Unexpected anti-alpha GalNAc antibodies in alpha-galactosyl transferase-deficient mice: complex relationship between genotype and the natural antibody repertoire.

Mice lacking the alpha-galactosyl transferase gene (GalT(-/-) mice) have been used extensively as a model for xenotransplantation. Unlike wild type (WT) mice, GalT(-/-) mice do not produce Gal alpha 1-3Gal and are known to produce natural IgM specific for Gal alpha 1-3Gal, as do humans and higher primates. In addition to natural anti-Gal alpha 1-3Gal IgM in GalT(-/-) mice, we identified natural IgM which bound alpha-N-acetylgalactosamine (alpha GalNAc) but not Gal alpha 1-3Gal or blood group A. Although unexpected, these antibodies were expressed at 10-fold greater concentrations in GalT(-/-) mice than in WT mice. One explanation for this unexpected observation is that the production of natural antibodies is affected by self-antigen(s) that are similar but not identical to targets recognized by the natural antibody. Thus, the natural humoral immune system may be unresponsive to "near-self" antigens even though the individual is not tolerant to those antigens. Another explanation for the unexpected results is that there may be unanticipated and uncharacterized differences between GalT(-/-) mice and WT mice. These studies underscore the need to extensively characterize phenotypes in KO mice and indicate that the relationship between genotype and the natural immune repertoire can be complex.

A prospective evaluation of health-related quality of life after ileal pouch anal anastomosis for ulcerative colitis.

OBJECTIVES: The ileal pouch anal anastomosis is a safe and effective procedure but is also associated with pouchitis, small bowel obstruction, and incontinence. We prospectively evaluated the health-related quality of life using generic and disease-specific measures in a cohort of patients with ulcerative colitis undergoing ileal pouch anal anastomosis. METHODS: Health-related quality of life measures included the Time Trade-off, Rating Form of IBD Patient Concerns, and the Short-Form 36. Assessments occurred preoperatively and 1, 6, and 12 months postoperatively. RESULTS: Time Trade-off scores had significantly improved at the 1-month postoperative assessment and approached perfect health at the 12-month postoperative assessment. The Rating Form of IBD Patient Concerns revealed a significant reduction in patient concerns at 1 month, and this difference persisted at 6 and 12 months. Seven of the eight subscales of the Short-Form 36 revealed improved health-related quality of life postoperatively. CONCLUSIONS: Health-related quality of life improved after ileal pouch anal anastomosis when assessed with both generic and disease-specific measures. Improvements were observed as early as 1 month postoperatively. These results may guide patients and physicians as they consider and prepare for the impact of ileal pouch anal anastomosis.

Organ procurement organization (OPO), best practices.

There are currently 59 organ procurement organizations (OPOs) in the United States which serve their assigned geographic areas with variable productivity. Knowledge of organizational characteristics, programs and practices of more successful OPOs may be useful to increase the productivity of less successful OPOs. A preliminary survey of all OPO executive directors in the United States ascertained the most important beneficial and detrimental factors affecting their success. Site visits were then conducted at OPOs based on a selection process utilizing population size, geographic location, minority population, donors per million population and donors per thousand deaths among potential donors. All OPOs were categorized and the highest ranking OPOs in each of seven categories, based on 4 years of national data, were selected for the site visits. Regression analysis and correlation analysis using Pearson's product-moment correlation were performed. The survey to identify the important factors was returned by 47 (77%) of 61 OPOs existent in 1999. The most important beneficial factors identified by responding OPOs were adequate staffing and experience, allocation of responsibilities, hospital development and leadership. The most important detrimental factors were inadequate staffing and experience, poor donor hospital/transplant center/ OPO relationships and failure in the consent process. Site visits of the highest-ranking OPOs demonstrated all had respected, experienced leadership focused on the donation process; efficient mechanisms for resolving allocation or transplant center conflicts; systems for monitoring activity and tracking outcomes; excellent communication between OPO and transplant centers; open internal communication at all levels of the OPO; immediate, on-site response to vascular donor referrals; and volunteer support of public and/or professional education. Regression and correlation analysis demonstrated that as minority population increases, OPO performance declines (P < 0.03). Moreover, independent OPOs were associated with poorer performance regardless of minority population (P < 0.05). All of the successful OPOs visited had strong leadership, excellent donor hospital and transplant center relationships, well-developed communication and innovative methods to deal with their minority populations. Application of these practices within all OPOs could significantly enhance organ donation.

Natural anti-carbohydrate IgM in mice: dependence on age and strain.

