Association of CXCL1 promoter polymorphism with ischaemic stroke in Korean population.

This is a pilot study analysing association of chemokine gene polymorphisms (CXCL1, rs3117604; CXCL2, rs3806792; CCL2, rs2857656 and rs3760396; CCL5, rs2107538) in Korean patients with ischemic stroke (IS) (n = 120) and age-matched controls (n = 267). The CXCL1 gene and particularly T allele of rs3117604 was associated with IS.

Lack of association between promoter polymorphisms of HLA-G gene and rheumatoid arthritis in Korean population.

The aim of this study was to determine whether the HLA-G gene was associated with susceptibility to rheumatoid arthritis (RA). Major histocompatibility complex, class I, G (HLA-G) is involved in immunoregulatory processes and particularly in pathogenesis of inflammatory disorders. To investigate possible association between HLA-G and RA, 296 RA patients and 468 healthy controls were enrolled in this study. Two-promoter single-nucleotide polymorphisms (SNPs) (rs1736936, -1202T/C and rs2735022, -586C/T) in HLA-G gene were analyzed using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). For analysis of data, Helixtree software, SNPAnalyzer, SNPStats, and Haploview version 4.2 were used. Multiple logistic regression models (codominant, dominant, and recessive) were performed for odds ratio (OR), 95% confidence interval (CI), and P value. There were no significant differences in distributions of genotypes and haplotypes between RA patients and control subjects. In clinical features of RA, we found differences between C-reactive protein levels (≥0.5 or <0.5 mg/dL) and two-promoter SNPs. Rs1736936 was significant in codominant (P = 0.028, OR = 0.66, 95% CI = 0.45-0.96) and dominant (P = 0.046, OR = 0.58, 95% CI = 0.34-0.99) models. Also, rs2735022 was significant in codominant (P = 0.038, OR = 0.67, 95% CI = 0.46-0.98) and dominant (P = 0.03, OR = 0.55, 95% CI = 0.33-0.94) models. However, these significant associations disappear after Bonferroni correction. Our results suggest that HLA-G promoter polymorphisms may be not associated with the development of RA in Korean population.

Association between interleukin-4 gene polymorphisms and intracerebral haemorrhage in Korean population.

Interleukin-4 (IL4) polymorphisms (rs2243250, rs2070874) were analysed in Korean patients with ischaemic stroke (IS) (n=119) and intracerebral haemorrhage (ICH) (n=79), and age-matched controls (n =267, IS; n=401, ICH) using direct sequencing. Both single nucleotide polymorphisms and their haplotypes were associated with ICH, but not IS.

The interleukin-1 family gene polymorphisms in Korean patients with rheumatoid arthritis.

OBJECTIVE: The interleukin (IL)-1 family and its related family members are primary inflammatory cytokines. The aim of this study was to assess the possible association between nine IL-1 family gene polymorphisms and rheumatoid arthritis (RA). METHODS: To investigate the genetic association between IL-1 family gene polymorphisms and the risk of RA in a Korean population, 69 single nucleotide polymorphisms (SNPs) of the nine IL-1 family gene regions were selected. A total of 806 subjects (498 controls and 308 RA patients) were included in the study. The genotypes of the selected SNPs in the IL-1 family genes were determined using Illumina Sentrix Array Matrix chips. SNP Stats, Haploview, and SNP Analyzer, and Helixtree programs were used for the analysis of the genetic data. RESULTS: We observed statistically significant associations between the SNPs of IL1F10 and IL1RN among the IL-1 family genes in the RA patients and the control population. When the patients were divided into two groups according to the parameters of disease activity, including C-reactive protein (CRP) level (> or = 0.5 or < 0.5 mg/dL), the erythrocyte sedimentation rate (ESR) (> or = 30 or < 30 mm/h), and parameters of severity, including rheumatoid factor (RF), anti-cyclic citrullinated peptide (anti-CCP), and bone erosion (positive or not), we found significant associations between the parameters, including CRP, ESR, and bone erosion, and SNPs of the IL-1 family genes in RA. CONCLUSION: This study suggests that IL-1 family gene (IL1F10 and IL1RN) polymorphisms may play an important role in the susceptibility to developing RA.

