Nf silicon hybrid plate acts 1) as a high-affinity substrate for phosphopeptides and 2) as a SALDI substrate. The feasibility of using the FDTS-Pt Nf silicon hybrid plate for SALDI-MS has been demonstrated by using a β-casein digest and various analytes, including small molecules, peptides, phosphopeptides, phospholipids, carbohydrates, and synthetic polymers. The hybridization of Pt Nfs with a scratched silicon substrate has been found to be important for achieving excellent SALDI activity.

Risk factors for short- and long-term survival of primary cadaveric renal allografts in pediatric recipients: a UNOS analysis.

Background. Pediatric kidney graft survival rates have improved in the United States. This study evaluates early and late risk factors for cadaveric graft loss in pediatric recipients. Methods. From January 1994 to December 2002, 2,597 primary cadaveric kidney-alone transplants (donor age 5-45 years, recipient age 2–20 years) were reported to the United Network for Organ Sharing (UNOS). The analysis includes follow-up information based on OPTN data as of October 14, 2003. Odds ratio of early graft loss and relative risk of late graft loss are estimated using logistic regression and Cox proportional hazards model, respectively. Results. Graft survival rates significantly improved during 1999–2002 (95% and 79% at 1-year and 3-years, respectively) compared with those of 1994–1998 (88% and 76% at 1-year and 3-years, respectively) (log rank P = 0.02). After adjusting for other variables, the factors that significantly affected early transplant outcome adversely within 3 months posttransplant were prolonged cold ischemia time (>36 hours, odds ratio [OR] = 3.38 vs. 0–36 hours) and young recipient age (2–5 years old, OR = 2.02 vs. 6–12 years). Beyond 3 months, significant risk factors were African-American recipients (relative risk [RR] = 1.93 vs. others), teenage recipients (13–20 yrs, RR = 1.50 vs. 6–12 yrs), and patients with focal glomerulosclerosis (FGS) (RR = 1.27 vs. others). Conclusions. The short-term graft survival rate of pediatric cadaveric kidney transplants has significantly improved, yet the long-term outcome has changed little. The long-term outcomes for teenagers (13–20 yrs), patients with FGS, and African-Americans lag significantly behind other groups. In order to improve long-term graft survival in these high-risk patients, newer preventive or treatment strategies must be developed.

Liver transplantation in positive cytotoxic crossmatch cases using FK506, high-dose steroids, and prostaglandin E1.

