Serial sodium Amytal interviews in the clinical setting

A prospective final crossmatch with patient serum and donor lymphocytes using the complement-dependent cytotoxicity assay to identify any performed anti-donor antibody is required for kidney transplantation. The presence of pre-existing antibody may lead to hyperacute rejection of the transplanted kidney. Certain anti-donor antibodies have previously been shown to be ineffective in promoting hyperacute rejection, such as IgM autoantibodies and non-specific IgM lymphocytotoxic antibodies. In this report, we present evidence that IgM HLA alloantibody specific to the donor does not lead to hyperacute rejection and produces graft survival results equivalent to transplants with negative pre-transplant final crossmatches. Forty-eight (48) of 402 patients transplanted over and 8 yr period were transplanted across a positive final crossmatch due to IgM antibodies alone. Three patients exhibited IgM autoantibodies and 26 patients demonstrated non-specific IgM antibodies to lymphocytes. In 15 patients, following a detailed serum screening analysis, a significant correlation (r > 0.9, p < 0.001) was observed between HLA Class I antigens and the presence of corresponding IgM alloantibodies. Five of these patients were subsequently transplanted despite a positive final crossmatch that was clearly demonstrated to be the result of IgM alloantibody to donor HLA Class I specificities. All of these patients continue to have graft function. These results suggest that hyperacute rejection is not mediated by any type of IgM antibody to donor lymphocytes and that kidney transplantation when only IgM antibody is present against donor lymphocytes represents a reasonable opportunity for a safe transplant and successful long-term graft survival.     

The clinical significance of flow cytometry crossmatching in heart transplantation

BACKGROUND: Flow cytometry crossmatching is more sensitive than cytotoxic methods in identifying preformed antibodies to donor alloantigens. However, the significance of a positive flow crossmatch remains unknown for a recipient of a heart transplant who has a negative anti-human globulin crossmatch. METHODS: Flow crossmatching was performed retrospectively for 92 recipients of a primary cardiac allograft who underwent transplantation with a negative AHG crossmatch. RESULTS: Forty-six patients were flow crossmatch-positive for alloantibody: 20 were positive on both T and B lymphocytes, 12 were positive only on B lymphocytes, and 13 were positive only on T lymphocytes. Eleven had autoantibody invalidating the flow crossmatch with donor cells. Thirty-six patients had negative flow crossmatch. A significantly higher incidence of graft dysfunction with vascular rejection by 6 months was found for patients who had a positive flow crossmatch on B lymphocytes. This group also had an increased incidence of mortality within this same period. Patients who were flow crossmatch-positive on T and B lymphocytes were more likely to experience greater than two episodes of treated cellular rejection within the first 6 months. Flow crossmatch-positive patients stayed longer in the hospital in comparison to the other two groups, although the increases were not statistically significant. There were no differences between groups with regard to time to first rejection, absence of rejection episodes, episodes of decreased cardiac index (<2.3 L/m2), depressed left and right ventricular ejection fraction, or development of transplant atherosclerosis. CONCLUSION: A positive flow crossmatch identified a subset of patients who are predisposed to development of vascular rejection or are more likely to have frequent cellular rejection.     

Comparative study of target antigens for primate xenoreactive natural antibodies in pig and rat endothelial cells

