Cs1, a Clonorchis sinensis-derived serodiagnostic antigen containing tandem repeats and a signal peptide.

BACKGROUND: Clonorchiasis, caused by the liver fluke Clonorchis sinensis, remains a serious public health issue in Asia, especially in China, and its relationship with cholangiocarcinoma has highlighted the importance of C. sinensis infection. Proteins containing tandem repeats (TRs) are found in a variety of parasites and, as targets of B-cell responses, are valuable for the serodiagnosis of parasite infections. Here, we identified a novel C. sinensis-specific antigen, Cs1, containing TRs, and investigated its diagnostic value, other immunological properties, and tissue distribution. METHODOLOGY/PRINCIPAL FINDINGS: A partial Cs1 cDNA sequence was cloned by screening an adult C. sinensis cDNA expression library. The full-length Cs1 cDNA was obtained by 5' rapid amplification of cDNA ends. The deduced Cs1 protein consists of a signal peptide and five TRs of 21 amino acids. The recombinant Cs1 (rCs1) was constructed and purified. rCs1 showed higher sensitivity (94.3%) and specificity (94.4%) than the C. sinensis excretory-secretory products (ESPs) according to ELISA of 114 serum samples. Native Cs1 was identified in C. sinensis ESPs and crude antigens of adult C. sinensis by western blotting using an anti-rCs1 monoclonal antibody. ELISA of recombinant peptides of different Cs1 regions demonstrated that the TR region was immunodominant in Cs1. Immunohistochemistry and confocal microscopy revealed that Cs1 is located in a granule-like structure surrounding the acetabulum of C. sinensis adults that has not previously been described. CONCLUSIONS/SIGNIFICANCE: We identified a novel C. sinensis-specific TR protein, Cs1, which is an antigen of high serological significance, compared with C. sinensis ESPs. The deduced features of Cs1 show a unique structure containing TRs and a signal peptide and the TR region is immunodominant in Cs1. This provides a basis for targeted screens of other antigens. The novel structure in which Cs1 is located also deserves further investigation.

Evaluation of patients with relapsing-remitting multiple sclerosis using tract-based spatial statistics analysis: diffusion kurtosis imaging.

BACKGROUND: Diffusion kurtosis imaging (DKI) has the potential to provide microstructural insights into myelin and axonal pathology with additional kurtosis parameters. To our knowledge, few studies are available in the current literature using DKI by tract-based spatial statistics (TBSS) analysis in patients with multiple sclerosis (MS). The aim of this study is to assess the performance of commonly used parameters derived from DKI and diffusion tensor imaging (DTI) in detecting microstructural changes and associated pathology in relapsing remitting MS (RRMS). METHODS: Thirty-six patients with RRMS and 49 age and sex matched healthy controls underwent DKI. The brain tissue integrity was assessed by fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (Da), radial diffusivity (Dr), mean kurtosis (MK), axial kurtosis (Ka) and radial kurtosis (Kr) of DKI and FA, MD, Da and Dr of DTI. Group differences in these parameters were compared using TBSS (P < 0.01, corrected). To compare the sensitivity of these parameters in detecting white matter (WM) damage, the percentage of the abnormal voxels based on TBSS analysis, relative to the whole skeleton voxels for each parameter was calculated. RESULTS: The sensitivities in detecting WM abnormality in RRMS were MK (78.2%) > Kr (76.7%) > Ka (53.5%) and Dr (78.8%) > MD (76.7%) > FA (74.1%) > Da (28.3%) for DKI, and Dr (79.8%) > MD (79.5%) > FA (68.6%) > Da (40.1%) for DTI. DKI-derived diffusion parameters (FA, MD, and Dr) were sensitive for detecting abnormality in WM regions with coherent fiber arrangement; however, the kurtosis parameters (MK and Kr) were sensitive to discern abnormalities in WM regions with complex fiber arrangement. CONCLUSIONS: The diffusion and kurtosis parameters could provide complementary information for revealing brain microstructural damage in RRMS. Dr and DKI_Kr may be regarded as useful surrogate markers for reflecting pathological changes in RRMS.

Sensory neuronopathy involves the spinal cord and brachial plexus: a quantitative study employing multiple-echo data image combination (MEDIC) and turbo inversion recovery magnitude (TIRM).

