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Objective: Statins reduce inflammation and risk of myocardial infarction (MI). Because the myeloid IgA Fc receptor encoded by FCAR mediates inflammation, we hypothesized that the FCAR Asp92Asn polymorphism is associated with risk of MI and that this risk would be modified by pravastatin., Methods and Results: In the placebo arm of the Cholesterol and Recurrent Events (CARE) study, male carriers of the 92Asn allele had an adjusted hazard ratio for incident MI of 1.68 (95% CI 1.10 to 2.57); relative risk reduction by pravastatin was 69% in carriers and 12% in noncarriers (P(interaction)=0.007). In the placebo arm of the all-male West of Scotland Coronary Prevention Study (WOSCOPS), carriers had an adjusted odds ratio for incident coronary heart disease (CHD) of 1.46 (90% CI 1.05 to 2.03); for pravastatin compared with placebo treatment, the adjusted odds ratios were 0.55 (95% CI 0.32 to 0.93) in carriers and 0.65 (95% CI 0.51 to 0.83) in noncarriers (P(interaction)=0.55)., Conclusions: Carriers of 92Asn had increased risk of MI in CARE and increased odds of CHD in WOSCOPS. Pravastatin significantly reduced risk in carriers in both CARE and WOSCOPS. A genotype by treatment interaction was observed in CARE but not in WOSCOPS.

Coronary heart disease (CHD) has a genetic component independent of traditional risk factors. However, the risk associated with individual genetic polymorphisms is likely modest, and therefore initial claims of associations with CHD require testing in other studies. We surveyed the literature for studies that retested polymorphisms previously reported to be associated with CHD, and found that these associations were reproduced for only 15 of the 172 polymorphisms considered. This review discusses how the modest risk associated with each individual polymorphism generates false-positive, as well as false-negative results. Common strategies to address these issues are described, including methods to aggregate risk estimates from several polymorphisms into a genetic risk score.

Objective: Identify gene variants associated with early-onset myocardial infarction (MI)., Methods and Results: We tested 11 647 single-nucleotide polymorphisms (SNPs) for association with early-onset MI in a case-control study (study 1 200 cases, 262 controls). To reduce the number of false positives among the 666 SNPs that were nominally associated with early-onset MI (P<0.05) in study 1, we tested these SNPs in study 2 (434 cases, 504 controls). We found that 8 of the 666 SNPs were associated with early-onset MI in study 2 (P<0.05) and had the same risk alleles as in study 1. These 8 SNPs were then tested for association with early-onset MI in study 3 (187 cases, 434 controls). We found that a VAMP8 variant (P = 0.025; odds ratio [OR], 1.75; CI, 1.17 to 2.62) and an HNRPUL1 variant (P = 0.0043; OR, 1.92; CI, 1.28 to 2.86) were associated with early-onset MI (nominal P<0.05; false discovery rate <10%) and had the same risk alleles in all 3 studies., Conclusions: Variants in 2 genes were associated with early-onset MI: VAMP8, which is involved in platelet degranulation, and HNRPUL1, which encodes a ribonuclear protein. The identification of these variants could improve understanding of disease mechanisms and suggest novel drug targets.

Family history is a major risk factor for myocardial infarction (MI). However, known gene variants associated with MI cannot fully explain the genetic component of MI risk. We hypothesized that a gene-centric association study that was not limited to candidate genes could identify novel genetic associations with MI. We studied 11,053 single-nucleotide polymorphisms (SNPs) in 6,891 genes, focusing on SNPs that could influence gene function to increase the likelihood of identifying disease-causing gene variants. To minimize false-positive associations generated by multiple testing, two studies were used to identify a limited number of nominally associated SNPs; a third study tested the hypotheses that these SNPs are associated with MI. In the initial study (of 340 cases and 346 controls), 637 SNPs were associated with MI (P<.05); these were evaluated in a second study (of 445 cases and 606 controls), and 31 of the 637 SNPs were associated with MI (P<.05) and had the same risk allele as in the first study. For each of these 31 SNPs, we tested the hypothesis that it is associated with MI, using a third study (of 560 cases and 891 controls). We found that four of these gene variants were associated with MI (P<.05; false-discovery rate <10%) and had the same risk allele as in the first two studies. These gene variants encode the cytoskeletal protein palladin (KIAA0992 [odds ratio (OR) 1.40]), a tyrosine kinase (ROS1 [OR 1.75]), and two G protein-coupled receptors (TAS2R50 [OR 1.58] and OR13G1 [OR 1.40]); all ORs are for carriers of two versus zero risk alleles. These findings could lead to a better understanding of MI pathophysiology and improved patient risk assessment.