BACKGROUND: A rat-to-primate cardiac xenograft model has been proposed as an alternative to the clinically relevant but more cumbersome pig-to-primate model for assessing the efficacy of strategies aimed at preventing xenograft hyperacute rejection. As in pig xenografts, the rejection of rat hearts was mediated by the binding of xenoreactive natural antibodies (XNA) and complement activation. The present study was conducted to identify target antigens recognized by cynomolgus and rhesus monkey IgM XNA on rat tissues and cells in comparison with pig cells. METHODS: The reactivity of rhesus or cynomolgus serum on pig and rat endothelial cells (ECs) was studied by flow cytometry, ELISA, and complement-dependent cytotoxicity, after removal of primate XNA by perfusion of pig livers, immunoadsorption on a Gal alpha(1,3)Gal affinity column, and enzymatic removal of alpha-galactosyl epitopes from the cell surface. Rat and pig EC extracts were also immunoprecipitated with primate serum and resolved in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The expression of the Gal alpha(1,3)Gal epitope was analyzed on rat tissues and ECs by immunohistochemistry, flow cytometry, and Western blot, using the isolectin B4 from Griffonia simplicifolia. RESULTS: Removal of primate XNA or of alphaGal epitopes resulted in a decrease in XNA binding to pig and rat cells, leaving a similar degree of residual reactivity in the two species. At least five proteins of 260, 210, 110, 56, and 50 kDa were immunoprecipitated on rat ECs, with molecular weight similar to several proteins identified on pig ECs. These results suggest that primate XNA recognize similar antigens on rat and pig ECs. Rat cells expressed lower levels of the Gal alpha(1,3)Gal epitope than pig cells. A large proportion, but not all, of primate XNA react with this epitope on pig and rat ECs. CONCLUSION: This study suggests that the rat is a valuable species for the evaluation of genetic engineering strategies on the vascular endothelium aimed at preventing hyperacute xenograft rejection.     

Frequency of dissociative disorders among psychiatric inpatients in a Turkish University Clinic

BACKGROUND: Intravenous gammaglobulin (i.v.IG) contains anti-idiotypic antibodies that are potent inhibitors of HLA-specific alloantibodies in vitro and in vivo. In addition, highly HLA-allosensitized patients awaiting transplantation can have HLA alloantibody levels reduced dramatically by i.v.IG infusions, and subsequent transplantation can be accomplished successfully with a crossmatch-negative, histoincompatible organ. METHODS: In this study, we investigated the possible use of i.v.IG to reduce donor-specific anti-HLA alloantibodies arising after transplantation and its efficacy in treating antibody-mediated allograft rejection (AR) episodes. We present data on 10 patients with severe allograft rejection, four of whom developed AR episodes associated with high levels of donor-specific anti-HLA alloantibodies. RESULTS: Most patients showed rapid improvements in AR episodes, with resolution noted within 2-5 days after i.v.IG infusions in all patients. i.v.IG treatment also rapidly reduced donor-specific anti-HLA alloantibody levels after i.v.IG infusion. All AR episodes were reversed. Freedom from recurrent rejection episodes was seen in 9 of 10 patients, some with up to 5 years of follow-up. Results of protein G column fractionation studies from two patients suggest that the potential mechanism by which i.v.IG induces in vivo suppression is a sequence of events leading from initial inhibition due to passive transfer of IgG to eventual active induction of an IgM or IgG blocking antibody in the recipient. CONCLUSION: I.v.IG appears to be an effective therapy to control posttransplant AR episodes in heart and kidney transplant recipients, including patients who have had no success with conventional therapies. Vascular rejection episodes associated with development of donor-specific cytotoxic antibodies appears to be particularly responsive to i.v.IG therapy.     

The metalloproteinase-mediated pathway is essential for generation of soluble HLA class I proteins by activated cells in vitro: proposed mechanism for soluble HLA release in transplant rejection

We and others have found donor-derived soluble beta2m-associated HLA class I proteins (sHLA/beta2m) in the serum of allograft recipients with acute and chronic rejection. Whether appearance of sHLA/beta2m and upregulated expression of donor cell-bound HLA/beta2m during allograft rejection are related events is unknown. Activation-induced upregulation of in vitro HLA/beta2m expression correlates with the surface expression of another form of HLA class I, namely beta2m-free HLA heavy chains (beta2m-free HC). We have shown that beta2m-free HC, but not beta2m-associated HC, are then cleaved by a specific membrane-bound metalloproteinase and released into supernatants as soluble 36 kDa proteins. We show now that activated peripheral blood lymphocytes produce predominantly the 36 kDa form of sHLA proteins which is present in supernatants as both beta2m-free HC and sHLA/beta2m. Importantly, the metalloprotease inhibitor BB-94 blocked not only the release of soluble beta2m-free HC, but also the appearance of sHLA/beta2m in cell supernatants. Low levels of 36 kDa beta2m-free HC were also present in human plasma of healthy donors. These data suggest an important role for the HLA class I-specific metalloproteinase in vivo in healthy individuals and during allograft rejection in the generation of soluble beta2m-free and beta2m-associated HLA proteins.     

