R. Randal Bollinger

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.

The effect of soluble complement receptor type 1 on hyperacute rejection of porcine xenografts

The use of xenografts (Xgs) from distantly related species to relieve the increasing shortage of organs for clinical transplantation is prevented by the occurrence of hyperacute rejection (HAR). This process, in which C activation plays a central role, cannot be inhibited with currently available immunosuppressants. In two clinically relevant xenotransplantation models, this study evaluated the effect of C inhibition using recombinant soluble complement receptor type 1 (sCR1) on HAR. In an ex vivo model in which porcine cardiac Xgs were perfused with human blood, cardiac function ceased within 34 min when the perfusate blood was untreated (n = 3). When the perfusate blood was treated with sCR1 (300 micrograms/ml), cardiac Xg function was maintained for up to 4 hr (n = 3). Immunohistologic examination of these Xgs demonstrated deposition of C3b/iC3b and C3d in Xgs perfused with untreated human blood but only C3d deposition in those Xgs perfused with sCR1-treated human blood. These findings are consistent with the cofactor activity of sCR1 for factor I-mediated degradation of deposited C3b/iC3b to C3d. Treatment with sCR1 also prevented the histopathologic changes of HAR observed when untreated blood was used as the perfusate. In an in vivo pig-to-primate heterotopic cardiac xenotransplantation model, in which porcine Xgs transplanted into untreated cynomolgus monkey recipients underwent HAR in 1 hr or less (n = 3), a single intravenous bolus of sCR1 (15 mg/kg) administered to the recipient immediately before Xg reperfusion markedly inhibited total and alternative pathway serum C activity and prolonged Xg survival to between 48 and 90 hr (n = 5). These studies confirm the important role of C activation in HAR of porcine cardiac Xgs by primates and indicate that sCR1 may be a useful agent for xenotransplantation.

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.

The effect of soluble complement receptor type 1 on hyperacute xenograft rejection

In the guinea pig-to-rat model of hyperacute xenograft (Xg) rejection, the effect of complement inhibition using systemically administered soluble complement receptor type 1 (sCRl) on discordant cardiac Xg survival was investigated. In PBS-treated control Xg recipients (n=13), hyperacute rejection was rapid, with a mean Xg survival of 17±4 min. Therapy with sCRl prolonged survival of cardiac Xgs in a dose-dependent manner. A 3 mg/kg bolus of sCRl (n=4) prolonged Xg survival to 64±29 min (not significant). Increasing the sCRl dose to 5.9 mg/kg (n=4) significantly delayed Xg rejection to 71±17 min (P-0.026, log-rank test vs. control). In 10 recipients treated with 15 mg/kg sCRl, mean Xg survival was further prolonged to 189±36 min (P-0.0004) with no adverse effects. While 2 of 8 recipients receiving 60 mg/kg sCRl died with functioning Xgs at 30 and 300 min due to anastomotic bleeding, Xg survival averaged over 12 hr (747±100 min, P-0.0004) in the remaining 6 recipients. sCRl administration significantly inhibited serum complement activity in a parallel dose-dependent fashion, with the 60 mg/kg dose reducing complement activity by 95±1 and 96±1% five and 30 min following Xg reperfusion, respectively. Immunofluorescence microscopy revealed rat IgM bound to all cardiac Xgs in control as well as sCRl -treated recipients. In addition, serial histologic examination of cardiac Xgs harvested within 21 min of graft reperfusion revealed occlusive platelet aggregates within the coronary vessels as well as interstitial hemorrhage and myocardial necrosis in Xgs from control recipients, all of which were only minimally present in Xgs from recipients treated with sCRl. These studies show that complement inhibition with sCRl significantly delays hyperacute cardiac Xg rejection in this discordant model and may be an important component in a therapeutic protocol for xenotransplantation.

Human secretory immunoglobulin A may contribute to biofilm formation in the gut

It is critical, both for the host and for the long‐term benefit of the bacteria that colonize the gut, that bacterial overgrowth with subsequent bacterial translocation, which may lead to sepsis and death of the host, be avoided. Secretory IgA (sIgA) is known to be a key factor in this process, agglutinating bacteria and preventing their translocation in a process termed ‘immune exclusion’. To determine whether human sIgA might facilitate the growth of normal enteric bacteria under some conditions, the growth of human enteric bacteria on cultured, fixed human epithelial cells was evaluated in the presence of sIgA or various other proteins. Human sIgA was found to facilitate biofilm formation by normal human gut flora and by Escherichia coli on cultured human epithelial cell surfaces under conditions in which non‐adherent bacteria were repeatedly washed away. In addition, the presence of sIgA resulted in a 64% increase in adherence of E. coli to live cultured epithelial cells over a 45‐min period. Mucin, another defence factor thought to play a key role in immune exclusion, was found to facilitate biofilm formation by E. coli. Our findings suggest that sIgA may contribute to biofilm formation in the gut.

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

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

The human antiporcine cellular repertoire. In vitro studies of acquired and innate cellular responsiveness.

