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.

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.

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.

Studies on a new proteolytic enzyme from A chromobacter lyticus M497-1. I. Purification and some enzymatic properties.

Achromobacter lyticus M497-1 produces three kinds of alkaline proteases (protease I, II and III) in culture medium along with the bacteriolytic enzyme (Masaki, T., Nakamura, K., Isono, M. and Soejima, M. (1978) Agric. Biol. Chem. 42, 1443--1445). Among these three proteases, Achromobacter protease I (EC 3.4.21.-) shows strict splitting for lysine residues at the carboxyl side of the splitting point. This enzyme was purified through a sequence of benzalkonium chloride treatment, acetone fractionation, CM-cellulose and DEAE-cellulose treatment chromatography on AH-Sepharose 4B and isoelectric focusing method. This form was shown to be homogeneous by polyacrylamide gel electrophoresis and ultracentrifugation analysis. The physicochemical properties of the enzyme were: Mr 30 500; partial specific volume (v), 0.717 ml/g; intrinsic viscosity (nu), 0.0385) dl/g; isoelectric point (pI) 6.9; and E1%1cm at 280 nm, 18.77. The enzyme was composed of 294 residues of amino acid per molecule, with glycine as NH2-terminal and lysine as COOH-terminal amino acids. The optimum pH values with casein, Bz-lys-pNA and Tos-Lys-OMe were 8.5--10.7, 9.0--9.5 and 7.8--8.2, respectively. The enzyme was inhibited by iPr2P-F, PhCH2SO2F and Tos-LysCH2Cl but not by Tos-ArgCH2Cl, EDTA, o-phenanthroline and PCMB.

Studies on a new proteolytic enzyme from Achromobacter lyticus M497-1. II. specificity and inhibition studies of Achromobacter protease I.

The unique specificity of Achromobacter protease I for lysine residue was investigated using synthetic and natural substrates, i.e., lysine derivatives, arginine derivatives, lysine vasopressin, substance P, ACTH and insulin. The enzyme cleaved only the -Lys-X- bonds in the above substrates. The binding affinity of alkylamines as determined by Ki was much stronger than that of the corresponding alkylguanidines.

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.

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.

Presence of H type 3/4 chains of ABO histo-blood group system in serous cells of human submandibular gland and regulation of their expression by the secretor gene (FUT2).

We have investigated by immunochemistry the distribution of H Type 3/4 chains of the ABO histo-blood group system in human submandibular gland using a monoclonal anti-H MBr1 antibody specific for H Type 3/4 chains, and have found the expression of H Type 3/4 chains was mainly in the serous cells. Serous cells from secretors were stained by MBr1 but not by anti-A and anti-B antibodies, whereas serous cells from nonsecretors exhibited a negative reaction with MBr1. Mucous cells were not stained by MBr1. Only a few striated duct cells showed a weak reaction with anti-H MBr1. These results suggested that the H Type 3/4 chains were distributed predominantly in the serous cells of the human submandibular gland and that secretor Type alpha(1,2)fucosyltransferase (Se enzyme) controlled the synthesis of H Type 3/4 chains in vivo. Saliva also contained H Type 3/4 chains, which were controlled by the secretor gene (FUT2). The differences in the distributions of H Type 1, H Type 2, and H Type 3/4 chains of the ABO histo blood group system in the submandibular gland are discussed.

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.

Rapid decline of residual renal function in patients on automated peritoneal dialysis.

OBJECTIVE: To determine the effect of peritoneal dialysis modalities such as nightly intermittent peritoneal dialysis (NIPD), continuous cyclic peritoneal dialysis (CCPD), and continuous ambulatory peritoneal dialysis (CAPD) on residual renal function. DESIGN: A six-month prospective, nonrandomized comparison study. SETTING: Outpatient CAPD unit of a university hospital. PARTICIPANTS: Eighteen end-stage renal disease patients treated by peritoneal dialysis (8 by NIPD, 5 by CCPD, and 5 by CAPD). INTERVENTIONS: Samples from the total dialysate, blood, and 24-hour urine collection were obtained monthly. MEASUREMENTS: Urea, creatinine, and beta2-microglobulin concentrations were measured. Renal and peritoneal clearances of each substance and KT/V urea were calculated. Residual renal function (RRF) was estimated by renal creatinine clearance (RCcr). RESULTS: No significant differences in age, sex, and primary renal disease among the three groups were noted. In all groups, anemic and hypertensive states were controlled identically, and mean weekly total (renal + peritoneal) KT/V urea (over 2.1/wk) and total creatinine clearance (over 60 L/wk/1.73 m2) were maintained during the whole experimental period. Starting mean RCcr was near 4.0 mL/min/1.73 m2 in all groups. Thereafter, a rapid and significant decline in RRF was demonstrated on NIPD and CCPD. The declining rates of RCcr values at 6 months after starting NIPD and CCPD were -0.29 and -0.34 mL/min/month, respectively, which were much greater than those of CAPD (+0.01 mL/min/month). CONCLUSION: Because of a possibly characteristic progressive loss of RRF in automated peritoneal dialysis (APD), strict regular assessment of RRF should be performed from the start of APD.

Single channel analysis of the inward rectifier K current in the rabbit ventricular cells.