Although the liver is resistant. to antibody-mediated rejection (1), a significant adverse effect has been reported of its transplantation into recipients with preformed cytotoxic antibodies (2). We report here a strategy that appears to convert the prognosis in such potentially disadvantaged patients to essentially the same if not better than that in conventionally treated crossmatch-negative recipients. The crossmatch status was determined with recipient sera treated with dithiothreitol (DTT) (3). Four cohorts of adult liver recipients were studied. In all, intravenous FK506 was started with doses of 0.075 to 0.15 mg/kg/day, and converted to oral dosing after resumption of diet. Treatment was guided by plasma FK506 levels (target approximately 1 ng/ml), toxicity (primarily renal), the clinical status of the patient, and the quality of postoperative liver function (4). The 4 study groups (Table 1) differed in the cytotoxic crossmatch state (negative in group 1 only), the use of prostaglandin E1 (group 4 only), and the prophylactic use of methylprednisolone (MP). Low-dose MP was with 20 mg/day starting on the day of transplantation (most of group 1 and all of group 2). High-dose MP was with 1 g i.v. during operation, followed by a “burst” with 200 mg the first day and daily decreases of 40 mg until 20 mg/day was reached on the 6th day if the course was uneventful (groups 3 and 4). In group 4, i.v. PGE1 (Prostin VR) was started after completion of the operation with 0.2 µg/kg/hr and gradually increased to 0.6 µg/kg/hr if tolerated without cardiovascular instability. One of the 14 patients in group 4 had transient hypotension. There were no other side effects of PGE1 which was stopped without weaning when the patients resumed diet after 5–7 days. TABLE 1 T lymphocytotoxic crossmatch state The crossmatch positive cases in groups 2–4 were consecutive, with no omissions. The incidences of hepatic malignancy, high UNOS urgency scores (50–78% III or IV), and cytotoxic titers > 1:8 were similar in the cytotoxic groups 2–4. Females were disproportionately represented in all of the crossmatch-positive groups compared with crossmatch-negative controls. In the crossmatch-positive cases, the outcome was remarkably different according to treatment (Table 1). With high-dose steroid therapy (group 3), the unacceptably high early and delayed graft loss seen with low-dose steroid therapy (group 2) was reduced by two-thirds. Graft loss was reduced further if PGE1 was added to the high-dose steroid regimen (group 4). The only death in the 14 patients of group 4 was caused by a biliary leak and subhepatic infection in a patient whose native gall bladder had empyema. The favorable course of the group 4 recipients relative even to group 3 and especially group 2 patients was reflected by an improvement in the perioperative renal function (Fig. 1), and superior postoperative liver function (Fig. 2). FIGURE 1 First-week FK506 doses and plasma concentrations and serum creatinine (Cr) in crossmatch-positive liver recipients (groups 2, 3, 4). (Cr)—group 2 vs. 3 P<0.0001, 2 vs. 4 P<0.0001, 3 vs. 4 = P = 0.007. FK506 dose—2 vs. 3 ... FIGURE 2 Serum total bilirubin (1st month) and serum GOT levels (2 weeks) in crossmatch-positive liver recipients. Bilirubin in first week—group 2 vs. 3 P = 0.007, 2 vs. 4 P<0.01, 3 vs. 4 P=0.36. SGOT in first week—group 2 vs. 3 P=0.36, ... The superior renal function in the liver recipients of group 4 was noteworthy. It is possible the PGE1 specifically protects the kidneys from FK506 nephrotoxicity by a mechanism similar to that described by Makowka et al. (5) for cyclosporine. However, protection of the liver graft from antibody-mediated rejection and consequent avoidance of prerenal complications may have been a significant or even more important factor. The pathogenesis of hyperacute rejection of kidney grafts, and the much slower manifestations of antibody-mediated rejection seen in liver grafts are not fully understood (6). Whatever the mechanisms, it has been known since the earliest work of Dempster (7) that intense vasoconstriction occurs of the graft microvasculature (8). PGE1 treatment added to FK506 and high doses of steroids could have ameliorated this consequence, aside from its inherent immunosuppressive (9) and cytoprotective (10) qualities. Amelioration by PGE1 of humoral rejection has not been reported clinically, but there have been clues of its potential value in xenograft transplant models that have heterospecific antibody barriers, including cat-to-dog (11), hamster-to-rat (12), and pig-to-dog (13). High-dose prednisone in conjunction with FK506 but without PGE1 also was effective. The steroid mitigation of humoral rejection presumably was by different pathways although both glucocorticoids and PGE suppress cytokines (14, 15). Accurate delineation of these drug actions and interactions will be of the greatest interest in providing clues for other therapeutic improvements. At a practical level, the experience reported herein indicates not only the feasibility but also the surprising ease with which the liver can be transplanted to sensitized recipients. If optimum therapy is given, a policy of excluding patients with widely reacting cytotoxic antibodies from candidacy for liver transplantation would seem unwarranted. The results in the high-dose steroid-PGE1 group 4 were at least as good as in the cases with negative crossmatches. Whether all liver transplantations, including those with crossmatch-negative donors should be given this treatment deserves consideration. PGE1 already has been used in this way in cyclosporine-based drug cocktails for crossmatch-negative kidney transplantation (16). There were no obvious artifacts that would undermine these conclusions, such as the use in later cases of a different titer cutoff for a positive-crossmatch reading. About the same percentage of titers greater than 1:8 were in groups 2–4, but the highest positive titer was not measured systematically at the time of the case accrual. In 6 subsequent patients who were given the optimal recommended treatment and who had titers equal to or greater than 1:256, only 1 had a humoral rejection. This was a female patient with a titer of 1:1024 who required retransplantation 2 days after primary graft nonfunction, using a crossmatch-positive donor liver (titer 1:2) on the second occasion. She is well 4 months later. The influence of titers, different sensitization schedules, and the nature of the antibody upon humoral rejection of the liver in rats has been described elsewhere.4 These experimental studies, combined with our clinical experience herein reported, have led to our present policy of not delaying urgently needed liver transplantation in order to search for a more appropriately crossmatched donor.

Thoracic duct fistula and renal transplantation.

