Anti-enolase１antibodies from a patient with systemic lupus erythematosus accompanied by pulmonary arterial hypertension promote migration of pulmonary artery smooth muscle cells.

OBJECTIVE: Pulmonary arterial hypertension (PAH) is an intractable complication in connective tissue diseases, but the pathological mechanisms responsible for progression remain obscure. This study aims to test whether patient IgG possesses biological activity promoting the migration of pulmonary artery smooth muscle cells (PASMCs). METHODS: Cell migration was estimated by lamellipodia formation and by utilizing a Boyden chamber method. The specificity of autoantibodies was established by western blotting, ELISA, and immunocytochemistry. The target antigen was investigated by mass spectrometry. RESULTS: IgG obtained from a patient with systemic lupus erythematosus (SLE) accompanied by PAH was found to promote lamellipodia formation and migration of PASMCs. The IgG bound to a ∼50 kDa protein expressed on the cell membrane, and in the cytoplasm and nucleus. This molecule was identified as enolase 1. Removal of enolase 1-binding antibodies from the IgG fraction, or treatment of the cells with an enolase inhibitor, significantly suppressed the migration of PASMCs. CONCLUSION: Patients with SLE may possess autoantibodies to enolase 1 which stimulate the migration of PASMCs and are likely to play a role in the progression of PAH.

The effect of targeted rheumatoid arthritis therapies on anti-citrullinated protein autoantibody levels and B cell responses.

Rheumatoid arthritis (RA) is a complex inflammatory disorder associated with synovitis and joint destruction that affects an estimated 1·3 million Americans and causes significant morbidity, a reduced life-span and lost work productivity. The use of biological therapies for the treatment of RA is costly, and the selection of therapies is still largely empirical and not guided by the underlying biological features of the disease in individual patients. The synovitis associated with RA is characterized by an influx of B and T cells, macrophages and neutrophils and the expansion of fibroblast-like synoviocytes, which form pannus and lead to cartilage and bone destruction. RA is associated with synovial production of rheumatoid factor (RF) and anti-citrullinated protein autoantibodies (ACPA) and with the production of inflammatory cytokines, including interleukin (IL)-1, IL-6, IL-17 and tumour necrosis factor (TNF)-α, which are targets for RA therapeutics. Recent ideas about the pathogenesis of RA emphasize a genetic predisposition to develop RA, a preclinical phase of disease that is associated with the production of ACPA and the development of symptomatic disease following inflammatory initiating events that are associated with expression of citrullinated epitopes in the joints of patients. However, we still have a limited understanding of the cytokine and intracellular pathways that regulate ACPA levels. In humans, therapy with biological agents affords a unique opportunity to better understand the cytokine and signalling pathways regulating ACPA levels and the impact of ACPA level changes on disease activity. In this study we summarize the effect of RA therapies on ACPA levels and B cell responses.

Selective expression of MHC class I in the affected muscle of a patient with idiopathic inflammatory myopathy.

The dominant clinical feature of polymyositis/dermatomyositis is weakness in proximal, rather than distal, musculature. Although rare, cases of focal/localized myositis in which polymyositis-like muscle inflammation is present in only one muscle or extremity have also been reported. The underlying mechanisms dictating involvement of specific muscle groups in polymyositis/dermatomyositis and focal/localized myositis have not been identified. Here, we describe a rare case of dropped-head syndrome due to localized idiopathic inflammatory myopathy (IIM) in the splenius capitis (neck extensor) muscle where major histocompatibility complex (MHC) class I expression was up-regulated in involved muscle fibers. Interestingly, the adjacent trapezius muscle was not affected, corresponding to muscle biopsy findings that did not show any sign of inflammation or MHC class I expression. Our case report therefore suggests that selection of affected muscle in IIM might be influenced by the MHC class I expression of the muscle.

Association study of a polymorphism of the CTGF gene and susceptibility to systemic sclerosis in the Japanese population.