Heterogeneity of antibody response to human platelet transfusion

To study the antibody response to human platelet transfusions, nine thrombocytopenia patients with bone marrow failure were given 6 U (3X10(11)) of random platelet concentrates twice a week. Before transfusion, none of the patients had preexisting antibodies detectable with lymphocytotoxicity, platelet aggregation, or capillary leukoagglutination techniques. After receiving 18-78 U of platelets, they became refractory to further transfusions of random platelets and alloantibodies were detectable. Two patterns of antibody response could be identified. In three patients, the sera were not lymphocytotoxic with a panel of standard cells in which all the known HLA antigens in the first and second series were represented at least once. Yet, they caused platelet aggregation with 30, 24, and 60%, respectively, of a donor population studied. The aggregating activities were inhibited by antihuman IgG but not by antihuman IgA or antihuman IgM antiserum. The aggregating antibodies could be absorbed out with donor platelets but not lymphocytes or granulocytes. Antibodies from two of these patients aggregated platelets of their respective siblings matched for both HLA haplotypes. Transfusion of platelets from these two siblings did not increase the platelet count while platelets obtained from aggregation-negative donors did. The sera from the remaining six patients were lymphocytotoxic with 15-100% of the panel of standard cells. They also had aggregating antibodies, which could be absorbed out by both platelets and lymphocytes, suggesting that they were HLA antibodies. These data suggest that the development of platelet-specific antibodies may play an important role in the immunological rejection of isologous platelets, and should be considered in the selection of donors for patients who are refractory to platelets from random donors.     

Specific interaction of HLA antibodies (eluates) with washed platelets

The mechanism of complement-independent action of HLA-A2 antibodies (eluates) on washed platelets was investigated. HLA-specific alteration was confirmed by serological (platelet micro-complement fixation), morphological (platelet spreading) and functional parameters (platelet aggregation, inhibition of collagen-induced platelet aggregation, [14C]serotonin release). In the presence of fibrinogen and calcium ions, HLA antibodies induced instantaneous platelet aggregation and release. Although no morphological (spreading) and functional changes (collagen-induded aggregation) were seen, these platelets did not aggregate or release when fibrinogen was subsequently added. When platelets--in the presence of fibrinogen--were incubated with antibody concentrations too low to induce platelet aggregation or release, specific reduction of platelet reactivity was observed by subsequent collagen aggregation. HLA-specific action of antibodies on washed platelets was inhibited by apyrase and acetyl-salicylic acid, indicating an active participation of platelets in HLA antibody-induced platelet alteration.     

The significance of a positive flow cytometry crossmatch test in primary kidney transplantation

This study was conducted to determine the efficacy of the T cell flow cytometry crossmatch (T-FCXM) test in 841 first cadaver donor transplants. Results showed one-year graft survival rates were 82% for T-FCXM-negative patients, compared with 75% for T-FCXM-positive patients (P = 0.01). Early one-month graft failure was 13 percentage points higher in those with a positive T-FCXM than those with a negative T-FCXM. The positive crossmatch patients also had more frequent immunological failures. A positive T-FCXM was found in 39% of the sensitized patients (PRA > 10%) and 8% of those who had not been sensitized. Patients with a positive T-FCXM in either category had a 74% graft survival rate. Thus, most of the T-FCXM-positive results occurred in patients with complement-fixing antibodies. It is suggested that flow cytometry crossmatching (FCXM) be used prospectively, despite the fact that many patients with a positive crossmatch did have successful transplants (TXs). In the current climate of a cadaver kidney scarcity and large recipient waiting pools, utilization of kidneys for patients with the highest probability of success seems a most prudent policy.     