Thoracic duct drainage (TDD) was established for 21 – 115 days in 40 kidney recipients with an average removal per patient day of 4.7 I lymph and 1.88 billion cells. Cellular and humoral immunity were depressed. TDD and immunosuppressive drugs were started at transplantation in 35 recipients of cross-match negative grafts. Although the results were better than in precedent non-TDD controls, eight patients rejected their grafts before a full TDD effect, and three of the eight developed predominantly anti-B lymphocyte cytotoxic antibodies which were probably responsible for positive cross-matches with their next donors. With continuing TDD, all eight patients had good initial function after early retransplantation. In five more “nontransplantable” patients with performed cytotoxic antibodies, TDD was started 30–56 days before transplantation. In these five pretreated patients, antibodies persisted with positive antidonor cross-matches. Hyperacute rejection occurred repeatedly in two patients with high anti-T (and anti-B) titers, but was surmounted in three patients with lower titers. From the clinical and immunologic data, we have concluded that TDD should be used for pretreatment of all cases with or without prior antibodies, and have suggested an adjustable management plan that takes into account new developments in antibody monitoring.

Evaluation of sequential serum interleukin-6 levels in liver allograft recipients

Control serum levels of IL-6 measured by ELISA in 30 healthy blood donors or volunteers were 18 +/- 34 pg/ml (mean +/- SD). Pretransplant serum levels of IL-6 in 169 adult candidates for liver transplantation were significantly higher than control in those with fulminant hepatitis (203 +/- 232 pg/ml), alcoholic cirrhosis (116 +/- 257 pg/ml), and hepatocellular carcinoma (82 +/- 105 pg/ml). With these data as background, plasma or serum levels of IL-6 were monitored in 24 adult patients after first OLT and correlated with the clinical courses and the histopathological diagnosis of rejection. Serum or plasma levels of IL-6 decreased after transplantation regardless of pretransplant value. Four patients with infection subsequently developed continuously high IL-6 values. In the 20 of 24 patients who did not have infection, significantly higher levels of IL-6 were consistently found 0-4 days before histopathological diagnosis of rejection (131 +/- 78 pg/ml) compared with significantly lower values in patients without rejection episodes (40 +/- 21 pg/ml). The elevations of IL-6 were spike shaped, did not correlate well with the histopathological grades of rejection, and were highly responsive to augmented immunosuppression. These 20 cases were classified as: group 1, no spikes of IL-6 after liver transplantation; group 2, single spike of IL-6 after liver transplantation; and group 3, multiple spikes of IL-6 after liver transplantation. The combined early and late graft loss of each group was 0% (group 1), 25% (group 2), and 67% (group 3). We conclude that daily monitored serum or plasma IL-6 levels can be a good premonitor of liver allograft rejection and also a useful predictor of long-term graft outcome.

B cell antibodies and crossmatching.

Over 200 cadaver donor transplants were crossmatched against T and B lymphocytes at 5 and 37 C. Of 54 positive B cell crossmatches, one-half had cold antibodies and one-half had warm antibodies. The 1-year transplant survival was 58% for B-cold-positive crossmatches and 42% for B-warm-positive crossmatches. The B-warm-positive crossmatches, when subdivided into those with strong HLA-DR antibodies showed a 27% 1-year transplant survival rate. We conclude that B cell-positive crossmatches with HLA-DR antibodies are deleterious and that those with cold anti-IgM antibodies may enhance graft survival.

HLA-DR Specifications in Japanese with Juvenile-onset Insulin-Dependent Diabetes Mellitus

Specific allelic associations vary among ethnic groups. We studied the distribution of HLA-A,-B,-C, and -DR antigens in 34 Japanese juvenile-onset diabetic patients. The focus of our current work was HLS-DR antigens because there have been few studies of Japanese with this disease. A significant increase in the frequency of HLA-DR4 was found in patients but not in unaffected persons: DR4 was found in 56.3% of the patients versus 32.6% of the unaffected persons. However, the negative correlation between DR2 and patients was not statistically significant.

Crossreactivity between human and canine Ia antigens using a mouse monoclonal antibody (CIA).

A mouse monoclonal antibody (CIA) produced against human Ia antigens that reacts with 20–30% of human peripheral blood lymphocytes was found to react with more than 90% of dog lymphocytes. Less than 41% of the la-positive dog lymphocytes expressed surface immunoglobulins. Immunoprecipitation studies with 125I- labeled cells precipitated heavily labeled Ia bands at 28K and 35K from human cells, but from dog cells the 29K β chain of Ia was much more heavily labeled than the α chain.