Characterizing the metabolic divide: distinctive metabolites differentiating CAD-T2DM from CAD patients.

OBJECTIVE: To delineate the metabolomic differences in plasma samples between patients with coronary artery disease (CAD) and those with concomitant CAD and type 2 diabetes mellitus (T2DM), and to pinpoint distinctive metabolites indicative of T2DM risk. METHOD: Plasma samples from CAD and CAD-T2DM patients across three centers underwent comprehensive metabolomic and lipidomic analyses. Multivariate logistic regression was employed to discern the relationship between the identified metabolites and T2DM risk. Characteristic metabolites' metabolic impacts were further probed through hepatocyte cellular experiments. Subsequent transcriptomic analyses elucidated the potential target sites explaining the metabolic actions of these metabolites. RESULTS: Metabolomic analysis revealed 192 and 95 significantly altered profiles in the discovery (FDR < 0.05) and validation (P < 0.05) cohorts, respectively, that were associated with T2DM risk in univariate logistic regression. Further multivariate regression analyses identified 22 characteristic metabolites consistently associated with T2DM risk in both cohorts. Notably, pipecolinic acid and L-pipecolic acid, lysine derivatives, exhibited negative association with CAD-T2DM and influenced cellular glucose metabolism in hepatocytes. Transcriptomic insights shed light on potential metabolic action sites of these metabolites. CONCLUSIONS: This research underscores the metabolic disparities between CAD and CAD-T2DM patients, spotlighting the protective attributes of pipecolinic acid and L-pipecolic acid. The comprehensive metabolomic and transcriptomic findings provide novel insights into the mechanism research, prophylaxis and treatment of comorbidity of CAD and T2DM.

Comprehensive Metabolic Profiling of Inflammation Indicated Key Roles of Glycerophospholipid and Arginine Metabolism in Coronary Artery Disease.

Background: Systemic immune inflammation is a key mediator in the progression of coronary artery disease (CAD), concerning various metabolic and lipid changes. In this study, the relationship between the inflammatory index and metabolic profile in patients with CAD was investigated to provide deep insights into metabolic disturbances related to inflammation. Methods: Widely targeted plasma metabolomic and lipidomic profiling was performed in 1,234 patients with CAD. Laboratory circulating inflammatory markers were mainly used to define general systemic immune and low-grade inflammatory states. Multivariable-adjusted linear regression was adopted to assess the associations between 860 metabolites and 7 inflammatory markers. Least absolute shrinkage and selection operator (LASSO) logistic-based classifiers and multivariable logistic regression were applied to identify biomarkers of inflammatory states and develop models for discriminating an advanced inflammatory state. Results: Multiple metabolites and lipid species were linearly associated with the seven inflammatory markers [false discovery rate (FDR) <0.05]. LASSO and multivariable-adjusted logistic regression analysis identified significant associations between 45 metabolites and systemic immune-inflammation index, 46 metabolites and neutrophil-lymphocyte ratio states, 32 metabolites and low-grade inflammation score, and 26 metabolites and high-sensitivity C-reactive protein states (P < 0.05). Glycerophospholipid metabolism and arginine and proline metabolism were determined as key altered metabolic pathways for systemic immune and low-grade inflammatory states. Predictive models based solely on metabolite combinations showed feasibility (area under the curve: 0.81 to 0.88) for discriminating the four parameters that represent inflammatory states and were successfully validated using a validation cohort. The inflammation-associated metabolite, namely, ß-pseudouridine, was related to carotid and coronary arteriosclerosis indicators (P < 0.05). Conclusions: This study provides further information on the relationship between plasma metabolite profiles and inflammatory states represented by various inflammatory markers in CAD. These metabolic markers provide potential insights into pathological changes during CAD progression and may aid in the development of therapeutic targets.

The dipeptidyl peptidase (DPP)-4 inhibitor trelagliptin inhibits IL-1ß-induced endothelial inflammation and monocytes attachment.