Bone allografts are immunogenic and may preclude subsequent organ transplants

The authors report a case of a 41-year-old woman with diabetes and chronic renal failure in whom antihuman leukocyte antigen antibodies developed after she received a frozen bone allograft that limited her access to organ donors. The patient had a chondrosarcoma of the right distal femur. A wide resection with segmental total knee arthroplasty was followed by a revision using a composite bone allograft prosthesis. After revision, broadly reactive lymphocytotoxic antibodies developed in the patient. The patient's panel reactive antibody level rose from 28% to a peak of 70%. Panel reactive antibody expresses the percentage of a panel of human leukocyte antigen type T lymphocytes from 40 individuals (representative of all human leukocyte antigen Class I histocompatibility antigens) to which antihuman leukocyte antigen Class I lymphocytotoxic antibodies have developed in the recipient as measured by the antiglobulin crossmatch method. The specificity of the patient's primary antibody is found in 45% of donors available in Illinois since 1988 (N = 1606). Because a positive crossmatch precludes kidney and pancreas transplantation, at least 45% of cadaver organ donors were excluded from use for this patient. This is an unusual case that focuses on the potential impact of bone allografts in patients who may need subsequent organ transplantation.     

Endothelial cell activation by sera containing HLA antibodies is mediated by interleukin-1

BACKGROUND: It has been suggested that antibodies which are associated with chronic pathological conditions such as chronic rejection and autoimmune diseases have the capacity to activate endothelial cells by induction and up-regulation of adhesion molecules. It has also been suggested that HLA antibodies formed by patients awaiting transplantation can activate endothelial cells. These antibodies include HLA and those that bind to endothelial cells. METHODS: We have further investigated this phenomenon using monoclonal antibodies against HLA class I determinants and sera from aortic valve graft recipients, containing strong HLA antibodies. The effect of 24-hr incubation of antibodies/serum with human umbilical vein endothelial cells (HUVECs) on adhesion molecule expression was measured by flow cytometry. RESULTS: HLA monoclonal antibodies had no effect on ICAM-1 expression on HUVECs. Five of 31 (16%) patients' sera caused strong up-regulation of adhesion molecules (ICAM-1, vascular cell adhesion molecule-1, and E-selectin) but this did not correlate with HLA specificity, IgG, or IgM binding to HUVECs. The activity, found in whole serum and IgG-depleted fractions was inhibited by neutralizing antibodies against interleukin (IL)-1beta and tumor necrosis factor-alpha. Examination of patient sera for presence of IL-1beta demonstrated high levels of IL-1beta in all five sera (range, 30 -500 U/ml) as well as in samples from an additional three patients. CONCLUSION: The ability to activate endothelial cells detected in our patient sera was caused by cytokines and not antibody. Our observation that addition of cytokines to sera before separation into large and low molecular weight fractions demonstrated retention of cytokines in both fractions may be a confounding issue when investigating endothelial cell activation by patients' sera.     

Immunologic monitoring in lung allograft recipients

To identify patients with increased risk of chronic lung allograft rejection, we assessed the utility of an in vitro biopsy-derived lymphocyte growth assay and serum anti-HLA antibody screening as a complement to currently available methods of monitoring lung allograft recipients. Lymphocyte growth assay was performed on bronchoscopic fragments of tissue cultured in medium with rIL-2. Seventy-nine biopsies from 31 lung transplant recipients were tested by lymphocyte growth assay, and results were correlated with histopathology findings. Positive lymphocyte growth was found in 12/26 (46%) episodes of acute rejection, 5/44 biopsies without rejection (11%), and 0/9 episodes of bronchitis. Positive lymphocyte growth was seen in 7/16 (44%) grade A1 rejections and in 5/10 (50%) grade A2 rejections, as opposed to only 5/44 (11%) grade A0 (no rejection) biopsies (P < 0.01 for both A1 and A2 with respect to A0). Actuarial probability of remaining free from obliterative bronchiolitis (OB)* tended to be higher in patients who did not exhibit lymphocyte growth in biopsies. Sequential samples of sera obtained at the time of the biopsy were screened for lymphocytotoxic anti-HLA antibodies. Twenty-two of 44 recipients (50%) developed anti-HLA antibodies during the first postoperative year, exhibiting greater than 10% reactivity to an HLA reference panel of lymphocytes in four or more consecutive serum samples. Actuarial survival of lung allograft recipients with anti-HLA antibodies (n = 22) was lower than in those without anti-HLA antibodies (n = 22; P = 0.03). Of the 22 antibody producers, 7/12 died as a consequence of OB. Of the 22 non-antibody-producers, 1/2 deaths occurred as a consequence of OB. Anti-HLA antibodies were present in 9/11 instances of OB (82% sensitivity) and in 13/33 patients without OB (61% specificity; P = 0.03). These data indicate that lung transplant recipients with positive lymphocyte growth and anti-HLA antibodies are at an increased risk of chronic allograft rejection.     

