Objective To produce an erythrocytic stage chimeric protein of Plasmodium berghei in Pichia pastoris and evaluate its immunogenicity. Methods The DNA sequences of AMA1 (Ⅲ) and MSP1-19 from P. berghei homologous to the corresponding sequences of P. falciparum chimeric antigen 2.9 (PfCP-2.9) were fused to generate a chimeric gene, designated as PbCP-2.9. The resulting gene was redesigned using Pichia preferential coden usage and expressed in P. pastoris in the secreted form. The recombinant protein was purified by Ni-NTA affinity chromatography. Three groups each with 10 BALB/c mice were im- munized subcutaneously with 20 ?g of purified PbCP-2.9 antigen formulated in Freund’s adjuvant, Montanide ISA720 and Montanide IMS 1 312, respectively. Three control groups each with 10 mice received only adjuvants emulsified with PBS. All the mice received three immunizations at 2-week intervals with the same dose of antigen. Serum samples were collected at preimmunization and one week after each immunization, and were analyzed for specific antibodies by ELISA and reaction with natural P. berghei proteins by IFAT. Results The PbCP-2.9 antigen with Mr 26 400 was successfully expressed in P. pastoris in secret- ed form. The recombinant protein can be recognized by the serum against blood stage parasites of P. berghei. High antibody responses were detected in all three PbCP-2.9-immune groups of mice by ELISA. However, mice immunized with PbCP-2.9 antigen in Freund’s adjuvant produced higher antibody titers than those with PbCP-2.9 antigen in Montanide ISA 206 and Montanide IMS 1312 adjuvants. The mean antibody titer in Freund’s adjuvant was 6.9-fold higher than in Montanide ISA 206 adjuvant and 5.6-fold higher than in Montanide IMS 1312 adjuvant after the second immunization (F=81.06, P

ObjectiveTo investigate the relationship between KAI1/CD82 protein expression in human hepatocellular carcinoma (HCC) and invasion and metastasis.MethodsSpecimens from primary hepatocellular carcinoma (155 cases), tumorous emboli (22 cases), intrahepatic satellite metastases (4 cases), extrahepatic metastases (16 cases), and normal livers (5 cases) for which a tissue microarray was constructed and used for immunohistochemical detection of KAI1/CD82 protein expression. ResultsImmunohistochemical analysis of tissue microarrays demonstrated KAI1/CD82 protein expression in 61%(95/155)of the primary HCCs and only in 32%(7/22) of the tumorous emboli ( P

Objective To express and evaluate the immunogenicity of ectodomain and its subdomains of Plasmodium berghei apical membrane antigen 1(PbAMA-1).Methods Sequence of PbAMA-1 gene was isolated from the genome of P.berghei,and was redesigned and divided into three overlapped fragments according to its subdomain structure.The codon-optimized DNA fragments of PbAMA-1 were synthesized and inserted into vector pET32a for expression in E.coli and the recombinant proteins were purified by Ni-NTA column,followed by refolding in vitro.Mice and rabbits were immu-nized with the recombinant proteins formulated with Freund adjuvant.Titer of the specific antibodies was detected by ELISA and IFA.The immunized mice were challenged by P.berghei to evaluate protective efficacy in vivo.Results The sequence of the PbAMA-1 gene was shown to be identical to that published before.PbAMA-1 sequence was redesigned via codon optimization and synthesized.Both ectodomain and its subdomains of PbAMA-1 were successfully expressed in E.coli after induction.The proteins were isolated with the purity of more than 90% after Ni column purification and refolding in vitro.Immunization of mice with the recombinant proteins induced high level of specific antibodies.The antibody titer to ectodomain E after the 3rd immunization showed a strong immunogenicity at(34.4?0.15)?10-4.The antibodies interacted with the parasites by indirect fluorescence.The immunized mice were partially protected from the challenge of P.berghei.Conclusion The recombinant PbAMA-1 is highly immunogenic and induces protective immunity against the challenge of P.berghei.