Cardiovascular diseases remain the major cause of death worldwide. Atherosclerosis is recognized as the common ground of cardiovascular diseases. Inflammatory cytokines-induced attachment of monocytes to endothelial cells is a significant event in the progression of atherosclerosis. As a highly selective dipeptidyl peptidase (DPP)-4 inhibitor, trelagliptin is used for the treatment of type 2 diabetes mellitus (T2DM). However, whether trelagliptin possesses an inhibitory effect on endothelial dysfunction and monocyte adhesion is unknown. In the current study, we tested the effect of trelagliptin in endothelial cells. We used human aortic endothelial cells (HAECs) exposed to interleukin (IL)-1ß to mimic the microenvironment of atherosclerosis. Our results showed that trelagliptin inhibited the expression of pro-inflammatory chemokines including monocyte chemoattractant protein 1 (MCP-1), CXCL-1, and IL-6. Furthermore, trelagliptin suppressed the expression of adhesion molecules such as vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Mechanistically, trelagliptin suppressed the activation of activator protein-1 (AP-1) and NF-κB signaling pathways, which modulate the inflammatory process and monocyte adhesion. Collectively, our results showed that trelagliptin had a powerful inhibitory effect on the attachment of monocytes to endothelial cells, indicating that trelagliptin might have a protective effect on cardiovascular diseases such as atherosclerosis.

Dual deficiency of angiotensin-converting enzyme-2 and Mas receptor enhances angiotensin II-induced hypertension and hypertensive nephropathy.

Angiotensin-converting enzyme-2 (ACE2) and Mas receptor are the major components of the ACE2/Ang 1-7/Mas axis and have been shown to play a protective role in hypertension and hypertensive nephropathy individually. However, the effects of dual deficiency of ACE2 and Mas (ACE2/Mas) on Ang II-induced hypertensive nephropathy remain unexplored, which was investigated in this study in a mouse model of hypertension induced in either ACE2 knockout (KO) or Mas KO mice and in double ACE2/Mas KO mice by subcutaneously chronic infusion of Ang II. Compared with wild-type (WT) animals, mice lacking either ACE2 or Mas significantly increased blood pressure over 7-28 days following a chronic Ang II infusion (P < .001), which was further exacerbated in double ACE2/Mas KO mice (P < .001). Furthermore, compared to a single ACE2 or Mas KO mice, mice lacking ACE2/Mas developed more severe renal injury including higher levels of serum creatinine and a further reduction in creatinine clearance, and progressive renal inflammation and fibrosis. Mechanistically, worsen hypertensive nephropathy in double ACE2/Mas KO mice was associated with markedly enhanced AT1-ERK1/2-Smad3 and NF-κB signalling, thereby promoting renal fibrosis and renal inflammation in the hypertensive kidney. In conclusion, ACE2 and Mas play an additive protective role in Ang II-induced hypertension and hypertensive nephropathy. Thus, restoring the ACE2/Ang1-7/Mas axis may represent a novel therapy for hypertension and hypertensive nephropathy.

Treatment of Hypertensive Heart Disease by Targeting Smad3 Signaling in Mice.

Transforming growth factor ß (TGF-ß)/Smad3 signaling plays a central role in chronic heart disease. Here, we report that targeting Smad3 with a Smad3 inhibitor SIS3 in an established mouse model of hypertension significantly improved cardiac dysfunctions by preserving the left ventricle (LV) ejection fraction (LVEF) and LV fractional shortening (LVFS), while reducing the LV mass. In addition, SIS3 treatment also halted the progression of myocardial fibrosis by blocking α-smooth muscle actin-positive (α-SMA+) myofibroblasts and collagen matrix accumulation, and inhibited cardiac inflammation by suppressing interleukin (IL)-1ß, tumor necrosis factor alpha (TNF-α), monocyte chemotactic protein 1 (MCP1), intercellular cell adhesion molecule-1 (ICAM1) expression, and infiltration of CD3+ T cells and F4/80+ macrophages. Interestingly, treatment with SIS3 did not alter levels of high blood pressure, revealing a blood pressure-independent cardioprotective effect of SIS3. Mechanistically, treatment with SIS3 not only directly inactivated TGF-ß/Smad3 signaling but also protected cardiac Smad7 from Smurf2-mediated proteasomal ubiquitin degradation. Because Smad7 functions as an inhibitor for both TGF-ß/Smad and nuclear factor κB (NF-κB) signaling, increased cardiac Smad7 could be another mechanism through which SIS3 treatment blocked Smad3-mediated myocardial fibrosis and NF-κB-driven cardiac inflammation. In conclusion, SIS3 is a therapeutic agent for hypertensive heart disease. Results from this study demonstrate that targeting Smad3 signaling with SIS3 may be a novel and effective therapy for chronic heart disease.