Function and specificity of human natural killer cell receptors

In humans, natural killer lymphocytes express HLA class I-specific inhibitory receptors belonging to at least two different molecular families. The first is represented by members of the Ig superfamily that are involved in the recognition of different groups of HLA class I alleles, and the second is represented by a molecular complex formed by CD94 and NKG2A that displays a broad specificity for various class I molecules including the 'non-classical' HLA-G molecules. In addition to the inhibitory receptors, a series of activating receptors has been identified. Some display the same specificities as the corresponding inhibiting receptors and can be viewed as HLA class I-specific activating receptors. Another group of activating receptors appear to be involved in the cytolytic activity against HLA-'negative' target cells. These receptors are clearly non-MHC specific and, under physiological conditions, their function is suppressed by the HLA class I-specific inhibitory receptors.     

Complement-fixing elicited antibodies are a major component in the pathogenesis of xenograft rejection

Hamster to rat cardiac xenografts undergo delayed rejection as compared with the hyperacute rejection of discordant xenografts. Elicited xenoreactive Abs (EXA) are thought to initiate hamster to rat cardiac xenograft rejection. In this study, we demonstrate that following transplantation of a hamster heart, rats generated high levels of EXA. Adoptive transfer into naive recipients of purified IgM, IgG2b, or IgG2c, but not IgG1 or IgG2a EXA, induced xenograft rejection in a complement-dependent manner. Ability of EXA to cause rejection correlated with complement activation, platelet aggregation, and P-selectin expression in the xenograft endothelium. Cyclosporin A (CyA) administration, after transplantation, totally suppressed IgG1, IgG2a, IgG2b, and IgG2c EXA, and inhibited IgM EXA production, but failed to overcome rejection. Administration of cobra venom factor (CVF), 1 day before and at the time of transplantation, resulted in complement inhibition during 3 days after transplantation, which failed to overcome rejection. Combination of CyA and CVF, which we have previously shown to overcome rejection, resulted in suppression of IgG EXA production and in the return of IgM XNA to preimmunization serum levels, 3 to 7 days after xenotransplantation, while complement remained inhibited. Thus, under CyA/CVF treatment, complement activation by hamster cells was suppressed following xenotransplantation, and presumably for this reason xenograft rejection did not occur. In conclusion, our data demonstrate that EXA play a pivotal role in the pathogenesis of xenograft rejection and that CyA and CVF suppress xenograft rejection by preventing exposure of xenograft endothelial cells to complement activation by EXA.     

Inhibition of bispecific monoclonal antibody (bsAb)-targeted cytolysis by human anti-mouse antibodies in ovarian carcinoma patients treated with bsAb-targeted activated T-lymphocytes