The inward rectifier K channel in rabbit ventricular cells was studied by the patch-clamp method. Single channel currents were recorded in giga-sealed cell-attached patches with 150 mM K+ in the pipette. The slope conductance in the membrane potential range from -140 to -40 mV was 46.6 +/- 6.7 pS (mean +/- S.D., n = 16), and was reduced by decreasing [K+] in the pipette (20 or 50 mM). The channel was blocked by an application of Cs+ or Ba2+ (0.04-1 mM) in the pipette. Outwardly directed current, recorded with 50 mM K+ in the pipette, revealed the inward rectification of the single channel current. The probability of the channel being open was 0.33 +/- 0.05 (n = 10) at the resting potential (RP=-81.7 +/- 1.7 mV, n = 16) with 150 mM K+ in the pipette, and it decreased with hyperpolarization. The mean open time of the channel was 178 +/- 25 msec (n = 6) at RP. The closed time of the channel seemed to have two exponential components, with time constants of 11.0 +/- 2.0 msec and 1.92 +/- 0.52 sec (n = 6) at RP. The slower time constant was increased with hyperpolarization. The averaged patch current recorded upon hyperpolarizing pulses demonstrated a time-dependent current decay as expected from the single channel kinetics. The results indicated that the inward rectifier K+ current has time- and voltage-dependent kinetics.

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.

A dialysis patient with systemic calciphylaxis exhibiting rapidly progressive visceral ischemia and acral gangrene.

Systemic calciphylaxis is a rare, poorly understood syndrome of progressive peripheral ischemic necrosis and medial arterial calcification in patients with end-stage renal disease. We report a patient with this syndrome which developed following corticosteroid administration and who ultimately required amputation of the four extremities. Furthermore, cerebral, myocardial, splenic, and intestinal infarctions also developed in parallel with the increment of visceral arterial calcification. No evidence of noticeable hyperparathyroidism or elevation of serum calcium-phosphate product was observed. We speculated that, in addition to diabetes mellitus and chronic renal failure while undergoing dialysis therapy, the administration of corticosteroids might act synergistically to cause calciphylaxis.

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.

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).

PCR analysis of Lewis-negative gene mutations and the distribution of Lewis alleles in a Japanese population.

Three mutations in the Lewis-negative gene, T59G, G508A and T1067A, have been detected by means of a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 149 unrelated Japanese individuals. We found three common Lewis alleles-Le (without the T59G, G508A, and T1067A mutations), le1 (with the T59G and G508A mutations), and le2 (with the T59G and T1067A mutations) in a Japanese population. In addition, we also found one rare Lewis-negative allele, le3 (only with the T1067A mutation). The allele frequencies of Le, le1, le2, and le3 were 0.607, 0.275, 0.114, and 0.003, respectively. Our results were in accordance with those expected by the Hardy-Weinberg equilibrium. Some statistical parameters of forensic interest were also calculated.

Lewis (FUT3) genotypes in two different Chinese populations.

The allelic frequencies of the alpha (1,3/4)fucosyltransferase gene (FUT3) in two different Chinese populations (138 individuals in Shenyang and 154 in Guangzhou) were investigated using PCR-RFLP and nucleotide sequencing methods. The common alleles in the Oriental population, Le (wild type allele), le59,508 (with the mutations at nucleotide (nt) 59T-->G and nt 508G-->A) and le59,1067 (with the mutations at nt 59T-->G and nt 1067T-->A) were encountered, and also the rare alleles, le1067 (with the mutation at nt 1067T-->A) and Le59 (with the mutation at 59T-->G), were observed in these Chinese populations. In addition, the common allele in Caucasians, le202,314 (with the mutations at nt 202T-->C and nt 314C-->T), was found in the Oriental population for the first time. The allelic frequencies of the Le, Le59, le59,508, le59,1067, le202,314, and le1067, were 0.750, 0.011, 0.145, 0.054, 0.036, and 0.004 in the Shenyang population and 0.675, 0.026, 0.14, 0.123, 0.026, and 0.010 in the Guangzhou population, respectively. The presence of the alleles containing either the 59 mutation (Le59) or the 1067 mutation (le1067) suggested that the allele le59,1067 may have originated by recombination between them.

[A study on uric acid excretion in patients with hypouricemia].

We studied four patients with hypouricemia and almost normal renal function. Two patients are idiopathic renal and familial. The other two cases are secondary to diabetes mellitus. The study of the uricosuric mechanism with pyrazinamide (PZA) and benzbromarone (Bb) test shows that two patients have a defect of pre-secretory reabsorption of urate, the other one a incomplete combined defect of pre-and post-secretory reabsorption, and the other one a enhanced secretion of urate. The fact that two patients have the history of acute renal failure (ARF) suggested that strenuous exercise or dehydration would be responsible for ARF in patients with renal hypouricemia.

[A case of hyperparathyroid bone disease and the review of renal osteodystrophy].

We report here a typical case of hyperparathyroid bone disease associated with CRF on maintenance HD and review on Renal Osteodystrophy. A 39 year-old female patient was admitted because of polyarthralgia and pruritus. She had a history of HD due to CGN for about 13 years. Laboratory data showed an increase in serum PTH and Alkaline phosphatase level. The evidence of osteitis fibrosa was revealed by bone Xp and scintigraphy. Enlarged solid masses were found in her neck by echogram and parathyroid scintigraphy. She was diagnosed as hyperparathyroid bone disease and total parathyroidectomy c autoplantation was done. Shortly after the surgical treatment, subjective symptoms were relieved and PTH level was normalized. The bone Xp findings improved gradually.