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.

Distinct but parallel evolutionary patterns between alcohol and aldehyde dehydrogenases: addition of fish/human betaine aldehyde dehydrogenase divergence.

Alcohol dehydrogenases (ADHs) of the MDR type (medium-chain dehydrogenases/reductases) have diverged into two evolutionary groups in eukaryotes: a set of 'constant' enzymes (class III) typical of basal enzymes, and a set of 'variable' enzymes (remaining classes) suggesting 'evolving' forms. The variable set has larger overall variability, different segment variability, and variability also in functional segments. Using a major aldehyde dehydrogenase (ALDH) from cod liver and fish ALDHs deduced from the draft genome sequence of Fugu rubripes (Japanese puffer fish), we found that ALDHs form more complex patterns than the ADHs. Nevertheless, ALDHs also group into 'constant' and 'variable' sets, have separate segment variabilities, and distinct functions. Betaine ALDH (class 9 ALDH) is 'constant,' has three segments of variability, all non-functional, and a limited fish/human divergence, reminiscent of the ADH class III pattern. Enzymatic properties of fish betaine ALDH were also determined. Although all ALDH patterns are still not known, overall patterns are related to those of ADH, and group separations may be distinguished. The results can be interpreted functionally, support ALDH isozyme distinctions, and assign properties to the multiplicities of the ADH and ALDH enzymes.

Nicotinoprotein (NAD+ -containing) alcohol dehydrogenase: structural relationships and functional interpretations.

The primary structure of nicotinoprotein alcohol dehydrogenase (ADH) from Amycolatopsis methanolica was determined and used for modelling against known ADH structures, and for evaluation of the coenzyme binding. The results establish the medium-chain dehydrogenase/reductase nature of the nicotinoprotein ADH. Its subunit model and that of the human class Ibeta ADH subunit structure are similar, with mean a carbon deviations of 0.95 A, but they differ in seven loops. Nicotinoprotein ADH occupies a phylogenetic position intermediate between the dimeric and tetrameric ADH families. Two of the differing loops are important for coenzyme binding in the nicotinoprotein model, where one (with a Thr271Arg exchange towards the traditional enzyme) may suggest a slight rotation of the coenzyme adenine ring in the nicotinoprotein, and the other, with an Asn288 insertion, may suggest an extra hydrogen bond to its nicotinamide ribose, favouring stronger binding of the coenzyme. Combined with previous data, this suggests differences in the details of the tight coenzyme binding in different nicotinoproteins, but a common mode for this binding by loop differences.

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)

Deprivation of leukemia inhibitory factor by its function-blocking antibodies augments T cell activation.

Leukemia inhibitory factor (LIF) is a cytokine that acts on a wide range of cell types in vitro, but knowledge of its physiological role is limited. High levels of LIF protein have been selectively detected in the thymus throughout postnatal development. LIF-deficient mice have shown impaired thymic T cell maturation, suggesting the possibility that T cells require LIF for maturation. We have used highly specific antibodies raised against native rat LIF to inhibit LIF function during a defined and restricted period of thymic T cell maturation (first postnatal week). Surprisingly, we observed increased T cell activation in the LIF-deprived wild-type rat. The increased T cell response is retained even 4 weeks after anti-LIF treatment when the level of LIF in the thymic microenvironment has returned to normal. Our results are in contrast to findings with LIF knockout mice, where decreased T cell activation was observed. These observations suggest that LIF may have alternative effects on various phases of T cell development and that LIF may be involved in the restriction of the T cell repertoire during maturation occurring in the first postnatal week.

Mycothiol-dependent formaldehyde dehydrogenase, a prokaryotic medium-chain dehydrogenase/reductase, phylogenetically links different eukaroytic alcohol dehydrogenases--primary structure, conformational modelling and functional correlations.

