Impact of human leukocyte antigen-DR mismatch status on kidney graft survival in a predominantly African-American population under the newer immunosuppressive era.

BACKGROUND: Human leukocyte antigen (HLA)-DR has been shown to be immunogenic and associated with poor long-term graft function. However, under potent induction immunosuppression with antithymocyte globulin, the impact of the HLA-DR remains unclear. METHOD: We reviewed 672 renal transplant recipients who received their transplants between 1998 and 2007. All patients received antithymocyte globulin as induction therapy followed by tacrolimus + prednisone + mycophenolate mofetil for maintenance immunosuppression. We divided the patients into three groups according to HLA-DR mismatch status (zero, one, or two mismatches). RESULTS: The three groups were different in total number of mismatches, deceased donor transplant, and delayed graft function, respectively. By Kaplan-Meier survival analysis, actuarial graft survival was significantly lower in the HLA-DR two mismatches group (72%) compared to HLA-DR zero mismatches group (78.5%) or HLA-DR one mismatch group (78.5%; P = .05, by log-rank test). Using Cox regression analysis, the risk of graft failure with two HLA-DR mismatches as compared with zero HLA-DR mismatches was 1.6 (95% confidence interval = 1.0-2.44, P = .049). When adjusted for age, wait time, race, type of transplant, retransplant status, T-cell flow crossmatch, delayed graft function, acute rejection, HLA-A and HLA-B, the effect of HLA-DR on survival was not significant (P = .55). CONCLUSION: The independent effect of HLA-DR mismatches on adverse graft survival is diminished under potent antibody induction and maintenance immunosuppression in our predominantly African-American population.

Genetic identity and differential expression of p38.5 (Haymaker) in human malignant and nonmalignant cells.

Previous studies from our laboratory revealed a novel protein of 38.5 kD on the surface of malignant cell lines of hematopoetic origin that exhibit susceptibility to naive natural killer (NK) cell-mediated lysis. In contrast, p38.5 was not detected on the surface of NK cell-resistant carcinoma cell lines or normal cells. We now report that this protein is differentially expressed, intracellularly, in malignant cell lines of both hematopoetic and epithelial origin compared with nonmalignant cells. To characterize p38.5 further, we used a previously developed antipeptide antibody (anti-11-mer) to probe cDNA expression libraries and subsequently performed 5' extension by rapid amplification of cDNA ends (RACE). Sequence analyses of these cDNA clones reveal open reading frames (ORFs) that include the previously identified 11-mer peptide from purified, native p38.5 and that have identical sequences to a gene of unknown function on chromosome 19. Nucleotide sequence data obtained from these cDNA clones, as well as analysis of the genomic sequence, permitted design of primers for reverse transcriptase-polymerase chain reaction (RT-PCR) that resulted in a cDNA clone encoding an ORF of 361 amino acids; the clone was identical to a sequence encoded by an unpublished mRNA in GenBank. Anti-p38.5 antibody against the 11-mer peptide encoded in exon 5 and against a 25-mer peptide encoded in exon 1 both reacted with the same protein in immunoprecipitation studies, providing further evidence of identity. RT-PCR and Northern blot analyses both demonstrated p38.5 gene transcripts in normal cells, nonmalignant cell lines and malignant cell lines of epithelial as well as hematopoietic origin. Semiquantitative studies revealed a greater level of p38.5 gene transcription in malignant cell lines compared with nonmalignant cells. Immunoblot analyses of protein expression confirmed and extended the latter studies by revealing substantially greater levels of the 38.5 kD protein in whole cell extracts of malignant cell lines compared with nonmalignant cells. Quantitative differences in detection of the 38.5 kD protein and mRNA in NK susceptible- hematopoietic malignancies compared with NK resistant-carcinomas were not observed. These experiments suggest that the p38.5 gene (Haymaker) is widely expressed in human cells of different tissue origins but that elevated expression is associated with the malignant phenotype.

Lack of cross-species transmission of porcine endogenous retrovirus infection to nonhuman primate recipients of porcine cells, tissues, or organs.