OBJECTIVES: To validate the association of a single nucleotide polymorphism (SNP) of the connective tissue growth factor gene (CTGF) with susceptibility to systemic sclerosis (SSc) in the Japanese population. METHODS: 395 Japanese patients with SSc, 115 patients with rheumatoid arthritis and 269 healthy Japanese volunteers were enrolled in the study. An SNP (rs6918698) at -945 bp from the start codon in the promoter region of the CTGF gene was determined by allelic discrimination with the use of a specific TaqMan probe. RESULTS: The G allele showed a significantly higher frequency in patients with SSc than in controls (p<0.001; odds ratio 1.5; 95% confidence interval 1.2 to 1.9). In particular, the clinical subsets of SSc showed a more significant association between the G allele and diffuse cutaneous SSc (p<0.001) and the presence of interstitial lung disease (p<0.001), the presence of anti-topoisomerase I antibody (p<0.001) and anti-U1RNP antibody (p = 0.010). Association analyses using the genotype of the SNP yielded results similar to those of analyses using the allele. CONCLUSIONS: This study confirms the association between an SNP in the CTGF gene and susceptibility to SSc, especially in the presence of diffuse cutaneous SSc, interstitial lung disease and anti-topoisomerase I antibody. The results strongly suggest that this SNP may be a powerful indicator of severe skin and lung involvement in patients with SSc.

Genetic polymorphisms in the surfactant proteins in systemic sclerosis in Japanese: T/T genotype at 1580 C/T (Thr131Ile) in the SP-B gene reduces the risk of interstitial lung disease.

OBJECTIVES: To investigate the association between single-nucleotide polymorphisms (SNPs) in the pulmonary surfactant protein (SP) genes and the presence or absence of interstitial lung disease (ILD) in SSc patients. METHODS: We studied 127 Japanese patients with SSc and 206 normal subjects. Investigated SNPs were C/T within amino acid (aa) 219, Arg219Trp in the SP-A1 gene (rs4253527), C/T within aa 131 (at nucleotide 1580) and Thr131Ile of the SP-B gene (rs1130866). Genotypes were determined by the TaqMan method. RESULTS: Genotype frequencies were not different between the SSc patients and normal controls for both loci. The patients were subsequently divided into two groups based on presence or absence of ILD. In the SNP in the SP-B gene, the frequency of the T/T genotype was significantly lower in the patients with ILD than in those without ILD. Limited in the patients who were positive for anti-Scl-70 antibody, the difference in the frequency of the T/T genotype between the ILD-positive and ILD-negative groups became more apparent. On the other hand, in the SNP in the SP-A1 gene, there was no significant skewing for a certain genotype. CONCLUSION: In SSc, where massive fibrosis occurs, possession of the T/T genotype in the SP-B gene would reduce the risk of ILD in Japanese.

Nucleotide sequence analyses of human complement 6 (C6) gene suggest balancing selection.

The sixth complement component (C6) has a common charge polymorphism, C6A and C6B, with similar gene frequencies in all major populations. In addition, C6B2 is also found in Japanese populations at a frequency of about 6%. Sequence analyses of the coding region of three human and ape C6 alleles indicated four nonsynonymous and three synonymous changes in C6*B2 relative to C6*A, suggesting that a recombination event occurred between C6*B2 and C6*A to give rise to C6*B. Sequence variation in a 3.86 kb region encompassing exon 3, where the causal base change of the common C6 polymorphism is found, indicated that several single nucleotide polymorphisms (SNPs) were in extensive linkage disequilibrium (LD), with little differentiation among populations. Sliding window estimates of two test statistics for neutrality revealed significant values in a subregion where the replacement coding polymorphism resides, in all three human populations. These results raise the possibility that the two common C6 alleles in human populations are maintained by balancing selection.

Population differences in DNA sequence variation and linkage disequilibrium at the PON1 gene.

Polymorphisms of the promoter region (-108C/T) and the coding region (192Q/R) of the paraoxonase 1 gene (PON1) showed differences in association with cardiovascular disease risk in various populations. To characterize the genetic variation underlying these important polymorphisms, we examined DNA sequence variation both in a 1.3-kb promoter region 16.5 kb from codon 192, and in a 1.7-kb region centered on the 192Q/R polymorphic site of the coding region of PON1, in 30 Africans, 30 Europeans and 64 Japanese. We found 10 polymorphic sites and 11 haplotypes in the 1.3-kb promoter region and 10 biallelic polymorphic sites and 10 haplotypes in the 1.7-kb region. From the PON1 sequences of chimpanzees and an orangutan, the ancestral type of codon 192 was found to be R. The number of pairs of polymorphic sites between the promoter and 1.7-kb regions that were in significant linkage disequilibrium was much higher in a Japanese population than in African and European populations. In addition, the pairs of polymorphic sites in linkage disequilibrium differed among the three populations. These results suggest that some of the population differences in association with risk for coronary heart disease can be explained by population differences in haplotype frequency of PON1 haplotypes.

A-61C and C-101G Hp gene promoter polymorphisms are, respectively, associated with ahaptoglobinaemia and hypohaptoglobinaemia in Ghana.