INTRODUCTION: Sensory neuronopathy (SNN) is a distinctive subtype of peripheral neuropathies, specifically targeting dorsal root ganglion (DRG). We utilized MRI to demonstrate the imaging characteristics of DRG, spinal cord (SC), and brachial plexus at C7 level in SNN. METHODS: We attempted multiple-echo data image combination (MEDIC) and turbo inversion recovery magnitude (TIRM) methods in nine patients with sensory neuronopathy and compared with those in 16 disease controls and 20 healthy volunteers. All participants underwent MRI for the measurement of DRG, posterior column (PC), lateral column, and spinal cord area (SCA) at C7 level. DRG diameters were obtained through its largest cross section, standardized by dividing sagittal diameter of mid-C7 vertebral canal. We also made comparisons of standardized anteroposterior diameter (APD) and left-right diameters of SC and PC in these groups. Signal intensity and diameter of C7 spinal nerve were assessed on TIRM. RESULTS: Compared to control groups, signal intensities of DRG and PC were higher in SNN patients when using MEDIC, but the standardized diameters were shorter in either DRG or PC. Abnormal PC signal intensities were identified in eight out of nine SNN patients (89 %) with MEDIC and five out of nine (56 %) with T2-weighted images. SCA, assessed with MEDIC, was smaller in SNN patients than in the other groups, with significant reduction of its standardized APD. C7 nerve root diameters, assessed with TIRM, were decreased in SNN patients. CONCLUSION: MEDIC and TIRM sequences demonstrate increased signal intensities and decreased area of DRG and PC, and decreased diameter of nerve roots in patients with SNN, which can play a significant role in early diagnosis.

[Evaluation of Clonorchis sinensis PPMP I antigen Cs2 recombinant protein for immunodiagnosis of clonorchiasis].

OBJECTIVE: To develop and preliminarily evaluate two immunodiagnostic methods for clonorchiasis using Clonorchis sinensis PPMP I antigen Cs2 recombinant protein (rCs2). METHODS: Using the soluble rCs2, an indirect ELISA and a colloidal-gold immuno-chromatography assay (GICA) dynamic flow strip was developed for detecting specific antibodies in serum. Serum samples from 35 egg-positive clonorchiasis patients, 33 healthy individuals, 15 schistosomiasis patients, 15 paragonimiasis westermani patients and 13 cysticercosis patients were examined by ELISA and GICA strip test. To further evaluate the diagnostic value of these two methods, eight New Zealand rabbits were randomly divided into infected group and treatment group. Each rabbit was infected with 600 C. sinensis metacercaria. Rabbits in treatment group were treated with praziquantel [150 mg/(kg x d) x 2d] individually at day 56 post-infection. ELISA and GICA strip test were used to observe the dynamic changes of specific antibodies against rCs2 in the two parallel groups during the period of 0-44 weeks. RESULTS: The sensitivity, specificity and total coincidence rate determined by the ELISA method were 71.4% (25/35), 93.4% (71/76), and 86.5% (96/111), respectively, and the cross reaction with schistosomiasis, paragonimiasis and cysticercosis patients were 1/15, 1/15, and 1/13, respectively. The sensitivity, specificity and coincidence rate in the GICA strip test were 85.7% (30/35), 92.1% (70/76), and 90.1%(100/111), respectively. In C sinensis infected rabbits, antibodies level began to increase at 4 weeks after infection, peaked at the 6th week, and declined rapidly to a lower level in the 20th week, while the changing pattern of antibodies level in the treatment group was similar with that of infected group (P > 0.05). In the GICA strip test, antibodies in two groups could be detected in 4-16 weeks. CONCLUSION: Indirect ELISA and the GICA dynamic flow strip developed in this study may be of value in the immunodiagnosis of clonorchiasis.

[Cloning, expression of PPMP antigen genes of Clonorchis sinensis and immunogenic identification of the recombinant proteins].

OBJECTIVE: To screen and identify new specific antigen gene from a cDNA library of adult Clonorchis sinensis, and investigate the immunogenicity of the recombinant proteins. METHODS: The lambdaZAP cDNA library of adult C. sinensis was immunoscreened with pooled sera of clonorchiasis patients. The positive clones were sequenced and analyzed. The sequence encoding the mature peptide was cloned into prokaryotic expression vector pET28b(+). The recombinant plasmid was transformed into E. coli BLR21 (DE3) or BLR21 (DE3) pLysS and followed by expression of the protein induced by IPTG. The recombinant protein was purified by His-bind-resin (Ni-NTA) affinity chromatography and identified by Western blotting. BALB/c mice were immunized with purified recombinant pET28b-Cs2 protein, and the sera from immunized mice were analyzed for specific antibodies by ELISA. RESULTS: A total of 44 positive clones were isolated from the C. sinensis cDNA library. Three clones containing specific tandem repeats of PPMP amino acid sequence were named as C. sinensis PPMP antigen genes. The genes containing KPPMPGDRDA, QPPMPGGRDA were named as type PPMP I and type PPMP II antigens, respectively. Sequence analysis revealed that these PPMP genes were a novel specific C. sinensis antigen gene family. Two new genes, PPMP I Cs2 and PPMP II Cs3, were expressed in E. coli, and SDS-PAGE showed that the two recombinant proteins were about M(r) 22 000 and M(r) 39 000. The two soluble recombinant proteins were recognized by pooled sera of clonorchiasis patients. A high level of specific IgG against the recombinant proteins (maximum dilution 1 : 64 000) was produced in immunized mice. CONCLUSION: A novel PPMP gene family of C. sinensis has been identified, and its recombinant proteins show high immunogenicity.