BACKGROUND: Nonhuman primates (NHPs) have been widely used in different porcine xenograft procedures inevitably resulting in exposure to porcine endogenous retrovirus (PERV). Surveillance for PERV infection in these NHPs may provide information on the risks of cross-species transmission of PERV, particularly for recipients of vascularized organ xenografts for whom data from human clinical trials is unavailable. METHODS: We tested 21 Old World and 2 New World primates exposed to a variety of porcine xenografts for evidence of PERV infection. These NHPs included six baboon recipients of pig hearts, six bonnet macaque recipients of transgenic pig skin grafts, and nine rhesus macaque and two capuchin recipients of encapsulated pig islet cells. Serologic screening for PERV antibody was done by a validated Western blot assay, and molecular detection of PERV sequences in peripheral blood mononuclear cells (PBMCs) and plasma was performed using sensitive polymerase chain reaction and reverse transcriptase-polymerase chain reaction assays, respectively. Spleen and lymph node tissues available from six bonnet macaques and three rhesus macaques were also tested for PERV sequences. RESULTS: All plasma samples were negative for PERV RNA suggesting the absence of viremia in these xenografted animals. Similarly, PERV sequences were not detectable in any PBMC and tissue samples, arguing for the lack of latent infection of these compartments. In addition, all plasma samples were negative for PERV antibodies. CONCLUSION: These data suggest the absence of PERV infection in all 23 NHPs despite exposure to vascularized porcine organs or tissue xenografts and the use of immunosuppressive therapies in some animals. These findings suggest that PERV is not easily transmitted to these NHP species through these types of xenografts.

Cloning of the human platelet F11 receptor: a cell adhesion molecule member of the immunoglobulin superfamily involved in platelet aggregation.

This study demonstrates that the human platelet F11 receptor (F11R) functions as an adhesion molecule, and this finding is confirmed by the structure of the protein as revealed by molecular cloning. The F11R is a 32-/35-kd protein duplex that serves as the binding site through which a stimulatory monoclonal antibody causes platelet aggregation and granule secretion. A physiological role for the F11R protein was demonstrated by its phosphorylation after the stimulation of platelets by thrombin and collagen. A pathophysiological role for the F11R was revealed by demonstrating the presence of F11R-antibodies in patients with thrombocytopenia. Adhesion of platelets through the F11R resulted in events characteristic of the action of cell adhesion molecules (CAMs). To determine the structure of this protein, we cloned the F11R cDNA from human platelets. The predicted amino acid sequence demonstrated that it is an integral membrane protein and an immunoglobulin superfamily member containing 2 extracellular C2-type domains. The structure of the F11R as a member of a CAM family of proteins and its activity in mediating adhesion confirm each another. We conclude that the F11R is a platelet-membrane protein involved in 2 distinct processes initiated on the platelet surface. The first is antibody-induced platelet aggregation and secretion that are dependent on both the FcgammaRII and the GPIIb/IIIa integrin and that may be involved in pathophysiological processes associated with certain thrombocytopenias. The second is an F11R-mediated platelet adhesion that is not dependent on either the FcgammaRII or the fibrinogen receptor and that appears to play a role in physiological processes associated with platelet adhesion and aggregation. (Blood. 2000;95:2600-2609)

Laboratory studies in cross-species lung transplantation.

The lack of sufficient suitable human donor lungs for the many patients requiring pulmonary transplantation as life-saving therapy for end-stage lung diseases has generated extensive interest in cross-species lung transplantation. Ethical concerns and those of animal rights advocates have prompted studies of nonprimate species as potential solid organ donors for humans. This paper provides an overview of some of the laboratory studies of cross-species pulmonary transplantation performed over the past 20 years and focuses, in particular, on more recent work (from our laboratory and others) in the area of porcine-to-primate pulmonary xenotransplantation.

Preferential interaction of a novel tumor surface protein (p38.5) with naive natural killer cells.