We have investigated the genetic basis for the Hp0 phenotype amongst 123 randomly selected Ghanaians. A total of 17 individuals were determined to be Hp0 phenotype, based on the classical method for Hp phenotyping of Hb-supplemented plasma. Out of the 17 Hp0 individuals, nine subjects were further classified as ahaptoglobinaemic and eight as hypohaptoglobinaemic by Western blots and double immunodiffusion. We identified three previously known base substitutions (A-55G, A-61C and T-104A) and three new ones (C-101G, T-191G and C-242T) within the 5' flanking region of the Hp gene. The A-61C base substitution significantly decreased transcriptional activity and was associated strongly with Hp2 allele and ahaptoglobinaemia. The C-101G substitution was similar in transcriptional activity to the wild-type and was associated with Hp1S allele and hypohaptoglobinaemia. The Hpdel allele seen in Asian populations was absent. We conclude that the Hp0 phenotype in Ghana has a genetic basis that differs significantly from that seen in Asia.

Polymorphisms of sorbitol dehydrogenase (SDH) gene and susceptibility to diabetic retinopathy.

The polyol pathway consists of two enzymes aldose reductase (AR) and sorbitol dehydrogenase (SDH); the former is the first enzyme in the polyol pathway, that catalyzes the reduction of glucose to sorbitol, the latter is the second one, that converts sorbitol to fructose using by NAD(+) as a cofactor. We along with others have recently found that SDH activity, the second step in the polyol pathway, might make a greater contribution to the etiology of diabetic retinopathy than does the first step involving AR. In this paper, we propose a novel hypothesis that polymorphisms of SDH gene may be correlated with SDH gene expression levels in diabetic retinas, thus being a valuable genetic marker for diabetic retinopathy.

Identification of human phosphoglucomutase 3 (PGM3) as N-acetylglucosamine-phosphate mutase (AGM1).

We performed phenotyping of human phosphoglucomutase 3 (PGM(3)) and screening for mutations in the human N-acetylglucosamine-phosphate mutase gene (AGM(1)) to identify PGM(3) as AGM(1). By sequencing the coding region of AGM(1), two alleles containing a G or A base at nucleotide 1396, that can respectively encode aspartic acid or asparagine at codon 466, were identified. Cell extracts of COS7 cells after transfection with the pcDNA 3.1(+) plasmid containing an AGM(1) allele with 1396G or 1396A showed similar electrophoretic patterns to the PGM(3) 1 or PGM(3) 2 protein, respectively, with the isozyme detection method used for PGM(3) phenotyping. The genotypes determined by the two alleles of AGM(1) coincided exactly with the PGM(3) phenotypes in 20 individuals. We also investigated the allele frequency of the AGM(1) nucleotide polymorphism in a Japanese population by DNA sequencing and found that the frequencies of alleles 1396G and 1396A were similar to previously reported PGM(3) *1 and PGM(3) *2 frequencies. Overall, the facts that the AGM(1) gene product shows PGM activity, AGM(1) is polymorphic, the electrophoretic mobility is similar between AGM(1) allele-specific products and PGM(3) 1 and 2 proteins, PGM(3) phenotypes and AGM(1) genotypes completely coincide in 20 individuals, and AGM(1) allele frequencies are similar to those of PGM(3) *1 and PGM(3) *2 in Japanese populations, suggest that PGM(3) is identical to AGM(1).

Pigment epithelium-derived factor Met72Thr polymorphism in patients with diabetic microangiopathy.

Pigment epithelium-derived factor (PEDF) has recently been shown to be the most potent inhibitor of angiogenesis in the mammalian eye. We, along with others, have very recently found that loss of PEDF is involved in the development and progression of diabetic retinopathy. However, there are no studies on the allelic effects of PEDF gene polymorphism in diabetic retinopathy. In this study, we investigated whether a functional amino acid change, a methionine to threonine polymorphism (Met72Thr polymorphism) of the PEDF gene, is associated with microangiopathy in 143 patients with diabetes. We found that there were no significant associations between PEDF Met72Thr gene polymorphism and diabetic microangiopathy. These observations suggest that these genetic variants might not be involved in the mechanism of diabetic microangiopathy in patients with diabetes.

Contrasting patterns of polymorphisms at the ABO-secretor gene (FUT2) and plasma alpha(1,3)fucosyltransferase gene (FUT6) in human populations.