Natural anti-carbohydrate antibodies in humans play a key role in natural immunity and in recognition of allogeneic and xenogeneic antigens. Presumably, natural anti-carbohydrate antibodies in mice have similar functions; but these antibodies have not been extensively characterized. An assay was developed and used to screen for anti-carbohydrate IgM in the serum of BDF-1 mice. Among the natural anti-carbohydrate IgM identified, anti-betaGlcNAc IgM were the most abundant. Anti-betaGlcNAc IgG was not detected. Levels of anti-betaGlcNAc IgM were very low in 3-week-old BDF-1 mice and increased until 5 to 7 months of age. Levels of serum anti-betaGlcNAc IgM similar to those in BDF-1 mice were found in the serum of some strains related to the BDF-1 strain (DBA and C57BL/6) and in BDF mice lacking the galactosyl transferase gene. However, in two strains unrelated to the BDF-1 strain (FVB and SJL), levels of anti-betaGlcNAc IgM were less than one-tenth of those found in BDF-1 mice. These results provide considerable insight into the effect of age on the production of natural anti-carbohydrate antibodies in mice and indicate that production of those antibodies is strongly dependent on the strain of mouse. These studies will help in future development of murine models for studying the biological and medical roles of natural anti-carbohydrate antibodies.

The role of antibodies in acute vascular rejection of pig-to-baboon cardiac transplants.

Long-term success in xenotransplantation is currently hampered by acute vascular rejection. The inciting cause of acute vascular rejection is not yet known; however, a variety of observations suggest that the humoral immune response of the recipient against the donor may be involved in the pathogenesis of this process. Using a pig-to-baboon heterotopic cardiac transplant model, we examined the role of antibodies in the development of acute vascular rejection. After transplantation into baboons, hearts from transgenic pigs expressing human decay-accelerating factor and CD59 underwent acute vascular rejection leading to graft failure within 5 d; the histology was characterized by endothelial injury and fibrin thrombi. Hearts from the transgenic pigs transplanted into baboons whose circulating antibodies were depleted using antiimmunoglobulin columns (Therasorb, Unterschleisshein, Germany) did not undergo acute vascular rejection in five of six cases. Biopsies from the xenotransplants in Ig-depleted baboons revealed little or no IgM or IgG, and no histologic evidence of acute vascular rejection in the five cases. Complement activity in the baboons was within the normal range during the period of xenograft survival. In one case, acute vascular rejection of a xenotransplant occurred in a baboon in which the level of antidonor antibody rose after Ig depletion was discontinued. This study provides evidence that antibodies play a significant role in the pathogenesis of acute vascular rejection, and suggests that acute vascular rejection might be prevented or treated by therapies aimed at the humoral immune response to porcine antigens.

Health-related quality of life after ileoanal pull-through evaluation and assessment of new health status measures.

BACKGROUND & AIMS: Health-related quality of life (HRQL) after proctocolectomy is a critical parameter for management decisions in patients with chronic pancolitis. The aim of this study was to evaluate the HRQL of patients with ileoanal pull-through and to validate new, easy-to-administer HRQL measures. METHODS: The Sickness Impact Profile (SIP), Short Form 36 (SF-36), Rating Form of Inflammatory Bowel Disease (IBD) Patient Concerns (RFIPC), and the time trade-off (TTO) were used to measure HRQL of pull-through patients. The SF-36 and the RFIPC were validated. RESULTS: HRQL of patients with ileoanal pull-through was better than that of a national sample of patients with IBD (SIP and RFIPC) and similar to that of a normal population (SF-36). Physical and psychosocial subscales of the SF-36 correlated with the SIP, affirming the construct validity of the SF-36. The RFIPC results correlated with the SIP and SF-36 results, suggesting that it is also a valid health status measure for these patients. TTO results correlated with the physical subscales of the SIP and SF-36, reflecting the impact of physical health on this group. CONCLUSIONS: HRQL of patients with ileoanal pull-through is excellent. The SF-36 and RFIPC are valid health status measures that can be used by clinicians and researchers in these patients.

Effect of continuous complement inhibition using soluble complement receptor type 1 on survival of pig-to-primate cardiac xenografts.

A single bolus of soluble complement (C) receptor type 1 (sCR1, TP-10) has been shown to delay hyperacute rejection (HAR) of porcine cardiac xenografts (Xgs) by primate recipients. In these recipients, C activity slowly returned and C deposition was noted in the Xgs at rejection. To evaluate the effect of sustained C inhibition using sCR1 on HAR, two additional cynomolgus monkeys received porcine cardiac Xgs and a continuous infusion of sCR1. In the first recipient, Xgs survival was 5 days (120+ hr), whereas in the second, Xg survival was 7 days (168+ hr). Serial biopsies of the Xgs were remarkable for an increasing cellular infiltrate composed predominantly of neutrophils and macrophages, and the development of edema, hemorrhage, and myocyte necrosis. These findings suggest that once C-mediated HAR has been inhibited, infiltration of the Xg by these cells may lead to accelerated acute rejection, which is an additional barrier to successful longer term Xg survival.