A genome-wide association study on lipoprotein (a) levels and coronary artery disease severity in a Chinese population.

Lipoprotein (a) [Lp(a)] is a genetically determined risk factor of coronary artery disease (CAD). Previous genome-wide association studies (GWASs), which were mostly carried out in Caucasians, have identified many Lp(a)-associated SNPs. Here, we performed a GWAS on Lp(a) levels and further explored the relationships between Lp(a)-associated SNPs and CAD severity in 1,403 Han Chinese subjects. We observed that elevated Lp(a) levels were significantly associated with the increased synergy between percutaneous coronary intervention with TAXUS and cardiac surgery (SYNTAX) score and the counts of heavily calcified lesions and long-range lesions (LRLs; P < 0.05), which are defined as lesions spanning >20 mm. Moreover, we identified four independent SNPs, namely, rs7770628, rs73596816, and rs6926458 in LPA, and rs144217738 in SLC22A2, that were significantly associated with Lp(a) levels. We also found that rs7770628 was associated with high SYNTAX scores [odds ratio (OR) (95% CI): 1.37 (1.05-1.80), P = 0.0213, false discovery rate (FDR) = 0.0852], and that rs7770628 and rs73596816 were associated with high risk of harboring LRLs [OR (95% CI): 1.53 (1.17-2.01), P = 0.0018, FDR = 0.0072 and 1.72 (1.19-2.49), P = 0.0040, FDR = 0.0080, respectively]. Our study was a large-scale GWAS to identify Lp(a)-associated variants in the Han Chinese population. Our findings highlight the importance and potential of Lp(a) intervention and expand our understanding of CAD prevention and treatment.

The comparison of short-term prognostic value of T1 mapping with feature tracking by cardiovascular magnetic resonance in patients with severe dilated cardiomyopathy.

To evaluate and compare the prognostic value of T1 mapping with feature tracking cardiovascular magnetic resonance (FT-CMR) imaging in patients with severe dilated cardiomyopathy (DCM) during short-term follow-up. A total of 46 patients with severe DCM (LVEF < 35%) underwent 3.0-T CMR with T1 mapping and FT-CMR analysis. The study end-point was defined as a combination of cardiac death, heart transplantation, and hospitalization due to cardiovascular events. The significance of the risk factors was mainly evaluated by univariate and multivariate Cox model analyses. During the median follow-up of 13 months (interquartile range 7-17 months), two patients died of heart failure, one received a heart transplantation, and six were hospitalized for heart failure. In the univariate analysis, extracellular volume fraction (ECV) showed significant predictive association with cardiovascular events (hazard ratio [HR] 1.35; 95% confidence interval [CI] 1.13-1.62; P = 0.001). No strain parameters in FT-CMR differed significantly between patients with or without events (all P > 0.05). In the multivariate analyses, ECV was the sole independent predictor of cardiovascular events (HR, 1.48; 95% CI 1.13-1.94; P = 0.005). The area under the curve of the time-dependent receiver operating characteristic in leave-one-out cross-validation (all > 0.70) further confirmed the predictive significance of ECV. In patients with severe DCM, ECV was not only a strong independent predictor of adverse cardiovascular events but also provided prognostic value prior to strain parameters of the FT-CMR in the short term.

Plasma miR-142 accounting for the missing heritability of CYP3A4/5 functionality is associated with pharmacokinetics of clopidogrel.