A series of potent and selective inducible nitric-oxide synthase (iNOS) inhibitors was shown to prevent iNOS dimerization in cells and inhibit iNOS in vivo. These inhibitors are now shown to block dimerization of purified human iNOS monomers. A 3H-labeled inhibitor bound to full-length human iNOS monomer with apparent Kd approximately 1.8 nm and had a slow off rate, 1.2 x 10(-4) x s(-1). Inhibitors also bound with high affinity to both murine full-length and murine oxygenase domain iNOS monomers. Spectroscopy and competition binding with imidazole confirmed an inhibitor-heme interaction. Inhibitor affinity in the binding assay (apparent Kd values from 330 pm to 27 nm) correlated with potency in a cell-based iNOS assay (IC50 values from 290 pm to 270 nm). Inhibitor potency in cells was not prevented by medium supplementation with l-arginine or sepiapterin, but inhibition decreased with time of addition after cytokine stimulation. The results are consistent with a mechanism whereby inhibitors bind to a heme-containing iNOS monomer species to form an inactive iNOS monomer-heme-inhibitor complex in a pterin- and l-arginine-independent manner. The selectivity for inhibiting dimerization of iNOS versus endothelial and neuronal NOS suggests that the energetics and kinetics of monomer-dimer equilibria are substantially different for the mammalian NOS isoforms. These inhibitors provide new research tools to explore these processes.

Potent and selective inhibitors of inducible nitric oxide synthase (iNOS) (EC ) were identified in an encoded combinatorial chemical library that blocked human iNOS dimerization, and thereby NO production. In a cell-based iNOS assay (A-172 astrocytoma cells) the inhibitors had low-nanomolar IC(50) values and thus were >1,000-fold more potent than the substrate-based direct iNOS inhibitors 1400W and N-methyl-l-arginine. Biochemical studies confirmed that inhibitors caused accumulation of iNOS monomers in mouse macrophage RAW 264.7 cells. High affinity (K(d) approximately 3 nM) of inhibitors for isolated iNOS monomers was confirmed by using a radioligand binding assay. Inhibitors were >1,000-fold selective for iNOS versus endothelial NOS dimerization in a cell-based assay. The crystal structure of inhibitor bound to the monomeric iNOS oxygenase domain revealed inhibitor-heme coordination and substantial perturbation of the substrate binding site and the dimerization interface, indicating that this small molecule acts by allosterically disrupting protein-protein interactions at the dimer interface. These results provide a mechanism-based approach to highly selective iNOS inhibition. Inhibitors were active in vivo, with ED(50) values of <2 mg/kg in a rat model of endotoxin-induced systemic iNOS induction. Thus, this class of dimerization inhibitors has broad therapeutic potential in iNOS-mediated pathologies.

Recombinant retroviral vectors are useful tools for gene transfer in both gene therapy and research applications. An enhanced form of green fluorescent protein has been incorporated into recombinant retroviruses as a marker to follow infected cells. In this paper, we extended the use of the fluorescent reporter to quantify protein expression using such analytical tools as fluorescent microscopy, flow cytometry and fluorescent plate reader analysis. These tools enabled us to rapidly assess the titer of recombinant retrovirus harvested from packaging cells and to optimize parameters for infection of different cell lines.

The ethnic origin of renal graft recipients is recognized as an important determinant of graft survival. In liver transplantation, the effect of racial origin has been studied in black American recipients and has suggested a trend toward inferior graft survival in this group. In this study, we have analyzed outcome of transplantation in a large multiethnic liver transplant program. Non-Caucasoid recipients had an inferior patient survival compared with Caucasoids and, in particular, European Caucasoids at 1, 3, and 5 years after transplantation (46.7% vs. 60.2% at 3 years, P = 0.05). Non-European recipients had an inferior graft survival compared with European recipients at 1, 2, and 3 years after transplantation (e.g., north Europeans 53.5%, south Europeans 48.5%, Middle Eastern 40%, and non-Caucasoids 27% at 3 years, P < 0.01). Different frequencies of chronic allograft rejection in the ethnic groups contributed to the rates of graft survival, with the non-European recipients developing chronic rejection at over twice the rate of European recipients (12.6% vs. 5.9%, respectively, P = 0.002). The findings in this study support the evidence from renal transplant programs that the ethnic origin of recipients is an important determinant of outcome after transplantation, with increasing frequency of chronic rejection in recipients nonindigenous to the donor population contributing to the variations in patient and graft survival rates.

We report two cases of recurrence of primary biliary cirrhosis (PBC) in the transplanted liver whilst maintained on a FK506-based immunosuppressive regime, the first to be described. One patient experienced symptoms in association with the development of cholestasis. In both there was a persistence of serological markers of PBC and liver histology revealed florid bile duct destruction and a granulomatous reaction.