Orthostatic acute renal failure in a renal transplant.

Complications due to ureteric obstruction are an occasional cause for renal transplant dysfunction. Here we report an unusual case of orthostatic renal failure in a renal transplant recipient. Our patient had the previously reported predisposing risk factors including: female sex, obesity, and lax abdominal musculature. It is important to recognize this unusual complication of renal transplantation early in order to preserve long-term graft function.

Renal transplantation in adults with thrombotic thrombocytopenic purpura/haemolytic-uraemic syndrome.

BACKGROUND: Thrombotic thrombocytopenic purpura/haemolytic-uraemic syndrome (TTP/HUS) is a rare cause of renal failure in adults. There is little data concerning the outcome of adult patients who receive a renal transplant for TTP/HUS: METHODS: We have carried out a survey of 22 transplant centres in the USA to determine the outcome of patients who developed ESRD from TTP/HUS and latter received a renal transplant. RESULTS: Twelve of the 22 centres responded to our inquiry. Seven centres had not transplanted any patients with TTP/HUS, and five centres had transplanted a total of 24 grafts in 17 patients with TTP/HUS: Thirty-three per cent of patients demonstrated definite clinical and pathological evidence of recurrence of TTP/HUS: An additional 16% of patients demonstrated pathological evidence of possible recurrence of TTP/HUS in the absence of clinical manifestations. The overall 1-year graft survival rate was 42% and the 2-year graft survival rate was 35%. In our experience recurrence TTP/HUS was associated with universal graft failure. Although cyclosporin A does occasionally cause a thrombotic angiopathy in patients with no history of TTP/HUS, we found no evidence that it should be avoided in patients with a previous history of ESRD from TTP/HUS who subsequently receive a renal transplant. CONCLUSIONS: TTP/HUS frequently recurres in adults who receive a renal transplant, with a 2-year graft survival rate of 35%.

Immunoglobulin prevents complement-mediated hyperacute rejection in swine-to-primate xenotransplantation.

Immunoglobulins regulate the complement system by activating complement on foreign surfaces and diverting reactive complement proteins away from autologous cell surfaces. Based on this model, we explored the ability of Ig to balance complement activation versus control in a pig-to-primate cardiac xenotransplantation model in which the binding of xenoreactive antibodies of the recipient to graft blood vessels and the activation of complement cause hyperacute rejection. Human IgG added to human serum caused a dose-dependent decrease in deposition of iC3b, cytotoxicity, and heparan sulfate release when the serum was incubated with porcine endothelial cells. This decrease was not caused by alteration in antibody binding or consumption of complement but presumably reflected decreased formation of C3 convertase on the endothelial cells. Infusion of purified human IgG into nonhuman primates prevented hyperacute rejection of porcine hearts transplanted into the primates. As expected, the transplants contained deposits of recipient Ig and C1q but not other complement components. The inhibition of complement on endothelial cell surfaces and in the xenotransplantation model supports the idea that IgG regulates the classical complement pathway and supports therapeutic use of that agent in humoral-mediated disease.

Differential expression of substance P receptors in patients with Crohn's disease and ulcerative colitis.

BACKGROUND & AIMS: Although clinical and pathological differences exist between Crohn's disease (CD) and ulcerative colitis (UC), distinguishing features are often absent, making diagnosis and treatment problematic. This study evaluated the differences in the expression of substance P (SP) receptors in patients with CD or UC. METHODS: Tissue samples from patients with inflammatory bowel disease or control patients were obtained at surgery, processed for 125I-SP binding, and analyzed by quantitative autoradiography. RESULTS: Patients with CD showed a massive increase in SP receptors in lymphoid aggregates, small blood vessels, and enteric neurons of the small and large bowel relative to controls. Six of 16 CD specimens had no pathological evidence of CD yet continued to express high concentrations of SP receptors. Pathologically positive patients with UC showed high concentrations of SP receptors on colonic lymphoid aggregates and small blood vessels but not enteric neurons. No increased SP binding was evident in clinically and pathologically quiescent UC colons and normal UC ileostomy samples. CONCLUSIONS: The increased expression of SP receptors on the enteric neurons of patients with CD distinguishes CD from UC. The persistent increased SP binding in pathologically normal CD tissue may indicate a subclinical disease state. SP receptor expression may have important diagnostic, etiologic, and therapeutic usefulness in inflammatory bowel disease.

Rapid, comprehensive analysis of human cytokine mRNA and its application to the study of acute renal allograft rejection.