A receptor-ligand interaction exclusive to natural killer (NK) cell-mediated recognition and triggering of tumor cell destruction has not yet been identified. In contrast, molecules that are involved in cellular adhesion and regulation of NK cytolysis have been well studied. In this report, a novel tumor surface protein is identified that exhibits characteristics of a recognition structure for naive NK cells. A tagged ligand-cell adsorption technique revealed a 38.5-kD plasma membrane protein (p38.5) from a prototypical NK-susceptible cell line (K562) that preferentially bound to NK cells (CD3(-)CD5(-)CD16(+)) relative to T lymphocytes (CD3(+)CD5(+) CD16(-)). The molecule was purified to apparent homogeneity for further characterization. An amino acid sequence of an 11-mer internal peptide of p38.5 did not exhibit homology to known proteins. Affinity-purified antibody generated against this peptide (anti-p38.5) reacted with a single protein of 38.5 kD on Western blots of whole cell extracts of K562. Flow cytometry and immunoprecipitation studies of surface-labeled tumor cells demonstrated expression of p38.5 on NK-susceptible tumor cell lines (K562, MOLT-4, Jurkat), whereas p38.5 was not detected on NK-resistant tumor cell lines (A549, Raji, MDA-MB-231). Significantly, p38.5 loss variants derived from wild-type Jurkat and Molt-4 cell lines exhibited decreased susceptibility to NK cell-mediated lysis demonstrating a strong association between cell surface expression of p38.5 and cytotoxicity. Purified p38.5 retained preferential binding to NK cells and inhibited NK activity in a dose-dependent manner, thereby providing direct evidence of a role in the lytic process. Binding studies identified a 70-kD membrane protein from NK cells as a possible receptor for the p38.5 tumor ligand. Consistent with cellular adsorption studies, the 70-kD, p38.5 binding protein was not detected on T lymphocytes. Based on studies demonstrating selective binding of p38.5 to NK cells, lack of expression on NK-resistant tumor cell lines and ability of the purified molecule to block cytolysis, we conclude that p38.5 may serve as a recognition/triggering ligand for naive human NK cells.

In vitro and in vivo inhibition of complement activity by a single-chain Fv fragment recognizing human C5.

Complement activation has been implicated in the pathogenesis of several human diseases. Recently, a monoclonal antibody, (N19-8) that recognizes the human complement protein C5 has been shown to effectively block the cleavage of C5 into C5a and C5b, thereby blocking terminal complement activation. In this study, a recombinant N19-8 scFv antibody fragment was constructed from the N19-8 variable regions, and produced in both mammalian and bacterial cells. The N19-8 scFv bound human C5 and was as potent as the N19-8 monoclonal antibody at inhibiting human C5b-9-mediated hemolysis of chicken erythrocytes. In contrast, the N19-8 scFv only partially retained the ability of the N19-8 monoclonal antibody to inhibit C5a generation. To investigate the ability of the N19-8 scFv to inhibit complement-mediated tissue damage, complement-dependent myocardial injury was induced in isolated mouse hearts by perfusion with Krebs-Henseleit buffer containing 6% human plasma. The perfused hearts sustained extensive deposition of human C3 and C5b-9, resulting in increased coronary artery perfusion pressure, end-diastolic pressure, and a decrease in heart rate until the hearts ceased beating approximately 10 min after addition of plasma. Hearts treated with human plasma supplemented with either the N19-8 monoclonal antibody or the N19-8 scFv did not show any detectable changes in cardiac performance for at least 1 hr following the addition of plasma. Hearts treated with human plasma alone showed extensive deposition of C3 and C5b-9, while hearts treated with human plasma containing N19-8 scFv showed extensive deposition of C3, but no detectable deposition of C5b-9. Administration of a 100 mg bolus dose of N19-8 scFv to rhesus monkeys inhibited the serum hemolytic activity by at least 50% for up to 2 hr. Pharmacokinetic analysis of N19-8 scFv serum levels suggested a two-compartment model with a T1/2 alpha of 27 min. Together, these data suggest the recombinant N19-8 scFv is a potent inhibitor of the terminal complement cascade and may have potential in vivo applications where short duration inhibition of terminal complement activity is desirable.

A novel ligand in lymphocyte-mediated cytotoxicity: expression of the beta subunit of H+ transporting ATP synthase on the surface of tumor cell lines.