T lymphocytes of 8 patients with ovarian cancer were targeted to the tumor cells using F(ab')2 fragments of a bispecific monoclonal antibody (bsAb), specific for CD3 (a component of the T lymphocyte receptor for antigen) and for the folate receptor MOv18 (overexpressed by ovarian carcinoma cells) as part of a phase I/II study. Phase I (days 0 to 3) consisted of increasing intraperitoneal (i.p.) numbers (10(6)-10(9)) of bsAb-targeted T lymphocytes plus low-dose interleukin-2 (IL-2). Phase II (days 6 to 13, and 27 to 33) consisted of daily i.p. infusions of 10(9) targeted T lymphocytes, 2 mg soluble bsAb, and low-dose IL-2. Using enzyme-linked immunosorbent assays (ELISA), human anti-mouse antibodies (HAMA) were detected in all patients: in the serum from day 13 onwards and in the peritoneal fluid from day 20 onwards. A significant proportion of the HAMA appeared to be directed against the idiotypes of the bsAb specific for CD3 and MOv18, as suggested by (1) the clearly higher ELISA titers against OC/TR bsAb as compared to those against a monoclonal antibody (MAb) with unrelated specificity, and (2) failure to abrogate the capacity of peritoneal fluid containing HAMA to block the binding of OC/TR bsAb to MOv18+ or CD3+ cells by absorption of human anti-mouse IgG-framework antibodies in peritoneal fluid to immobilized mouse IgG. The OC/TR-targeted cytolysis of the MOv18+ ovarian carcinoma cell line Igrov-I by autologous T lymphocytes was inhibited by peritoneal fluid samples containing relatively high HAMA titers. Such inhibitory activity was never detected at the start of phase II, but coincided with the last series of i.p. infusions of targeted T lymphocytes in 2 patients.     

Evidence of human non-alpha-galactosyl antibodies involved in the hyperacute rejection of pig lungs and their removal by pig organ perfusion

BACKGROUND: Human natural xenoantibodies represent a major hurdle to the clinical application of pig lungs in transplantation by initiating hyperacute rejection within minutes to hours. OBJECTIVE: The object was to compare pig organ perfusion and specific depletion of anti-alpha-galactosyl xenoantibodies for prevention of hyperacute rejection in the pig to human lung combination. METHODS: Large White pig (20-25 kg) left lungs were removed and continuously ventilated and reperfused ex vivo either with (1) whole human blood previously perfused in situ through pig right lung (group I), liver (group II), or spleen (group III) or with (2) human plasma in vitro immunoabsorbed on columns containing alpha-galactosyl disaccharide (Gal-alpha-(1-3)Gal-beta-(CH2)3NH2; B disaccharide) (group IV). Each study group included 6 animals. RESULTS: The in situ and in vitro preperfusions depleted anti-alpha-galactosyl xenoantibodies and all in situ perfused pig organs showed histologic signs of hyperacute rejection. After the ex vivo reperfusion, group I xenografts had a significantly (P < .001) longer functional and histologic survival than did xenografts in groups II, III, and IV. Human blood reperfusing group I xenografts had a significantly (P < 0.05) lower (1) decline of clotting factors and total circulating immunoglobulins, (2) total and membrane attack complex (C5b,6,7,8,9) complement activation, and (3) hemolysis. By Western blot analysis, the in situ lung preperfusion removed antibodies against non-alpha-galactosyl proteins of low molecular weight that were not eliminated by the alpha-galactosyl column. CONCLUSIONS: Results demonstrate that specific depletion of anti-alpha-galactosyl antibodies alone incompletely protects pig lungs from hyperacute rejection. It is speculated that the more complete prevention of this rejection afforded by pig lung preperfusion relates to the removal of other, non-alpha-galactosyl antibodies.     

The role of apoptosis in the thymic involution during growth of the syngeneic transplanted tumor in mice]