Cytokine mRNA analysis was performed on human renal allograft needle core biopsies by a PCR-based assay. The assay was specifically developed to be capable of simultaneous analysis of multiple interleukin transcripts (IL-1-IL-12), as well as those of other relevant cytokines, by one person in less than 1 day from cultured cells or directly from tissue samples. It was initially used on preparations containing known amounts of plasmid DNA encoding individual cytokine cDNA sequences, confirming that the sensitivity of this technique was both well defined and comparable for all target sequences tested. Analysis of human PBLs prior to stimulation, after polyclonal stimulation with PHA and after simultaneous treatment with PHA and MP or CyA, was also performed to show a proportional relationship between mRNA levels measured by PCR and protein release measured by ELISA (R2 = 0.86). This correlation was not adversely altered by pharmacologic immunosuppression by MP or CyA. Thus, this method of PCR primer design and usage was appropriate for the clinical study of cytokine mRNA levels during allograft rejection. Direct study of cytokine mRNA in allograft biopsy tissue showed that IL-2 was specifically and significantly (p = 0.006) elevated during ACR when compared to other causes of graft dysfunction. Transcripts from the IFN-gamma and IL-6 genes were also increased in ACR (p = 0.001 and 0.017, respectively), whereas increased IL-8 mRNA was correlated with irreversible loss of graft function (p = 0.02). TNF-alpha, IL-1 beta, and IL-10 gene transcripts were also detected during ACR, but were not quantitatively increased compared to other forms of graft injury (p > 0.2). We conclude that acute cellular rejection is associated with intragraft mRNA from the IL-2 gene. Other transcripts, including those from the IFN-gamma, IL-6, and IL-8 genes, are detected in increased amounts during this process. Messenger RNA from the TNF-alpha, IL-1 beta and IL-10 genes is also detected during ACR, but the presence of these transcripts is not exclusive to this process.

Recipient Kupffer cell influx into xenografted liver.

Previously we reported that the combination of total lymphoid irradiation (TLI), cyclosporine A (CsA), and splenectomy have an immunosuppressive effect sufficient to significantly prolong liver xenograft survival in the LVG hamster to the LEW rat model. Using this model, we have investigated recipient Kupffer cell influx into the xenografted liver. In the rat liver, the Kupffer cells are heavily stained with Ki-M2R, a rat anti-macrophage monoclonal antibody, whereas sinus endothelial cells and liver parenchymal cells are invariably negative. Kupffer cells of the hamster liver are not stained with Ki-M2R. In the xenografted animals, we found cells in the sinusoidal wall of the xenograft stained, with progressively increased activity, in animals 45-60 days after transplant. The morphological pattern of these Ki-M2R-positive cells in the hepatic xenograft resembled the Kupffer cells of the normal rat liver. These findings indicate recipient macrophage influx into the xenografted liver.

Mechanisms of injury in porcine livers perfused with blood of patients with fulminant hepatic failure.

Hyperacute rejection of renal and cardiac xenografts is initiated by the reaction of recipient natural antibodies and complement with endothelial cell antigens of the donor organ. The liver is thought to be less susceptible to this form of rejection; however, the mechanisms underlying its decreased susceptibility are not known. We investigated the organ injury occurring in porcine livers perfused with blood from 4 human subjects with fulminant hepatic failure. Nine porcine livers were perfused via an extracorporeal circuit in order to provide temporary metabolic support. Each porcine liver exhibited metabolic function, and the duration of xenoperfusion ranged from 2 to 5 hr. Histologic examination of the xenoperfused livers revealed focal hepatocellular necrosis, prominent infiltration of neutrophils, and, in 7 of 9 cases, periportal and centrilobular hemorrhage and thrombosis. Immunopathology demonstrated minimal or no human IgM and IgG along the small vessels and sinusoidal surfaces. Trace deposits of human IgM were observed along the luminal surfaces of large blood vessels in most cases. Trace deposits of C3 were noted in 2 of 9 livers; however, C4, iC3b, C5b, properdin, and the membrane attack complex were not detected. Human anti-porcine natural antibody titers decreased less than expected during the perfusions. Serum CH50, C3, and C4 levels were low before each procedure and decreased slightly with perfusion. One patient perfused 2 porcine livers and a human liver. The human liver had focal hepatocellular necrosis, trace deposits of IgM, no deposits of complement, and an infiltrate consisting of neutrophils; however, the neutrophil influx was less than that observed in the xenoperfused livers. To further evaluate the effects of alloperfusion, venovenous bypass was established in 2 pigs and the extracorporeal circuit was utilized to perfuse 2 porcine livers. The alloperfused porcine livers had focal hepatocellular necrosis and a minimal infiltrate of neutrophils. There were no deposits of porcine IgM, IgG, or complement components. In conclusion, although the porcine livers perfused by human blood sustained structural damage, the time course, the absence of immune deposits, and the findings of similar, albeit less severe, lesions in the alloperfused livers suggest that the pathogenesis of tissue injury in the xenoperfused livers differs from that of hyperacute rejection and may be related to the action of recipient neutrophils.