Extracellular adenosine triphosphate (eATP) has been suggested to play a role in lymphocyte-induced tumor destruction. We now provide evidence that a protein responsible for ATP synthesis in mitochondria may also play a physiologic role in major histocompatibility complex-independent, lymphocyte-mediated cytotoxicity. A 51.5-kD protein (p51.5) bearing structural and immunologic characteristics of the beta subunit of H+ transporting ATP synthase (E.C. 3.6.1.34, beta-H+ATPase, published molecular mass of 51.6 kD) was detected on the plasma membrane of three different human tumor cell lines studied. NH2-terminal amino acid sequence analysis of purified p51.5 from K562 tumor cells revealed 100% homology of 16 residues identified in the first 21 positions to the known sequence of human mitochondrial beta-H+ ATPase. Antibody directed against a 21-mer peptide in the ATP binding region of beta-H+ ATPase (anti-beta) reacted with only one band on Western blots of whole tumor extracts and tumor membrane extracts suggesting that the antiserum reacts with a single species of protein. Anti-beta reacted with the cell membranes of tumor cells as determined by fluorescence-activated flow cytometry and immunoprecipitated a 51.5-kD protein from surface-labeled neoplastic cells (but not human erythrocytes and lymphocytes). Purified p51.5 bound to human lymphocytes and inhibited natural killer (NK) cell-mediated cytotoxicity. Furthermore, anti-beta treatment of the K562 and A549 tumor cell lines inhibited NK (by > 95%) and interleukin 2-activated killer (LAK) cell (by 75%) cytotoxicity, respectively. Soluble p51.5 upon binding to lymphocytes retained its reactivity to anti-beta suggesting that the ATP binding domain and the lymphocyte-receptor binding domain reside in distinct regions of the ligand. These results suggest that beta-H+ ATPase or a nearly identical molecule is an important ligand in the effector phase (rather than the recognition phase) of a cytolytic pathway used by naive NK and LAK cells.

GAD antibody negative NIDDM in adult black subjects with diabetic ketoacidosis and increased frequency of human leukocyte antigen DR3 and DR4. Flatbush diabetes.

The objective of this study is to understand the metabolic and immunologic basis of diabetes in adult blacks with diabetic ketoacidosis (DKA). Twenty-one black adults presenting with DKA ([mean +/- SD] blood pH = 7.18 +/- 0.09, plasma glucose = 693 +/- 208 mg/dl, and positive serum ketones) had a subsequent clinical course of non-insulin-dependent diabetes mellitus (NIDDM). Human leukocyte antigens (HLAs) DR and DQ and antibodies to glutamic acid decarboxylase (GAD) and islet cell cytoplasmic proteins (ICP) were measured to assess autoimmunity. Insulin action was evaluated by the euglycemic insulin clamp, and insulin secretion was measured by C-peptide responses to oral glucose. Ketoacidosis was treated with insulin. Two subjects had a precipitating illness; four had a history of NIDDM. At the time of study, subjects' glycemic control was good (HbA1c = 5.7 +/- 1.6%). Nine subjects were treated with insulin, and 12 were on either sulfonylurea treatment or diet alone. Men (n = 12) were younger than women (n = 9) (40.8 +/- 9.8 and 51.1 +/- 6.3 years of age, respectively, P < 0.05) but similar in body mass index (27.8 +/- 2.7 and 29.98 +/- 4.1 kg/m2, respectively). Antibodies to GAD and ICP were absent. All but one subject was insulin resistant compared with normal subjects (glucose disposal 3.56 +/- 0.04 vs. 6.86 +/- 0.02 mg.kg-1.min-1), and insulin secretion was lower. HLA DR3 and DR4 frequency was higher than in nondiabetic black control subjects (65 vs. 30%, P < 0.012).(ABSTRACT TRUNCATED AT 250 WORDS)

HLA-DQ associations distinguish insulin-resistant and insulin-sensitive variants of NIDDM in black Americans.

OBJECTIVE: NIDDM in black Americans exists as two variants: one with a primary defect in insulin action (insulin-resistant variant) and the other with normal insulin action and a primary defect in insulin secretion (insulin-sensitive variant). The objective of this study was to determine whether these two variants were genetically distinct from each other and from normal control subjects as determined by HLA typing. RESEARCH DESIGN AND METHODS: Insulin action was measured with the euglycemic insulin clamp with a 1 mU.kg-1.min-1 insulin infusion with [3-3H]glucose. A glucose disposal of < 278 mumol.kg-1.min-1 was considered insulin resistant, and a value greater than this was considered insulin sensitive. The study population consisted of 21 insulin-resistant and 25 insulin-sensitive black NIDDM patients and 89 normal, nondiabetic black control subjects from an urban hospital. HLA typing was performed with serological methods. RESULTS: The frequency of HLA-DQW7 in the insulin-resistant population (76%) was significantly greater than that in the insulin-sensitive population (32%, corrected P < 0.018) and the normal control population (21%, corrected P < 0.001). The frequency of HLA-DQW6 was increased in the insulin-sensitive population (76%), corrected P < 0.023, as compared with the normal control subjects (33%). The relative risk of HLA-DQW7 in identifying insulin-resistant NIDDM patients compared with control subjects was 7. CONCLUSIONS: At least one component that differentiates insulin-resistant and insulin-sensitive NIDDM in black Americans is under different genetic control. One or more loci responsible for insulin-resistant and insulin-sensitive NIDDM are likely to be in linkage disequilibrium with the DQ locus of the human MHC region of chromosome 6.