BACKGROUND: Natural antibodies (NAbs) against a terminal alpha1-3 galactosyl (alphaGal) epitope have been identified as the major human anti-pig NAbs. METHODS AND RESULTS: We used two synthetic alphaGal trisaccharides--type 6 (alphaGal6) and type 2(alphaGal2)--linked to an inert matrix to remove NAbs from human plasma in vitro. Flow cytometry indicated that an average of 85% of the NAb binding activity was depleted by adsorption with alphaGal6. By measuring the binding of NAbs to pig peripheral blood mononuclear cells and bone marrow cells, we demonstrated that alphaGal6 was more effective than alphaGal2 in removing NAbs, and the combination of alphaGal6 + alphaGal2 did not further increase removal of NAbs. The specificity of the removal of NAbs (IgM and IgG) reactive with the alphaGal epitope by alphaGal6 matrix was shown by enzyme-linked immunosorbent assay. In vivo studies in nonhuman primates compared plasma perfusion through a alphaGal6 immunoaffinity column with hemoperfusion through a pig liver for changes in blood pressure, hematocrit, platelets, and NAb adsorption. CONCLUSIONS: Both methods reduced the level of anti-pig IgM and IgG xenoreactive antibodies to nearly background, but column perfusion caused less hypotension and reduction in platelets than liver perfusion. Four pig kidneys transplanted into monkeys after column perfusion did not undergo hyperacute rejection, remaining functional for 2-10 days, with a mean functional period of 7 days, demonstrating that a pig kidney can support renal function in a primate.     

Human preformed IgG combining with membrane-bound porcine serotransferrin lyse porcine endothelial cells through antibody-dependent cellular cytotoxicity

Preformed antibodies are involved in xenograft rejection. The purpose of this work was to characterize porcine xenoantigens recognized by human preformed IgG (hpIgG), and to investigate the role of hpIgG in xenogeneic rejection. IgG eluted from porcine livers perfused with human plasma, human sera and total human IgG were immunoblotted on porcine aortic endothelial cell extracts. The amino acid sequence of a 76-kDa antigen constantly revealed was 100% homologous with porcine serotransferrin (psTf). hpIgG from human sera, human IgG1 and IgG2 and F(ab')2gamma specifically bound to psTf. Neutralization by psTf abolished that binding. Although alpha1,3-linked galactose residues (Gal(alpha)1,3Gal) is the dominant epitope recognized by preformed antibodies in the swine-to-human combination, the analysis of carbohydrate composition of psTf showed that the molecule was devoid of Gal(alpha)1,3Gal moieties and that preformed anti-psTf IgG bound to epitopes localized on the peptide core of the molecule. Purified human anti-psTf IgG antibodies were able to bind to psTf linked to its receptor on porcine endothelial cells, and to kill those cells through antibody-dependent cellular cytotoxicity.     

Developmental expression of CYP2C and CYP2C-dependent activities in the human liver: in-vivo/in-vitro correlation and inducibility

Discordant xenografts surviving the initial hyperacute rejection phase may be subject to cellular rejection processes mediated by infiltrating leukocytes including T cells, NK cells and monocytes. The stable adhesion of these cell types to endothelial cells is due to the molecular interaction of the integrins VLA-4 and LFA-1 with their ligands vascular cell adhesion molecule (VCAM) and ICAM-1 present on the endothelial cells. Human VLA-4 binds to porcine VCAM, and blocking mAbs specific for porcine VCAM have been developed. We have localized the epitope of the anti-porcine VCAM blocking mAbs 2A2 and 3F4 to domains 1 and 2, respectively. Humanized antibodies (IgG4 isotype) were constructed from these anti-porcine VCAM antibodies and demonstrated to inhibit adhesion of Ramos, Jurkat and YT cells, as well as purified resting and activated human T cells, to porcine aortic endothelial cells (PAEC). These cell types express both LFA-1 as well as VLA-4, suggesting blockade of human VLA-4 interaction with porcine VCAM may alone be sufficient to significantly impair adhesion of human leukocytes to porcine endothelial cells. The chimeric anti-porcine VCAM (pVCAM) HuG4 antibodies promoted increased adhesion of Fc receptor (FcR) positive cells such as U937 monocytic cells to PAEC. In contrast, chimeric anti-porcine VCAM antibodies created using the CH1 and hinge region from human IgG2 and the CH2 and CH3 regions from human IgG4 (HuG2/G4 antibodies) inhibited binding of FcR positive cells to PAEC. These chimeric anti-pVCAM antibodies should allow delineation of the in vivo role of VLA-4/VCAM interaction in porcine-to-primate xenotransplants. Further, the design of the HuG2/G4 antibodies should render them efficacious in multiple settings requiring elimination of FcR binding.     