The coding sequences ( approximately 1 kb) of FUT2 [ABO-Secretor type alpha(1,2)fucosyltransferase] and of FUT6 [plasma alpha(1,3)fucosyltransferase] were analyzed for allelic polymorphism by direct sequencing in five populations. The nucleotide diversities of FUT2 estimated from pairwise sequence differences were 0.0045, 0.0042, 0.0042, 0.0009, and 0.0008 in Africans, European-Africans, Iranians, Chinese, and Japanese, respectively. The nucleotide diversities of FUT6 were 0.0024, 0.0016, 0.0015, 0.0017, and 0.0020 in Africans, European-Africans, Iranians, Chinese, and Japanese, respectively. At FUT2, excesses in pairwise sequence differences compared to the number of polymorphic sites as indicated by a significantly positive Tajima's D were observed in European-Africans and in Iranians. The data do not fit expectations of the equilibrium neutral model with an infinite number of sites. On the other hand, Tajima's D's at FUT6 in each of the five populations and at FUT2 in Africans, Chinese, and Japanese were not significantly different from zero. F(ST) between the Asians and the others measured at FUT2 was higher than at FUT6. These results suggest that natural selection was responsible for the generation of the FUT2 polymorphism in European-Africans and in Iranians.

Caveolae in mesangial cells and caveolin expression in mesangial proliferative glomerulonephritis.

BACKGROUND: Caveolae are plasma membrane invaginations that have a diameter of 40 to 60 nm. Recent evidences have demonstrated that caveolae contain a variety of signal transduction molecules. Caveolin is a marker protein of caveolae and has been proposed to play a negative regulatory role in signal transduction. The aim of this study was to investigate the behavior of caveolae and caveolin in experimental glomerulonephritis, the localization of both platelet-derived growth factor (PDGF) and transforming growth factor-beta (TGF-beta) receptors in the caveolae membrane, and the regulation of caveolin expression in cultured mesangial cells. METHODS: The expression of caveolin-1 was examined by immunoblotting and immunohistology using anti-caveolin antibody in anti-Thy-1 nephritis. The caveolae membrane fraction of mesangial cells was isolated by sucrose gradient method and expression of PDGF receptor and TGF-beta receptor were detected by immunoblotting. The effects of mitogens such as phorbol 12-myristate 13-acetate (PMA) and PDGF on the expression of caveolin-1 protein and mRNA were also examined in cultured mesangial cells. RESULTS: Caveolin-1 was mainly expressed in glomeruli and was significantly up-regulated in anti-Thy-1 nephritis rat kidney. In cultured mesangial cells, the membrane invaginations of caveolae were revealed by electron microscopy. PDGF receptors abounded in the caveolae membrane and rapidly changed their subcellular distribution after ligand stimulation. In contrast, TGF-beta receptors abounded in the non-caveolae membrane and did not change after ligand stimulation. Decreases in caveolin-1 protein, which were associated with increases in mRNA expression after the exposure of PMA or PDGF-BB, suggested an increased turnover of caveolin-1 in mesangial cells stimulated by mitogens. CONCLUSION: To our knowledge, this electron microscopical study is the first to demonstrate the presence of caveolae in cultured mesangial cells. Caveolae integrate PDGF receptors, and caveolin-1 may play a role in the pathogenesis of the mesangial proliferative glomerular diseases through PDGF signaling.

The polymorphisms of fucosyltransferases.

The alpha(1,2)fucosyltransferase Se enzyme regulates the expression of the ABH antigens in secretion. Secretors, who have ABH antigens in their saliva, have at least one functional Se allele in the FUT2 locus, while non-secretors, who fail to express ABH antigens in saliva, are homozygous for the non-functional se allele. Molecular analyses of the FUT2 polymorphism of various populations have indicated the ethnic specificity of null alleles: the null allele se(428) is a common Se enzyme-deficient allele in Africans and Caucasians but does not occur in Asians, whereas the null allele se(357,385) is specific to Asians. The gene frequency of se(428) or se(357,385) is about 0.5 in each respective population. Why the se(428) is absent in Asians is of interest. Also here, we describe the polymorphisms of the fucosyltransferase genes (FUT1, FUT3 and FUT6).

Polymorphism of the human ABO-Secretor locus (FUT2) in four populations in Asia: indication of distinct Asian subpopulations.