Libraries of random peptide sequences were constructed and screened to identify peptides that specifically bind to proteins. In one of these about 2 X 10(7) different 15-residue peptide sequences were expressed on the surface of the coliphage M13. Each phage encoded a single random sequence and expressed it as a fusion complex with pIII, a minor coat protein present at five molecules per phage. Phage encoding nine different streptavidin-binding peptide sequences were isolated from this library. The core consensus sequence was His-Pro-Gln and binding of these phage to streptavidin was inhibited by biotin. This type of library makes it possible to identify peptides that bind to proteins (or other macromolecules) that have no previously known affinity for peptides.

DNA restriction maps of the major histocompatibility complex and hybridization with low copy probes have previously revealed strong homology between the Q6-Q7 and the Q8-Q9 class I gene pairs in the Qa2 region of the C57BL/10 mouse. After DNA sequence analysis of the Q7, Q8 and Q9 genes, we have compared the Q7 gene with its apparent allele, 27.1, from the BALB/c mouse; the 99% homology between Q7 and 27.1 indicates that this is a non-polymorphic gene. Comparison of Q7 with Q9, its homologue in the Q8-Q9 gene pair, revealed greater than 99% homology, thus supporting our proposal that the Qa2 region has evolved by the duplication of gene pairs. Q7 was also found to be homologous (93%) to Q8, the second member of the Q8-Q9 pair. However, the first exon (encoding the leader sequence) as well as the first intron of Q7 and Q8, which are presumably not subject to strong selective pressure, are essentially identical in nucleotide sequence (having only one mismatch), which suggests that greater than 200 bp of DNA may have been exchanged by gene conversion. Furthermore, transcripts of both Q7 and Q8 would have termination codons derived from the exon that normally encodes the transmembrane domain, thus these genes could encode either membrane-bound class I proteins that lack a cytoplasmic protein domain or class I proteins that are secreted.

The DNA sequence of the Q10 genes appears to be highly conserved amongst strains of mice and has only been found to be transcribed in the liver. An examination of the nucleotide sequence of the exon that normally encodes the transmembrane domain of class I molecules suggested that the Q10 gene encodes a secreted protein. We have established this by showing that L cells transformed with an expression vector containing the Q10 gene secrete a class I molecule which was identified with an antiserum raised against a peptide predicted by the Q10 transmembrane exon. Both the L cell-derived Q10 molecule and a class I protein immunoprecipitated from serum with this anti-peptide antiserum have mol. wts. of approximately 38 000; the Q10 molecule secreted by L cells is heterogeneous in mol. wt. This heterogeneity was drastically reduced after endoglycosidase F treatment, suggesting that Q10 molecules secreted into the serum by the liver may be glycosylated differently from those secreted by L cells. Endoglycosidase F treatment of both the L cell and serum forms of the soluble molecule yielded two products with mol. wts. of approximately 32 000 and 35 000; this is consistent with the observation that the predicted Q10 protein sequence has two potential glycosylation sites. In contrast to previous published results, the Q10 molecule reacted with rabbit anti-H-2 antisera which is consistent with its greater than 80% homology to the classical transplantation antigens.

We have isolated a human melanoma line (LD-1) from a patient with melanoma and unexplained leukocytosis. The LD-1 cells produced a colony-stimulating factor (CSF) which stimulated primarily granulocytic colonies in human and murine bone marrow cultures. Erythroid burst and mixed colony-stimulating activity was not detected. A single CSF species with a molecular weight of 21,000 was detected in LD-1-conditioned media by G-200 chromatography. Nude mice transplanted with LD-1 tumors developed granulocytosis and had increased blood CSF levels. Messenger RNA from LD-1 cells directed the synthesis of CSF by Xenopus oocytes. Northern blots of LD-1 RNA hybridized strongly with oligonucleotide probes based on the published sequences for human G-CSF, but not with a probe based on the human GM-CSF sequence. Northern blots hybridized with an oligonucleotide probe based on the CSF-1 sequence showed a high-molecular weight band; however, low-molecular weight CSF-1 mRNAs, which are present in the CSF-1-producing cell line MIA-PaCa-2, were not detected in the LD-1 mRNA. The CSF activity of LD-1 cells is best described as human granulocyte CSF.

