Evidence for a gene controlling the induction of transplantation tolerance.

Class I mismatched kidney transplantation in Massachusetts General Hospital MHC-defined miniature swine has been studied extensively as a model for induction of systemic allograft tolerance. In a large series of juvenile swine, long-term graft acceptance has been observed consistently following a 12-day course of cyclosporine. It was therefore surprising when three of five recipients in one of our studies rejected their grafts. Examination of the origins of the rejecting animals revealed that they were derived from a subline of the SLA(dd) miniature swine herd that was intentionally being inbred toward full homozygosity and had been inbred for eight generations prior to these experiments. A blinded study of additional class I mismatched renal transplants into animals from this subline confirmed the genetic basis of this rejection. We present here preliminary evidence suggesting that a likely explanation for this phenomenon is that the rejectors in this subline are homozygous for a recessive mutant allele of a gene normally involved in the induction of tolerance. Subsequent studies will be directed toward identification and characterization of the gene(s) involved, since existence of a similar genetic locus in humans might have implications for assessing an individual's likelihood of graft rejection versus tolerance induction prior to organ transplantation.

Predictors of organ allograft tolerance following hematopoietic cell transplantation.

Using the miniature swine large animal model we have attempted to determine the relationship between tolerance and the presence of donor cells in the bone marrow, thymus and lineages of peripheral blood in a series of hematopoietic cell transplant recipients receiving delayed donor allografts without immunosuppression. Twenty-two animals receiving hematopoietic cell transplantation and a delayed organ allograft were analyzed. Assays for presence of donor CFUs in bone marrow (by PCR), thymic chimerism (by FACS and PCR/Southern Blot), peripheral blood chimerism (by FACS), and in vitro responsiveness to donor MHC were performed. Presence of donor BM CFUs, thymic chimerism and multilineage peripheral blood chimerism at the time of organ transplantation all correlated precisely with subsequent allograft tolerance (p < 0.001, p < 0.001, p < 0.005 respectively). These parameters were therefore accurate predictors (Positive Predictive Value (PPV) = 100% in all) of tolerance. In vitro assays of responsiveness were also highly associated (p < 0.002, p < 0.002 respectively), but were not as accurate predictors of subsequent organ tolerance (CML PPV = 80%). Engraftment, as indicated by the presence of donor derived CFU in the bone marrow, detectable thymic chimerism and multilineage peripheral blood chimerism are reliable predictors of subsequent donor allograft acceptance in hematopoietic cell transplant recipients.

Laparoscopic antireflux surgery for the treatment of esophageal strictures refractory to medical therapy.

OBJECTIVE: The response of esophageal strictures to laparoscopic antireflux surgery remains controversial. The aim of this study was to examine the outcome of patients with medically refractory esophageal strictures caused by severe gastroesophageal reflux disease and treated surgically. METHODS: A prospective follow-up analysis was completed using data obtained from detailed specific questioning by an independent observer. Responses were rated for symptoms, dysphagia (range 1-19), satisfaction with treatment, well-being (1 = best, 10 = worst), and need for further therapy. RESULTS: Of 102 patients, 74 (72.5%) responded to follow-up. There were 31 women, mean age 59.6 yr, and 43 men, mean age 55.2 yr. Mean follow-up was 25 months (range 4-68 months). A total of 252 dilations before surgery decreased to 29 after surgery (p < 0.0001) in the mean observation period of 26 months before and 25 months after surgery (mean/patient 5.3 and 1.8, respectively, p < 0.001). The mean dysphagia score was 6.8 +/- 3.6 preoperatively and 3.7 +/- 1.4 postoperatively (p < 0.0001). Nine (12%) patients required continuous postoperative H2-blockers or proton pump inhibitors. Seven of these had gastritis or peptic ulcer disease. Before antireflux surgery, 10 (13.5%) had frequent pneumonia. No pneumonia was observed after surgery. Sixty-eight (91.9%) patients were satisfied with their decision to have surgery. Among these, the well-being score was 1.8 +/- 0.4 postoperatively vs 5.5 +/- 1.2 (p < 0.001) preoperatively. CONCLUSIONS: Laparoscopic surgery in patients with medically refractory esophageal strictures results in a good clinical outcome with minimal complications. Patients are very satisfied with relief of dysphagia, and there is a diminished need for further dilation, with good quality of life.

Radioimmunoassay of regulatory peptides in the presence of acetonitrile: marked improvement of cholecystokinin assays.

Radioimmunoassay has made it possible to measure the levels of many hormones. However, samples for some hormones, such as cholecystokinin (CCK), need to be purified by reverse phase chromatography before assay. Usually, samples are eluted from cartridges or HPLC columns in about 50% acetonitrile, dried on a vacuum centrifuge, and then reconstituted in buffer. Drying and reconstituting samples is time consuming and introduces additional sources of error and peptide loss. The present study investigated the effect of acetonitrile on radioimmunoassays for CCK to see if samples containing acetonitrile could be assayed directly. The non-specific binding of a radiolabeled peptide, the zero binding (B0), and the fall in the presence of 2.5 fmol unlabeled CCK were determined in the presence of various proportions of acetonitrile with 0.1% TFA. Additionally, standard curves were compared in the presence and absence of 200microl of 50% acetonitrile, (n = 5). For assays using two separate CCK antisera, increasing amounts of acetonitrile gave progressively higher zero binding and fall, thereby increasing sensitivity and antibody titer. The use of 200microl 50% acetonitrile, chosen to represent typical sample conditions, increased antiserum titers by three to four-fold, as well as increasing sensitivity considerably. For one antiserum (CCK2), the IC20 was 0.36+/-0.02 fmol CCK/tube in the presence of acetonitrile and 1.45+/-0.08 fmol/tube in its absence (P< 0.001). For the other antiserum (Dino 7), the IC20 was 0.40+/-0.02 fmol CCK/tube in the presence of acetonitrile and 0.63+/-0.01 fmol/tube in its absence (P<0.001). A similar increase in sensitivity was seen with a gastrin assay. However, no significant change in the gastrin antibody titer was evident. Assays for several other hormones were unaffected by 200 microl of 50% acetonitrile. At volumes encountered in samples following chromatography, acetonitrile did not adversely affect radioimmunoassays for a number of hormones, and the sensitivity and antibody titer of the CCK assays were improved. Measurement of CCK samples without drying and reconstitution increases assay efficiency and sensitivity.

Effects of high-fat diet and cholecystokinin receptor blockade on promotion of pancreatic ductal cell tumors in the hamster.

The mechanism by which high-fat diets potentiate pancreatic cancer is not known, but pancreaticotrophic hormones such as cholecystokinin (CCK) may be involved. The effect of CCK receptor blockade on carcinogenesis during the entire promotion period was investigated in Syrian Golden hamsters fed a high- or low-fat diet and treated with N-nitrosobis(2-oxopropyl)amine (3 x 10 mg/kg at weekly intervals). One-half of the hamsters fed a high-fat diet received the CCK-A receptor antagonist devazepide (25 nmol/kg/hr) for the duration of the experiment. At 39 weeks the incidence of pancreatic malignancies was significantly higher in hamsters fed the high-fat diet than in those fed the low-fat diet (p < 0.05). Tumor incidence was not changed by CCK receptor blockade. Potentiation of pancreatic cancer by a high-fat diet in hamsters does not appear to be influenced by endogenous CCK during the tumor promotion period.