AIM: To investigate whether plasma miRNAs targeting CYP3A4/5 have an impact on the variance of pharmacokinetics of clopidogrel. MATERIALS & METHODS: The contribution of 13 miRNAs to the CYP3A4/5 gene expression and activity was investigated in 55 liver tissues. The association between plasma miRNAs targeting CYP3A4/5 mRNA and clopidogrel pharmacokinetics was analyzed in 31 patients with coronary heart disease who received 300 mg loading dose of clopidogrel. RESULTS: Among 13 miRNAs, miR-142 was accounting for 12.2% (p = 0.002) CYP3A4 mRNA variance and 9.4% (p = 0.005) CYP3A5 mRNA variance, respectively. Plasma miR-142 was negatively associated with H4 Cmax (r = -0.5269; p = 0.0040) and associated with H4 AUC0-4h (r = -0.4986; p = 0.0069) after 300 mg loading dose of clopidogrel in coronary heart disease patients. CONCLUSION: miR-142 could account for a part of missing heritability of CYP3A4/5 functionality related to clopidogrel activation.

The independent contribution of miRNAs to the missing heritability in CYP3A4/5 functionality and the metabolism of atorvastatin.

To evaluate the independent contribution of miRNAs to the missing heritability in CYP3A4/5 functionality and atorvastatin metabolism, the relationships among three levels of factors, namely (1) clinical characteristics, CYP3A4/5 genotypes, and miRNAs, (2) CYP3A4 and CYP3A5 mRNAs, and (3) CYP3A activity, as well as their individual impacts on atorvastatin metabolism, were assessed in 55 human liver tissues. MiR-27b, miR-206, and CYP3A4 mRNA respectively accounted for 20.0%, 5.8%, and 9.5% of the interindividual variations in CYP3A activity. MiR-142 was an independent contributor to the expressions of CYP3A4 mRNA (partial R(2) = 0.12, P = 0.002) and CYP3A5 mRNA (partial R(2) = 0.09, P = 0.005) but not CYP3A activity or atorvastatin metabolism. CYP3A activity was a unique independent predictor of variability of atorvastatin metabolism, explaining the majority of the variance in reduction of atorvastatin (60.0%) and formation of ortho-hydroxy atorvastatin (78.8%) and para-hydroxy atorvastatin (83.9%). MiR-27b and miR-206 were found to repress CYP3A4 gene expression and CYP3A activity by directly binding to CYP3A4 3'-UTR, while miR-142 was found to indirectly repress CYP3A activity. Our study indicates that miRNAs play significant roles in bridging the gap between epigenetic effects and missing heritability in CYP3A functionality.

Genome-wide screen of promoter methylation analysis of ES cells and ES derived epidermal-like cells.

Embryonic stem cells (ESCs) are a population of pluripotent cells which can differentiate into different cell types. However, there are few reports with regard to differentiate ESCs into epidermal cells in vitro. In this study, we aimed to investigate differentially methylated promoters involved in process of differentiation from ESCs into epidermal-like cells (ELCs) induced by human amnion. We successfully induced ESCs into ELCs, which expressed the surface markers of CK19, CK15 and ß1-integrin. With MeDIP-chip arrays, we identified 3435 gene promoters to be differentially methylated, involving 894 HCP (high CpG-containing promoter), 974 ICP (intermediate CpG-containing promoter) and 1567 LCP (low CpG-containing promoter) among all the 17,500 DNA methylation regions of gene promoters in both ESCs and ELCs. Gene oncology and pathway analysis demonstrated that these genes were involved in all the three categories of GO enrichment analysis, including biological process, molecular function and cellular component. All these data suggested that embryonic stem cells can differentiate into epidermal-like cells and promoter methylation is of great importance in this process.

Association of ß-adrenergic receptor genes polymorphisms with incidence of subsequent cardiovascular events in Han Chinese patients with coronary artery disease.