Discordant xenogeneic transplantation offers a potentially unlimited source of donor organs from easily bred, nonendangered, physiologically compatible animals, but has been limited by the inevitable occurrence of hyperacute rejection (HAR). The potential existence of cell-mediated discordant graft rejection has remained obscured by HAR, and hence is incompletely understood. To define the cellular elements capable of recognition of and subsequent response against discordant tissue in a clinically applicable species combination, we have studied the in vitro interaction of human peripheral blood lymphocytes against 3 porcine B lymphoblastoid cell lines and 6 primary porcine endothelial cell populations. PBL from all individuals tested (n = 10) proliferated in response to culture for 72 hr in xenogeneic mixed lymphocyte culture (XMLC) with cell lines expressing porcine MHC (SLA) class II antigens, while endothelial cultures lacking SLA class II generally failed to evoke a response. The proliferative response to class II-positive cells was attenuated by addition of anti-SLA class II antibody but not by anti-SLA class I antibody. Two endothelial populations expressing class II stimulated an inhibitable proliferative response. The magnitude of the short-term proliferative xenogeneic response was similar to that evoked by fully mismatched allogeneic human B lymphoblastoid stimulators. Additionally, extended XMLC was performed with PBL from 3 individuals. All populations responded with continued proliferation when repeatedly stimulated by porcine cells. This was characterized not only by T cell growth, but by prominent NK cell growth as well. Elucidation of the TCR V beta chain usage patterns by semiquantitative PCR documented selection of TCR transcripts from gene family V beta 2 in each group, complemented by a heterogeneous mixture of other transcripts including V beta 17.1, 20.1, and 6.1, suggesting that direct human TCR binding of porcine cells occurs, and that it is likely to be an individualistic response complemented by a more homogeneous NK response. A 51Cr release assay was utilized to demonstrate that unprimed PBL could also lyse porcine target cells. This cytotoxic response was maintained despite the complete removal of T cells, suggesting that porcine-directed NK cell activity is present prior to the maturation of any T cell response. Cytolysis was also demonstrated in serum-free medium and thus was not mediated solely by antibody-dependent cellular cytotoxicity. Chinese hamster ovary cells transfected with the human T cell receptor accessory molecule CD4 were used to study the ability of this molecule to stabilize the interaction between the human TCR and SLA class II.(ABSTRACT TRUNCATED AT 400 WORDS)

Prospective analysis of strategies for diagnosing renovascular hypertension.

Renovascular hypertension is a potentially curable form of high blood pressure. However, it is unclear how best to select patients who are likely to have renovascular hypertension, what diagnostic strategy to use in these selected patients, and how to predict the hemodynamic significance of a renal artery stenosis. We determined the prevalence of renovascular hypertension in adults who exhibited suggestive clinical features. In these clinically selected patients, we then determined the test characteristics of various diagnostic and potential screening tests. Renovascular hypertension was diagnosed if correction of renal artery stenosis resulted in decreased blood pressure. Of the 66 hypertensive adults evaluated, 11 (16.7%) had renovascular hypertension. Captopril-stimulated peripheral renln activity detected renovascular hypertension with 73% sensitivity, 72% specificity, 38% positive predictive value, and 92% negative predictive value. Less optimal combinations of sensitivity and specificity were found for differential glomerular filtration rate renography, differential effective renal plasma flow renography, and selective renal vein renin ratios, each performed after a single dose of captopril. Intravenous digital subtraction renal angktgraphy detected all patients with renovascular hypertension and was normal in 71% of patients with essential hypertension. To evaluate potential screening tests for renovascular hypertension, we calculated predictive values applied to a low prevalence population. If the observed sensitivities and specificities apply to a population with 5% prevalence of renovascular hypertension, captopril-stimulated peripheral renin would have a positive predictive value of 12% and a negative predictive value of 98%. In 16 patients with known renal artery stenosis, neither the captopril-stimulated renal vein renin ratio nor captopril-stimulated differential renography accurately predicted blood pressure response to correction of the stenosis. We conclude that clinical criteria can identify a subgroup with 16.7% prevalence of renovascular hypertension. In this high prevalence group, intravenous digital subtraction renal angiography will identify virtually all patients with renovascular hypertension, and a normal study will be sufficient to exclude renovascular hypertension. In unselected hypertensive patients, screening with captopril-stimulated peripheral renin activity may be the most useful and efficient procedure for identification of patients with renovascular hypertension. Functional tests do not accurately predict the hemodynamic significance of a renal artery stenosis.

Fate Of Four Cadaveric Donor Renal Allografts With Mesangial Iga Deposits

In the course of routine pretransplant cadaveric donor kidney biopsy examination, specimens from two donors were found to exhibit intense mesangial localization of IgA by immunofluorescence, with the presence of large immune complex-type deposits in these areas confirmed ultrastructurally. Both kidneys from each donor were transplanted with the ultimate result that three of the four kidneys underwent early irreversible rejection and were removed within 3 months, while the fourth kidney has maintained normal function for a period of 8 months. Morphological and immunofluorescent evaluation of the rejected kidneys at the time of nephrectomy showed minimal residual IgA mesangial deposits, but all had changes indicative of severe acute allograft rejection. These findings suggest that glomerular lesions involving mesangial IgA deposition can resolve fairly quickly following transplantation, but that the risk of irreversible acute rejection might be greater in the recipients of such kidneys.

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