Presence of anti-FKBP12 autoantibodies in patients with liver allografts: its association with allograft rejection

BACKGROUND: It was reported that autoantibodies against cyclophilin are present in sera from systemic lupus erythematosus. We hypothesized that autoantibodies against FKBP12, another immunophilin, may be present in the plasma of liver allograft recipients, which may affect the clinical outcome of liver allografts. METHODS: We investigated the relationship between the presence of anti-FKBP12 autoantibodies and rejection episodes in 47 patients treated with FK506 after living-related partial liver transplantation (LRLT). The patients consisted of two groups: 22 with rejection [R(+) group] and 25 without rejection [R(-) group]. The autoantibodies were measured by an indirect ELISA, and the specificity was confirmed by absorption with antigen and immunoblotting. RESULTS: The autoantibodies were detected in 13 of 22 in the R(+) group (IgG: 5; IgM: 6; both: 2) and in 6 of 25 in the R(-) group (IgG: 2; IgM: 3; both: 1) before LRLT (P=0.0193). After LRLT, they were also detected more frequently in the R(+) group (12 of 22; IgG: 1; IgM: 8; both: 3) than in the R(-) group (2 of 25; IgG: 1; IgM: 1) (P=0.001). In the R(+) group, the mortality of the patients who were positive and negative for the autoantibodies was 6 of 12 and 2 of 10, respectively. The autoantibodies were detected in all four patients with chronic or refractory acute rejection. The autoantibodies were not detected in any of the 34 healthy subjects. CONCLUSIONS: These results suggest that the presence of the autoantibodies in patients before transplantation is related to rejection, and the presence after transplantation may be associated with patient outcome.     

Virological diagnosis of an outbreak of fever and rashes caused by parvovirus B19, Cuba, 1995

We have previously described a form of xenograft rejection, mediated by natural killer (NK) cells, occurring in pig-to-primate organ transplants beyond the period of antibody-mediated hyperacute rejection. In this study, two distinct NK activation pathways were identified as mechanisms of pig aortic endotheliual cell (PAEC) lysis by human NK cells. Using an antibody-dependent cellular cytotoxicity (ADCC) assay, a progressive increase in human NK lysis of PAEC was observed following incubation with human IgG at increasing serum titer. In the absence of IgG, a second mechanism of PAEC lysis by human NK cells was observed following activation with IL-2. IL-2 activation of human NK cells increased lysis of PAEC by over 3-fold compared with ADCC. These results indicate that IL-2 activation of human NK cells induces significantly higher levels of lytic activity than does conventional ADCC involving IgG and FcRIII. We next investigated the role of MHC class I molecules in the regulation of NK lysis following IL-2 activation. PAEC expression of SLA class I molecules was increased by up to 75% by treatment with human TNFa. Following treatment with TNFa at 1 u/ml, IL-2 activated human NK lysis of PAEC was inhibited at every effector:target (E:T) ratio tested. Maximal effect occurred at an E:T ratio of 10:1, with TNFa inhibiting specific lysis by 59% (p < 0.01). Incubation with an anti-SLA class I Mab, but not IgG isotype control, abrogated the protective effects of TNFa on NK lysis of PAEC, suggesting direct inhibitory effects of SLA class I molecules on human NK function. To investigate whether human MHC class I molecules might have similar effects on human NK lysis of PAEC, further experiments were performed using a soluble peptide derived from the alpha-helical region of HLA-B7. Incubation with the HLA-B7 derived peptide significantly reduced the IL-2 activated NK lytic activity against PAEC in a dose-dependent fashion. Maximal effect occurred at a concentration of 10 mg/ml, where an 8-fold reduction in IL-2 augmented NK lysis was observed (p < 0.01). These results suggest that IL-2 activated human NK lysis of porcine xenografts may be inhibited by strategies which increase PAEC expression of SLA class I molecules, introduce HLA class I genes into PAEC, or use soluble HLA class I peptides.