The polymorphic alleles of the human ABO-Secretor locus (FUT2 or Se) show high heterogeneity and overt ethnic specificity. To provide additional data for analysis to elucidate the origins of populations, we have investigated the allelic polymorphism of FUT2 in 40 unrelated Tibetan and 53 Tamang individuals from Nepal, 42 Indonesian individuals from Surabaya, and 55 Uygur individuals from Urumqi, using DNA sequencing. In Tibetan, Tamang and Indonesian populations, the frequency of a nonfunctional allele, se 357,385, which is found only in Asian populations, was 0.638, 0.509 and 0.631, respectively. In Uygur, the se 428, which is common in Caucasian populations, and the se 357,385 consisting of two common nonfunctional FUT2 alleles, had frequencies of 0.3 and 0.145, respectively. The fixation index (F ST) based on genetic differentiation was obtained pairwise among the four populations in this study and six populations in our previous studies. The results suggested that genetic differentiation among Tibetan, Tamang, Indonesian and East Asian populations is very low, while the distribution feature of the FUT2 alleles in the Uygur population implied an admixture of European with Asian. The distribution of nonfunctional alleles at the FUT2 locus provided further evidence of human migration among the Asian populations.

Two distinct Alu-mediated deletions of the human ABO-secretor (FUT2) locus in Samoan and Bangladeshi populations.

The human secretor alpha(1,2) fucosyltransferase encoded by the FUT2 determines the production of ABO(H) antigens in secretions. Recent studies demonstrated the presence of several nonfunctional alleles in the FUT2. During the analysis for inactivating mutations at the FUT2 locus from 24 Samoan and 47 Bangladeshi individuals, we found two distinct Alu-mediated deletions of FUT2. The FUT2 deletion in a Bangladeshi population was identical with that found in Indian individuals with the Bombay phenotype (se(del)), but not associated with the null allele (T725G) of the H gene (FUT1). The FUT2 deletion in Samoans is a novel null allele (se(del2)). The junction region of se(del2) was successfully amplified using the same primers for the se(del) amplification. DNA sequencing of the junction region of the se(del2) indicated that there was a 32-bp sequence identity between DNA sequences surrounding the 5' and 3' breakpoints. The size of the deletion of the se(del2) was 9.3 kb, including the full coding region of FUT2. The frequency of the se(del) in a Bangladeshi population was 0.074, and that of the se(del2) in a Samoan population was 0.104. Hum Mutat 16:274, 2000.

An Alu-mediated large deletion of the FUT2 gene in individuals with the ABO-Bombay phenotype.

Recently, we have found an allelic deletion of the secretor alpha(1,2)fucosyltransferase (FUT2) gene in individuals with the classical Bombay phenotype of the ABO system. The FUT2 gene consists of two exons separated by an intron that spans approximately 7 kb. The first exon is noncoding, whereas exon 2 contains the complete coding sequence. Since the 5' breakpoint of the deletion has previously been mapped to the single intron of FUT2, we have cloned the junction region of the deletion in a Bombay individual by cassette-mediated polymerase chain reaction. In addition, the region from the 3' untranslated region of FUT2 to the 3' breakpoint sequence has been amplified from a control individual. DNA sequence analysis of this region indicates that the 5' breakpoint is within a free left Alu monomer (FLAM-C) sequence that lies 1.3 kb downstream of exon 1, and that the 3' breakpoint is within a complete Alu element (AluSx) that is positioned 1.5 kb downstream of exon 2. The size of the deletion is estimated to be about 10 kb. There is a 25-bp sequence identity between the reference DNA sequences surrounding the 5' and 3' breakpoints. This demonstrates that an Alu-mediated large gene deletion generated by unequal crossover is responsible for secretor alpha(1,2)fucosyltransferase deficiency in Indian Bombay individuals.

C(2)-Symmetric bis-sulfoxide: A novel chiral auxiliary for asymmetric desymmetrization of cyclic meso-1,2-diols

A new heterocyclic compound, C(2)-symmetric bis-sulfoxide 1, has been found to be an efficient chiral auxiliary for asymmetric desymmetrization of cyclic meso-1,2-diols via diastereoselective acetal fission. Both (R,R)- and (S,S)-1 are readily synthesized with high optical purity via asymmetric oxidation of 1, 5-benzodithiepan-3-one (2). After acetalization of meso-1,2-diols 6a-e and a mono-TMS ether 6f with this chiral auxiliary 1, the resulting acetals 7a-f were subjected to base-promoted acetal fission upon treatment with potassium hexamethyldisilazide (KHMDS) followed by acetylation or benzylation to give the desymmetrized diol derivatives 8a-f with high diastereoselectivity. The chiral auxiliary 1 is readily removed by acid-promoted hydrolysis and can be recovered without a loss in enantiomeric excess.