BACKGROUND: Sequence variants in the ß-adrenergic receptor (ADRB) genes have a close relationship with the development of coronary artery disease (CAD) and the patient's prognosis. However, there is a lack of data on the role of the variants in ADRBs genes in Han Chinese patients with CAD. We aimed to investigate the association of genetic variants in the ADRB1 and ADRB2 genes with the incidence of major adverse cardiac event (MACE) in Han Chinese patients with CAD. METHODS: A total of 545 Han Chinese patients with CAD undergoing percutaneous coronary intervention (PCI) were recruited to the study and followed for one year. Three variant sites in ADRB1 (rs1801253) and ADRB2 (rs1042713 and rs1042714) were genotyped. The effect of the ADRB1 and ADRB2 genotypes on MACE within one year was assessed. RESULTS: There were 47 cases of MACE during follow-up. There was no significant difference in the incidence of MACE among patients carrying different genotypes of the three variants in ADRB1 and ADRB2 (Log-rank, all P > 0.05). Cox regression analysis showed no association between three variants in ADRB1 and ADRB2 genes and the incidence of MACE during one-year follow-up, the adjusted hazard ratios (95% confidence interval) for rs1801253, rs1042713 and rs1042714 were 1.05 (0.54-2.02), 1.24 (0.58-2.64) and 1.66 (0.81-3.42), respectively. CONCLUSION: Our data did not support a relationship between the three polymorphisms of ADRB1 (rs1801253) and ADRB2 (rs1042713 and rs1042714) genes and risk of subsequent cardiovascular events after PCI in Han Chinese patients with CAD.

[Use of parallel factor and two dimensional fluorescence spectroscopy correlation technique for measurement of reaction between MDA and cooked ground meat].

The fluorescence characteristics of oxidation reaction between MDA and cooked ground meat were analyzed by front face three dimensional synchronous fluorescence spectroscopy, parallel factor and two dimensional correlation technique. The results showed that the reaction system has two synchronous fluorescence peaks, one is Ex 292 nm and deltalambda 50 nm, assigned to the fluorescence characteristics of tryptophan residues in proteins; the other is Ex 400 nm, delta 70 nm, corresponding with the fluorescence characteristics of MDA-protein adducts formed during oxidation; The synchronous fluorescence landscape was analyzed using PARAFAC. The loading profiles of 1st and 2nd components had an optimal lambda 50 and 70 nm, respectively. During oxidation reaction, the synchronous fluorescence intensity of tryptophan gradually decreased, while the synchronous fluorescence intensity of MDA-protein adducts gradually increased. Two dimensional correlation synchronous fluorescence spectroscopy technique showed that the variation ratio of fluorescence intensity of tryptophan preceded that of MDA-protein adducts.

[Screening and identification of auto-antigen RHDAG1 of rheumatic heart disease].

OBJECTIVE: To identify the candidate auto-antigen of rheumatic heart disease as a molecular marker for this disease. METHODS: The total RNA of the heart tissue of patients with rheumatic heart disease was extracted and reverse-transcribed into long cDNA to construct the phage expression library. The library was screened using the serum from patients with active rheumatic fever, and the positive clone was identified and analyzed by bioinformatics and expressed in vitro. The expressed products were evaluated with Western blotting and its cross-reactivity was assessed. RESULTS: The phage expression library of the heart tissue of patients with rheumatic heart disease was constructed, with the titer of the primary library of 3.3×10(6) pfu/ml, recombinant rate of 99%, and 81% of the inserted segments were larger than 1 kb. An auto-antigen RHDAG1 was identified by screening, which was homologous to keratin 18. RHDAG1 was detected in the serum of patients with active rheumatic fever and of those with rheumatic heart disease, but not in the serum of healthy subjects. CONCLUSION: Phage display library can be an effective strategy to screen the auto-antigens of rheumatic heart disease. The auto-antigen RHDAG1 can be a candidate molecular biomarker of rheumatic heart disease and/or rheumatic fever.

Molecular characterization of a Chinese pedigree with beta-thalassemia intermedia.