Mechanism of cyclosporine-induced overgrowth in gingiva.

UNLABELLED: Cyclosporine A (CsA) is a widely used immunosuppressant but with significant side-effects, such as gingival overgrowth. This study investigates how CsA induces gingival proliferation and shows the effects of the CsA-associated signaling messengers, IL-6 and TGF-beta1, on gingival proliferation. CsA increased both IL-6 and TGF-beta1 levels. In addition to CsA, an IL-6 or TGF-beta1 treatment also induced gingival fibroblast proliferation. Inhibiting the cytokine resulted in the suppression of CsA-induced proliferation. MAPKs and PI3K are known to be involved in cell proliferation. Therefore, the effect of CsA on the kinase activities was examined. The results showed that both p38 MAPK and PI3K are essential for gingival fibroblast proliferation. TGF-beta1 and IL-6 and their associated signaling transduction may be novel bona fide molecular targets for the prevention of gingival overgrowth in CsA-treated patients. ( ABBREVIATIONS: MAPK, mitogen-activated protein kinase; P13K, phosphatidylinositol 3-kinase.)

Prevention of bone loss in ovariectomized rats: the effect of Salvia miltiorrhiza extracts.

The preventive effect of Salvia miltiorrhiza extracts (SMEs) on the progress of bone loss induced by ovariectomy (OVX) was studied in rats. We measured body weight and bone histomorphometry in sham, OVX or SMEs-administered OVX rats. From light microscopic analyses, a porous or erosive appearances were observed on the surface of trabecular bone of tibia in OVX rats, whereas those of the same bone in sham rats and in SMEs-administered rats were composed of fine particles. The trabecular bone area and trabecular thickness in OVX rats decreased by 50% from those in sham rats, these decreases were completely inhibited by administration of SMEs for 7 weeks. In this study, the mechanical strength in femur neck was significantly enhanced by the treatment of SMEs for 7 weeks. In OVX rats, free T3 was normal in all cases, whereas free T4 was significantly increased. Although there was no difference between OVX and SMEs-administered rats in T3 level, we have found significant difference between them in T4 level. These results strongly suggest that SMEs are effective in preventing the development of bone loss induced by OVX in rats.

A cathepsin F of adult Clonorchis sinensis and its phylogenetic conservation in trematodes.

A novel 28 kDa cysteine protease (Cs28CF) secreted by the hepatobiliary trematode, Clonorchis sinensis was identified. The protease was purified from the excretory-secretory products (ESP) of the adult worm using DEAE-ion exchange and Arginine-Sepharose 4B chromatography. It showed a high activity between pH 6.5 and 7.5 in a dithiothreitol (DTT)-dependent manner. Inhibitors specific to cysteine proteases down-regulated the activity. Addition of Cs28CF to monkey cholangiocyte cultures resulted in approximately 95% cell death after 7 days. The full-length cDNA (1078 bp) encoded a single peptide of 328 amino acids (aa) with an N-terminal hydrophobic sequence, an ERFNAQ motif in the propeptide and a mature domain. Expression of mRNA transcripts of Cs28CF was observed in both the metacercaria and adult stages. Bacterially expressed recombinant protein exhibited a specific antibody reaction with clonorchiasis sera. Deduced aa exhibited 52-76% sequence identity with the cathepsin F analogues from other organisms. A novel E/DXGTA motif was recognized in the propeptide region. Phylogenetic analysis of 63 papain family members revealed that the trematode cysteine proteases formed 2 major clades of cathepsins F and L. The trematode cysteine proteases classified as cathepsin F shared higher homology among themselves than those classified as cathepsin L. Cathepsin F is phylogenetically conserved in the trematode parasites as well as in mammals.

A 78 kDa glucose-regulated protein gene of Spirometra erinacei plerocercoid induced by chemical and physiological stresses.