We have isolated and expressed a cDNA clone that encodes a human granulocyte colony-stimulating factor from the MIA PaCa-2 cell line. A genomic clone of this factor has been isolated from the CHU-2 cell line and is reported to encode two alternative transcripts [The EMBO J. 5,575, 1986]; one transcript predicts an amino acid sequence identical to that predicted by our MIA PaCa-2 cDNA clone; the other transcript predicts a similar protein containing a three amino acid residue insertion. To investigate which types of this colony-stimulating factor are produced by other cell lines, we used specific oligonucleotides to determine which types of transcripts were present in MIA PaCa-2, 5637, and LD-1 cells, all of which have been reported to produce a factor that can stimulate the growth of predominantly granulocyte colonies in human bone marrow cell cultures. Northern analysis with these probes revealed MIA PaCa-2-like transcripts in all of these cell lines and failed to detect transcripts that would encode the colony-stimulating factor that contained the three-amino-acid-residue insertion.

Transformation of murine thymocytes by radiation leukemia virus is associated with reduced expression of the class I antigens encoded in the major histocompatibility complex (MHC) and increased methylation and altered restriction enzyme patterns of MHC DNA. These changes may play a role in host susceptibility to virus-induced leukemogenesis and accord with the notion that viral genomes play a regulatory function when they integrate adjacent to histocompatibility genes.

We have improved the expression of recombinant human granulocyte-colony-stimulating factor (G-CSF), produced by either pL or trpP expression vectors in Escherichia coli, by altering the sequence at the 5' end of the G-CSF-coding region. Initial attempts to express G-CSF resulted in neither detectable G-CSF mRNA nor protein in the trpP system, and only G-CSF mRNA was detectable in the pL system. We modified both expression vectors to decrease the G + C content of the 5' end of the coding region without altering the predicted amino acid sequence. This resulted in expression of detectable G-CSF mRNA and protein in both systems. Expression reached 17% and 6.5% of the total soluble cellular protein in the pL and trpP expression systems, respectively. The N-terminal sequence of the recombinant G-CSF from the pL system was Met-Thr-Pro-Leu-Gly-Pro-. G-CSF isolated from several human cell lines (including the LD-1 cell line reported here), does not have an N-terminal methionyl residue. Deletion of the threonine codon at the beginning of the coding region for the mature G-CSF resulted in efficient removal of the N-terminal methionine residue during expression in E. coli.

The major histocompatibility complex (MHC) encodes several classes of protein vital to the regulation of the immune response. We have isolated 26 class I genes that map to this region in the C57BL/10 mouse and linked these into three gene clusters. The number of genes differs from the number found in the BALB/c strain and comparison of the organization of the class I genes in these two strains shows conserved regions and polymorphic regions which probably result from deletions, insertions and translocations within the MHC.

We have cloned 26 different class I genes that are located in the major histocompatibility complex of the C57BL/10 mouse. Two of the three class I genes found in the H-2 complex encode the H-2Kb and H-2Db antigens; the other 23 class I genes map to the adjacent Tla complex. We have grouped the cosmids containing these genes into three clusters: one cluster links the H-2K and I-A regions, one cluster links the H-2D and Qa-2 regions, and the final cluster maps to the TL region. The class I gene organizations in the Qa-2 and TL regions of the C57BL/10 and BALB/c mice are generally similar, but there are several polymorphic segments. The Qa-2 region of both mice seems to have evolved by the duplication of gene pairs; furthermore, the H-2K region may have been generated by the translocation of a gene pair from the Qa-2 region. We have evidence that several of the genes in the Qa-2 region are expressed.

We have cloned lines of IL 2-dependent human T cells derived from alloantigen, soluble antigen (tetanus toxoid), mitogen, or IL 2-stimulated peripheral blood lymphocytes and characterized their surface marker expression and cytolytic activity. The surface phenotype and cytolytic function was compared with the ability of these T cell clones to release cytotoxic lymphokines in response to mitogenic lectins. The cytotoxins released by these CTL clones were detected on the murine L929 target cells in a 16-hr assay. All of the T cell clones, whether stimulated by HLA alloantigens, tetanus toxoid, or mitogens, exhibited killer cell activity and the capacity to secrete a soluble cytotoxin(s). Specific polyclonal antisera to recombinant human tumor necrosis factor (rTNF) and human alpha-lymphotoxin (alpha LT) were unable to neutralize the cytotoxic activity released by most of these CTL clones. These results indicate that human CTL produce a novel antigenic form(s) of cytotoxin that we have termed CTL-toxin. Supernatants from several CTL clones yielded a cytotoxic activity that was partially neutralized (10 to 40%) by saturating levels of anti-TNF (but not anti-alpha LT) indicating that human CTL may be capable of producing a TNF-like molecule. Only two out of 60 CTL clones studied thus far produced a cytotoxic activity that was partially neutralized by anti-alpha LT (20 to 40%). Collectively, these results suggest that although both the CD4 and the CD8 subpopulations of human cytotoxic T cells may be capable of releasing several types of cytotoxins in response to mitogenic signals, the predominant cytotoxin is distinct from alpha LT and TNF.