One-year monkey heart xenograft survival in cyclosporine-treated baboons. Suppression of the xenoantibody response with total-lymphoid irradiation.

The shortage of cadaveric human organs for transplantation could be alleviated by the use of xenografts. Long-term (> one-year) survival of xenografts in humans or experimental animals has not been achieved with previous immunosuppressive protocols. Poor results in xenotransplantation compared with allotransplantation have been attributed to a more potent antibody response rather than to cell-mediated responses. To investigate these issues a concordant heterotopic cardiac xenograft model was developed in conjunction with cyclosporine and/or total-lymphoid irradiation. Concordant models permit examination of xenoantigen induced antibody and cell-mediated responses since preformed humoral factors (in discordant models) do not cause hyperacute rejection. Four groups of baboon recipients received cervical heart transplants from cynomolgous monkeys. Group I (n = 2), untreated, mean survival (MS) = 6 days; group II (n = 5), CsA and methylprednisolone, MS = 25 days; group III (n = 3), preoperative TLI, MS = 29 days; group IV (n = 6), preoperative TLI and CsA+MP, MS = 255 days (> 77, > 108, > 142, 184, > 480, 540 days). Heart xenografts of CsA-MP-treated recipients appear to be destroyed predominantly by antibody (IgM)-mediated processes whereas cell-mediated rejection is likely the major reaction in TLI-treated recipients. CsA-MP-treated recipients had early immunohistochemical evidence of antibody and complement-mediated rejection (deposition of IgM and complement but not IgG on heart xenografts). In contrast IgM and complement deposits were not detected on heart xenografts in TLI- and TLI-CsA-treated recipients. IgG xenoantibodies were only detected on the two rejected heart grafts of TLI-CsA-treated recipients. CsA-MP-treated recipients rapidly developed high xenoantibody titers (1:256 to 1:512) that immediately preceded rejection. In contrast, TLI-treated animals developed lower levels of xenoantibody (< or = 1:8) and TLI-CsA-treated recipients had no detectable xenoantibody during the initial three months after transplantation (and titers no greater than 1:8 thereafter.) The lack of xenoantibody was likely not due to a generalized inhibitory effect of the immunosuppressants on B cell function since all classes of serum immunoglobulins were in the normal range. Intragraft cytolytic lymphocyte activity was detected in rejecting TLI- and TLI-CsA-treated recipients but could not be detected in xenografts of CsA-MP-treated recipients. One explanation for these data is that TLI (either directly or indirectly) induces a state of specific B cell unresponsiveness to monkey xenoantigens, thereby preventing IgM mediated rejection in the third week after transplantation.(ABSTRACT TRUNCATED AT 400 WORDS)

Suppression of C3H/HeJ cell activation by lipopolysaccharide endotoxin.

Earlier studies in our laboratory showed that the lipopolysaccharide (LPS) of Salmonella typhi, which fails to activate B lymphocytes of C3H/HeJ mice, can suppress proliferation and polyclonal antibody synthesis by these cells when they are stimulated by polyclonal activators. In order to determine what stage of the cell cycle was blocked, resting B cells from C3H/HeJ spleens were activated by using different mitogens in the presence of inhibitory concentrations of LPS and analyzed by flow cytometry, using acridine orange to stain DNA and RNA. LPS was found to inhibit the progression of cells into the G1 stage of the cell cycle. Furthermore, [3H]uridine uptake studies showed that RNA synthesis is inhibited during the early phase of activation. These results indicate that inhibition by LPS of the signalling process occurs during a critical period of the cell cycle when the cells become susceptible to the inhibitory effects of LPS. To examine whether LPS acts only on B cells or whether it can suppress other immunocompetent cells from C3H/HeJ mice, studies were carried out on activated thymocytes and macrophages. LPS was found to inhibit thymocyte proliferation stimulated by concanavalin A or the combination of phorbol myristate acetate and ionomycin. Prostaglandin E2 synthesis by macrophages was also blocked by LPS. Thus, LPS is a potent inhibitor of the functioning of the major immunocompetent cells of C3H/HeJ mice.