To adapt to different environmental conditions between poikilothermic and homeothermic hosts, the plerocercoid of Spirometra erinacei (sparganum) might express a variety of biologically active molecules. We have identified a 78 kDa glucose-regulated protein of the sparganum (SpGrp78) by differential display of mRNA, employing RNAs each from sparganum adjusted at 9 degrees C and 37 degrees C. A full-length cDNA of 2148 bp encodes for a protein of 651 amino acids with a predicted molecular mass of 71 610 Da and shares molecular characteristics with heat-shock protein 70, including a putative ATP binding site, signal peptide cleavage site and endoplasmic reticulum retention signal. Phylogenetic analysis revealed that SpGrp78 was mostly related to those of Echinococcus multilocularis and E. granulosus. Expression of SpGrp78 mRNA increased approximately 7-fold by inhibition of glycosylation by tunicamycin, 2-fold by temperature-shift from 9 degrees C to 37 degrees C and slightly by pH-shift to 4.0 or 5.5. These results suggested that induction of SpGrp78 mRNA is related to the functional role of SpGrp78 as a molecular chaperone when the parasite adapts to a new host environment.

Structural and immunological characteristics of a 28-kilodalton cruzipain-like cysteine protease of Paragonimus westermani expressed in the definitive host stage.

A complete cDNA sequence encoding a 28-kDa cruzipain-like cysteine protease of adult Paragonimus westermani, termed Pw28CCP, was isolated from an adult cDNA library. The cDNA contained a single open reading frame of 975 bp encoding 325 amino acids, which exhibited the structural motif and domain organization characteristic of cysteine proteases of non-cathepsin Bs including a hydrophobic signal sequence, an ERFNIN motif, and essential cysteine residues as well as active sites in the mature catalytic region. Analysis of its phylogenetic position revealed that this novel enzyme belonged to the cruzipain-like cysteine proteases. The sequence of the first 13 amino acids predicted from the mature domain of Pw28CCP was in accord with that determined from the native 28-kDa enzyme purified from the adult worm. Expression of Pw28CCP was observed specifically in juvenile and adult worms, with a location in the intestinal epithelium, suggesting that this enzyme could be secreted and involved in nutrient uptake and immune modulation. The recombinant protein expressed in Escherichia coli was used to assess antigenicity by immunoblotting with sera from patients with active paragonimiasis and from those with other parasitic infections. The resulting sensitivity of 86.2% (56 of 65 samples) and specificity of 98% (147 of 150 samples) suggest its potential as an antigen for use in immunodiagnosis.

The 10 kDa protein of Taenia solium metacestodes shows genus specific antigenicity.

Genus specific antigenicity of the 10 kDa protein in cyst fluid (CF) of Taenia solium metacestodes was demonstrated by comparative immunoblot analysis. When CFs from taeniid metacestodes of T. saginata, T. solium, T. taeniaeformis and T. crassiceps were probed with specific monoclonal antibody (mAb) raised against 150 kDa protein of T. solium metacestodes, specific antibody reactions were observed in 7 and 10 kDa proteins of T. solium and in 7/8 kDa of T. saginata, T. taeniaeformis and T. crassiceps. The mAb did not react with any protein in hydatid fluid of Echinococcus granulosus and E. multilocularis. This result revealed that the 10 kDa peptide of T. solium metacestodes and its equivalent proteins of different Taenia metacestodes are genus specific antigens that are shared among different Taenia species.

Differential expression of the 27 kDa cathepsin L-like cysteine protease in developmental stages of Spirometra erinacei.

The 27 kDa cathepsin L-like cysteine protease of Spirometra erinacei plerocercoid is known to play an important function in tissue penetration, nutrient uptake and immune modulation in human sparganosis. In the present study, the expression of this enzyme was examined at different developmental stages of S. erinacei including immature egg, coracidium, plerocercoid in tadpole and rat, and adult. Proteolytic activity against carboxybenzoyl-phenylalanyl-arginyl-7-amino-4-methylcoumarin was detected in the extracts of coracidia and plerocercoid while no activity was observed in those of immature egg and adult. The specific activity in coracidial extracts was lower than that in the plerocercoid. Reverse transcription-polymerase chain reaction and Northern blot analysis demonstrated that the gene was expressed in the coracidium and plerocercoid but not in immature egg and adult. These results suggest that the 27 kDa cysteine protease is only expressed in the stages involving active migration of the parasite in the host tissue.