Hereditary persistence of fetal hemoglobin (HPFH), often associated with mutations in the beta-globin gene cluster, is normally benign, but a person carrying both HPFH and another beta-thalassemia (beta-thal) mutation will develop serious anemia. These people might be erroneously diagnosed as having homozygous beta-thal with common reverse dot-blot methods. Here we report a 5-year old boy with thalassemia intermedia, who is a compound heterozygote for the rare HPFH-6 deletion with codons 41/42 (-TCTT) beta(0)-thal, who inherited the deletion from his mother and the beta(41/42) mutation from his father.

[siRNAs targetting sphingomyelin phosphodiesterase 1 protect mouse oocytes from apoptosis].

OBJECTIVE: To investigate the potential of siRNAs targeting sphingomyelin phosphodiesterase 1 (SMPD1) in protecting the oocytes from apoptosis, and explore new approaches to female fertility preservation. METHODS: Chemically synthesized siRNA targeting SMPD1 were introduced into mouse oocytes retrieved by hyperstimulation, and the cell apoptosis was analyzed by comic assay 48 and 72 h later. RESULTS: In the oocytes without any siRNA injection, oocyte DNA damage occurred after 24 h, and large amount of DNA fragments migrated from the cells 48 h later. In oocytes injected with siRNA003, DNA migration decreased significantly as compared with the control and the other two groups injected with siRNA001 and siRNA002 (P<0.01). CONCLUSION: siRNA targeting SMPD1 may protect the oocytes from apoptosis, and has the potential for use in future female fertility preservation.

Preliminary molecular characterization of the human pathogen Angiostrongylus cantonensis.

BACKGROUND: Human angiostrongyliasis is an emerging food-borne public health problem, with the number of cases increasing worldwide, especially in mainland China. Angiostrongylus cantonensis is the causative agent of this severe disease. However, little is known about the genetics and basic biology of A. cantonensis. RESULTS: A cDNA library of A. cantonensis fourth-stage larvae was constructed, and approximately 1,200 clones were sequenced. Bioinformatic analyses revealed 378 cDNA clusters, 54.2% of which matched known genes at a cutoff expectation value of 10(-20). Of these 378 unique cDNAs, 168 contained open reading frames encoding proteins containing an average of 238 amino acids. Characterization of the functions of these encoded proteins by Gene Ontology analysis showed enrichment in proteins with binding and catalytic activity. The observed pattern of enzymes involved in protein metabolism, lipid metabolism and glycolysis may reflect the central nervous system habitat of this pathogen. Four proteins were tested for their immunogenicity using enzyme-linked immunosorbent assays and histopathological examinations. The specificity of each of the four proteins was superior to that of crude somatic and excretory/secretory antigens of larvae, although their sensitivity was relatively low. We further showed that mice immunized with recombinant cystatin, a product of one of the four cDNA candidate genes, were partially protected from A. cantonensis infection. CONCLUSION: The data presented here substantially expand the available genetic information about the human pathogen A. cantonensis, and should be a significant resource for angiostrongyliasis researchers. As such, this work serves as a starting point for molecular approaches for diagnosing and controlling human angiostrongyliasis.

[Identification and tissue localization of intermediate filament protein in Angiostrongylus cantonensis].

OBJECTIVE: To identify the type of the intermediate filament (IF) protein of Angiostrongylus cantonensis and analyze its tissue localization. METHODS: Recombinant pET-IF of antigen IF was expressed in E.coli with IPTG induction, and the expression products were purified by His.Bind column and identified for determining the type of the IF protein by Western blotting. Anti-IF antibody was prepared by multi-spot subcutaneous injection into mouse and used to detect the tissue slices of A. cantonensis by immunohistochemical analysis. RESULTS: The antigen IF were correctly expressed and purified, and identified as a keratin located in the intestine wall and cytoplusma. CONCLUSION: The antigen IF is distributed in the intestine wall of A. cantonensis.

[Induced differentiation and signaling factor PTEN expression of 3T3-L1 adipocytes].