[Asymptomatic Leishmania infection in human population of Wenxian County, Gansu Province].

OBJECTIVE: To analyze the status of Leishmania infantum asymptomatic infection in human population of a Kala-azar endemic area in Wenxian County, Gansu Province, and to evaluate the tests used. METHODS: Blood samples were tested by PCR using two pairs of primers, RV1-RV2 and K13A-K13B, for detecting Leishmania-specific DNA. ELISA and rK39-dipstick were used to detect Leishmania-specific antibodies. RESULTS: The positive rate of PCR, ELISA and rK39-dipstick was 30.9%(83/269). 24.2%(65/269) and 0 (0/269) respectively. CONCLUSION: The prevalence of asymptomatic infection of L. infantum in humans is high in the area. PCR test based on RV1-RV2 and K13A-K13B primer pairs is a sensitive and specific method for detecting the asymptomatic infection.

[Establishment and evaluation of colloid gold labeled immunochromatographic strip test for rapid diagnosis of malaria].

OBJECTIVE: To establish and evaluate a gold immunochromatographic strip test for detection and differentiation of Plasmodium vivax and P. falciparum. METHODS: The monoclonal antibodies, F4H12, G4C9 and D8F7, were conjugated with colloid gold as detecting reagent; monoclonal antibody B2G10 (against P. vivax/ P. falciparum) and D6A7 (only against P. falciparum) were immobilized on nitrocellulose in proper position. Blood samples from 107 febrile patients from endemic area of malaria and 17 patients with visceral leishmaniasis were used for evaluating the specificity. Blood samples of malaria patients (110 with P. vivax and 54 with P. falciparum) were used for evaluating the sensitivity. RESULTS: 5 samples out of 107 febrile patients and 17 patients with visceral leishmaniasis showed false positive reaction with a specificity of 96.0% (119/124), all the 17 samples from patients with visceral leishmaniasis were negative. 164 blood samples of malaria patients showed a sensitivity of 92.3% (153/164), 92.7% (102/110)and 94.4% (51/ 54) for patients infected with P. vivax or P. falciparum, respectively. CONCLUSION: The immunochromatographic strip test based on antigen-capturing is a sensitive, specific, simple and rapid assay for malaria diagnosis.

[Study on PCR method for detecting the asymptomatic infection of Leishmania infantum].

OBJECTIVE: To establish PCR method for the detection of the asymptomatic infection of Leishmania infantum. METHODS: Six primer pairs were selected for detecting Chinese strain of L. infantum by optimizing conditions which affect amplification. Their sensitivity and specificity were compared by using DNAs extracted from human blood seeded with cultured L. infantum promastigotes (MHOM/CN/86/GS) as template. Blood samples of the inhabitants without symptoms of visceral leishmaniasis in the endemic area were analyzed with two selected primer pairs with good sensitivity and specificity. RESULTS: The specificity of all six primer pairs reached 100%, and the sensitivity varied among the primer pairs. The primer pairs RV1-RV2 (0.1 parasite/ml blood) and K13A-K13B (1 parasite/ml blood) were most sensitive. Leishmania DNA was detected in 33% (33/100) and 30% (30/100) human blood samples by RV1-RV2 and K13A-K13B primer pairs respectively. CONCLUSION: This study suggests that RV1-RV2 and K13A-K13B primer pairs are suitable in detecting the asymptomatic infection of L. infantum, and the prevalence of the asymptomatic infection is high in human population in the endemic area.

[Preparation of monoclonal antibodies specific to lactate dehydrogenase of Plasmodium falciparum].

OBJECTIVE: To prepare monoclonal antibodies specific to lactate dehydrogenase of Plasmodium falciparum. METHODS: The Plasmodium falciparum lactate dehydrogenase (pLDH) gene was amplified from whole blood of malaria patients by PCR and cloned into expression vector pGEX-3X. Recombinant pLDH protein was expressed and purified, and used for immunizing mice to prepare monoclonal antibodies (McAbs). The McAbs were characterized by Western blotting analysis. RESULTS: The Plasmodium falciparum lactate dehydrogenase gene was amplified and cloned into ex pression vector pGEX-3X. The recombinant pLDH plasmid was expressed in E. coli) BL-21 cells. 15 cell lines of McAbs with high titer against pLDH were obtained using the recombinant pLDH as immunogen. Western blotting analysis showed that these McAbs recognized a Mr 33,000 of native Plasmodium falci parvum protein without cross reaction with constituents of red blood cell of febrile patients from endemic area of malaria. CONCLUSION: Fifteen hybridoma cell lines secreting high titer of McAb specific to Plasmodium falciparum LDH were established based on the recombinant pLDH.