Persisting antibody reaction in paragonimiasis after praziquantel treatment is elicited mainly by egg antigens.

Antibody responses in serum and cerebrospinal fluid (CSF) samples from patients with active and chronic paragonimiasis and in sera from patients on whom follow-up studies were done after praziquantel treatment were analyzed using antigens of Paragonimus westermani prepared from eggs, metacercariae, juveniles of 4- and 7-week old, adult worms and recombinant protein of 28 kDa cruzipain-like cysteine protease (rPw28CCP). The patient sera/CSFs of active and chronic paragonimiasis revealed strong antibody reactions against the crude extracts of 4- and 7-week old juveniles as well as against those from egg and adult. rPw28CCP also showed specific reaction to the sera with active paragonimiasis. After the treatment, levels of specific antibodies in the sera gradually decreased to negative range in most patients. In some cases with persisting high antibody levels, however, the reactions at 27 kDa egg protein were sustained throughout the observation period of 34 months. The reactions at 35 and 32 kDa in adult extract and rPw28CCP disappeared rapidly after the treatment. Persistent antibody reactions even after successful treatment are provoked by continuous antigenic challenge from eggs which were not resolved by treatment.

Variation of antigenic proteins of eggs and developmental stages of Paragonimus westermani.

Diagnosis of early paragonimiasis is difficult because parasitological evidence is not easily obtained. Antibody tests have been proposed as a good substitute for classical diagnostic techniques. Using the crude extracts of Paragonimus westermani eggs, metacercariae, 4- and 7-week juveniles, and 16-week adults as antigens, we observed the early antibody responses. Sera were obtained from 4 experimental cats, fed 50 metacercariae each, at intervals until 13 weeks post-infection. Antibody (IgG) responses were identified by ELISA using extracts of 4-week juveniles, followed by those of 7- and 16-week worms. Antibody responses were minimal against the metacercarial extracts. Antibodies to P. westermani egg extracts were elevated after 10 weeks post-infection. In immunoblot analysis, more than nine protein bands in 4-week juveniles reacted with the early infection sera. Antigenic proteins in adult worms were different from those of juveniles. After four weeks of infection, 32 and 35 kDa bands in the adult extracts were increasingly reactive. Egg specific proteins at 28, 46 and 94 kDa were reactive only after 10 weeks. Antigenic components reacting to the early infection sera changed during the maturation stages of P. westermani; almost all juvenile antigens were replaced by adult antigen components.

The promoter activity of the phospholipase C-gamma2 gene is regulated by a cell-type-specific control element.

We have cloned and characterized a genomic DNA spanning the 5'-flanking region, the first and second exons, and the first intron of the human PLC-gamma2 gene. The proximal upstream region is highly GC-rich and lacks a TATA box, whereas the distal region contains several AT-rich tracts. Multiple transcription initiation sites were identified by primer extension analysis. Based on the transient transfection assays, the major transcriptional activation element was identified between -183 and +43 (G2SE) and a transcriptional repressive element was found between -303 and -184 (G2RE). The expression of PLC-gamma2 in various cell lines was examined using monoclonal anti-PLC-gamma2 antibody. PLC-gamma2 was highly expressed in B-cell lines such as Daudi, SP2, and Ramos cells, whereas it existed at very low levels in Jurkat, 3T3-L1, NBL-7, and C6Bu-1 cells. Moderate levels of PLC-gamma2 were also detected in C2C12, P19, U937, HL60, A431, and PC12 cells. The 4-kb genomic fragment upstream of -1,654 was able to activate transcription from the PLC-gamma2 promoter in Daudi and C2C12 cells, but not in Jurkat cells, which is consistent with the PLC-gamma2 protein expression levels in those cell lines. These results suggest that the cell-type-specific expression of PLC-gamma2 might be attributed to the transcriptional regulation by the upstream cis-element.