OBJECTIVE: To optimize the condition for inducing the differentiation of 3T3-L1 preadipocytes into adipocytes and study the expression of PTEN tumor suppression gene in this process, aiming to understand the regulatory role of PTEN in normal adipocyte differentiation and collect laboratory evidence for developing drugs targeting PTEN. METHODS: The differentiation of 3T3-L1 preadipocytes cultured in high-glucose DMEM were induced according to 2 protocols with different combinations of dexamethasone, isobutylmethylxanthine (IBMX) and insulin, and the resultant adipocytes were identified by oil red O staining. The total proteins of 3T3-L1 were extracted and analyzed by Western blotting, and PTEN homology between mice and human was analyzed by bioinformatic method. RESULTS: For optimized 3T3-L1 differentiation, 3T3-L1 cells were initially induced with the combination of 1 micromol/L dexamethasone, 0.5 mmol/L IBMX and 5 microg/ml insulin for 48 h, followed by treatment with 5 microg/ml insulin in 4.5 g/L glucose DMEM for 48 h, which resulted in high differentiation rate of 3T3-L1 cells (up to 90% on the 10th day) with unified morphology and size. PTEN expression varied quantitatively in the process of differentiation, especially low on the 12th day as compared with those measured on days 4, 6 and 9. The mice PTEN mRNA shared 96% homology and PTEN amino acid 100% homology with their human counterparts. CONCLUSION: Endogenous PTEN expression is down-regulated during 3T3-L1 differentiation, suggesting that PTEN may enhance insulin sensitivity and promote adipogenesis under physiological conditions.

Schistosoma japonicum: construction of phage display antibody library and its application in the immunodiagnosis of infection.

BACKGROUND: A monoclonal antibody would be an effective tool for the detection of circulating antigens in the serum of patients with schistosomiasis, but the traditional way of producing monoclonal antibodies is not cost-effective. The objective of this study was to find a new method for the large-scale production of monoclonal antibodies against Schistosoma japonicum (Sj). METHODS: A phage display antibody library for Sj was constructed. To obtain a single-chain variable fragment antibody (scFv) against Sj, the library was screened with metabolic antigens from adult Sj worms (Sj-MAg) using enzyme-linked immunosorbent assay. The soluble scFvs selected were used to detect Sj antigens in the serum of acute and chronic schistosomiasis patients. RESULTS: Six positive clones with good reactivity to Sj-MAg were obtained from the phage display antibody library of about 1.07 x 10(6) individual clones. Only two of these six clones bound specifically to Sj-MAg and were chosen for further analysis. Specific soluble anti-Sj-MAg scFvs were produced by inducing the 2 clones with isopropyl-D-thiogalactopyranoside. The characteristics of the scFvs were then determined. The results of Western blot showed that these scFvs could bind to Sj-MAg specifically and had a molecular weight of about 31 kD. When testing serum from schistosomiasis patients with one of the two specific scFvs, its sensitivity was found to be 60% and 37% in acute and chronic patients, respectively, with a specificity of 90%. When the two specific scFvs were combined, their sensitivity was found to be 75% and 57% in acute and chronic patients, respectively, with a specificity of 85%. CONCLUSIONS: The results indicate that the scFvs are potentially useful for the diagnosis of schistosomiasis. The library construction also provides a useful tool for the further screening of other antibodies for both diagnostic and immunotherapeutic applications and for epitope analysis and vaccine design.

Inhibiting severe acute respiratory syndrome-associated coronavirus by small interfering RNA.

OBJECTIVE: To evaluate the effectiveness of small interfering RNA (siRNA) on inhibiting severe acute respiratory syndrome (SARS)-associated coronavirus replication, and to lay bases for the future clinical application of siRNA for the treatment of viral infectious diseases. METHODS: Vero-E6 cells was transfected with siRNA before SARS virus infection, and the effectiveness of siRNA interference was evaluated by observing the cytopathic effect (CPE) on Vero-E6 cells. RESULTS: Five pairs of siRNA showed ability to reduce CPE dose dependently, and two of them had the best effect. CONCLUSION: siRNA may be effective in inhibiting SARS-associated coronavirus replication.