Prokaryotic mycothiol-dependent formaldehyde dehydrogenase has been structurally characterized by peptide analysis of the 360-residue protein chain and by molecular modelling and functional correlation with the conformational properties of zinc-containing alcohol dehydrogenases. The structure is found to be a divergent medium-chain dehydrogenase/reductase (MDR), at a phylogenetic position intermediate between the cluster of dimeric alcohol dehydrogenases of all classes (including the human forms), and several tetrameric reductases/dehydrogenases. Molecular modelling and functionally important residues suggest a fold of the mycothiol-dependent formaldehyde dehydrogenase related overall to that of MDR alcohol dehydrogenases, with the presence of the catalytic and structural zinc atoms, but otherwise much altered active-site relationships compatible with the different substrate specificity, and an altered loop structure compatible with differences in the quaternary structure. Residues typical of glutathione binding in class-III alcohol dehydrogenase are not present, consistent with that the mycothiol factor is not closely similar to glutathione. The molecular architecture is different from that of the 'constant' alcohol dehydrogenases (of class-III type) and the 'variable' alcohol dehydrogenases (of class-I and class-II types), further supporting the unique structure of mycothiol-dependent formaldehyde dehydrogenase. Borders of internal chain-length differences between this and other MDR enzymes coincide in different combinations, supporting the concept of limited changes in loop regions within this whole family of proteins.

Class 2 aldehyde dehydrogenase. Characterization of the hamster enzyme, sensitive to daidzin and conserved within the family of multiple forms.

Mitochondrial (class 2) hamster aldehyde dehydrogenase has been purified and characterized. Its primary structure has been determined and correlated with the tertiary structure recently established for this class from another species. The protein is found to represent a constant class within a complex family of multiple forms. Variable segments that occur in different species correlate with non-functional segments, in the same manner as in the case of the constant class of alcohol dehydrogenases (class III type) of another protein family, but distinct from the pattern of the corresponding variable enzymes. Hence, in both these protein families, overall variability and segment architectures behave similarly, with at least one 'constant' form in each case, class III in the case of alcohol dehydrogenases, and at least class 2 in the case of aldehyde dehydrogenases.

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.

Class III alcohol dehydrogenase from Saccharomyces cerevisiae: structural and enzymatic features differ toward the human/mammalian forms in a manner consistent with functional needs in formaldehyde detoxication.

Alcohol dehydrogenase class III (glutathione-dependent formaldehyde dehydrogenase) from Saccharomyces cerevisiae was purified and analyzed structurally and enzymatically. The corresponding gene was also analyzed after cloning from a yeast genome library by screening with a probe prepared through PCR amplification. As with class III alcohol dehydrogenase from other sources, the yeast protein was obtained in two active forms, deduced to reflect different adducts/modifications. Protein analysis established N-terminal and C-terminal positions, showing different and specific patterns in protein start positions between the human/mammalian, yeast, and prokaryotic forms. Km values with formaldehyde differ consistently, being about 10-fold higher in the yeast than the human/mammalian enzymes, but compensated for by similar changes in kcat values. This is compatible with the different functional needs, emphasizing low formaldehyde concentration in the animal cells but efficient formaldehyde elimination in the microorganisms. This supports a general role of the enzyme in formaldehyde detoxication rather than in long-chain alcohol turnover.

Pulmonary fibroblast function in an acute lung injury model.

The role of pulmonary fibroblasts (PFBs) in early adult respiratory distress syndrome is poorly understood. To investigate PFB cellular function in acute lung injury, New Zealand rabbits (2 to 3 kg) were given either three daily doses of phorbol myristate acetate (PMA; 65 micrograms/kg, IV), a potent stimulator of oxygen radical formation, or saline (control). On day 4, the lungs were harvested, subjected to enzymatic digestion, and PFBs isolated via serial subculture. Proliferation was assessed via 6-hour pulsed [3H]thymidine incorporation and by creating 5-day growth curves. Confluent PFB cultures were assessed for collagen production and total protein production, as well as interleukin (IL)-1 alpha secretion. Qualitative comparisons using transmission electron micrography were also made. There were no differences between PFBs harvested from control versus PMA-treated animals in terms of growth rates, total protein, and IL-1 alpha production. However, there was a significant difference in collagen production, with the PMA-treated animals' PFBs producing 35% more collagen than controls. Transmission electron micrography revealed PMA fibroblasts to be smaller (two to three times), have more dark staining granules, and have hypertrophied smooth endoplasmic reticulum--all consistent with increased metabolic activity. This suggests that pulmonary fibrosis, a late development in adult respiratory distress syndrome, may be triggered during the acute phase of lung injury. The increase in collagen synthesis is not related to PFB proliferation or the secretion of IL-1 alpha.

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.