Objective To explore the diagnostic value of video bronchoscope-guide transbronchial needle aspiration(TBNA) combined with transbronchial lung biopsy(TBLB) on pulmonary sarcoidosis.Methods Twenty-two patients,definitely diagnosed as stage Ⅰ pulmonary sarcoidosis,were enrolled into the study and accepted TBNA and TBLB.Results The positive diagnostic rates of TBNA and TBLB were 63.6%(14/22) and 40.9%(9/22),but the rate increased to 90.9%(20/22) when the two methods were used together,which showed significant differences(x2=12.24,4.66,P＜0.01 or ＜0.05).Conclusion TBNA combined TBLB test is a safety method with high diagnostic accuracy for pulmonary sarcoidosis.

Objective To investigate the expression of MRP1/CD9 protein in human hepatocellular carcinoma (HCC),and its relationship to carcinoma invasion and metastasis. Methods The specimens of tissue microarray from 152 primary hepatocellular carcinomas with paracancerous liver tissue, 22 tumor emboli , 4 intrahepatic satellite metastases, 17 extrahepatic metastases ,and 5 normal livers, respectively, were constructed and used for detection of MRP1/CD9 expression by immunohistochemistry. Results Immunohistochemical analysis of tissue microarrays demonstrated MRP1/CD9 protein expression in 27.0%(41/152)of the primary HCCs. The expression of MRP1/CD9 protein was higher in HCCs without cancer thrombi than in those with cancer thrombi (40.48%vs21.82%,P10cm, P20?g/L, P=0.029). Conclusions Loss of MRP1/CD9 protein expression may be associated with invasion and metastases of hepatocellular carcinoma.

Objective: To study the difference of the cell proliferation activity and microvessel density (MVD) between hepatic benign and malignant lesions for further demonstrating the biological features of tumor. Methods: There were 290 specimens of hepatocellular carcinoma (HCC), 128 specimens of cirrhosis tissues and 25 specimens of hepatic benign lesions were detected for PCNA, Ki 67 and MVD by immunohistochemistry on tissue microarray, respectively.Results: The expression level of PCNA and Ki 67 in HCC were 90.2% and 43.1%, which was obviously higher than that in cirrhosis (48.5% and 3.9%, P 0.05). MVD counting in HCC pathological grade Ⅰ Ⅱ(29.9?18.6) was higher than those in gradeⅢ (22.2? 18.2) and Ⅳ(22.9?19.0, P

New strategies are needed for prevention of Pseudomonas aeruginosa (P. aeruginosa) infections, a widespread disease caused by P. aeruginosa with strong drug resistance. The immunoprotective capacity of the receptor of autoinducers protein LasR/RhlR was examined in the BALB/c mice. At first, specialized expression plasmids were developed to facilitate expression of LasR/RhlR proteins in Escherichia coli (E. coli). Then, biofilms were grown from clinical isolated P. aeruginosa PA0305 to investigate the relative contributions of cell signaling for biofilm formation. Morphological characters of biofilm were quantified using Image-Pro Plus software. Fluorescence analysis demonstrated that cell signal molecule LasR/RhlR significantly (P ＜ 0.05) influenced development of P. aeruginosa biofilm. Active immunization of mice with LasR/RhlR was found to provide significant protection against homologous challenge with P. aeruginosa in mice lungs. In 10 days after lungs inoculation, the bacterial clearance rate of the immunized mice was clearly higher than that of non-immunized groups on the basis of microbiological and histological assays. The protective effects of immunization with LasR and Rh1R together were the same as the result of LasR or Rh1R immunized mice alone. These data indicate that the manner ofLasR, Rh1R or both is an important determinant of immunoprotection in mice lungs infection.

Objective To analyse the clinical manifestations of adrenoleukodystrophy (ALD) pedigree and the background of the associated genes. Methods The clinical data of an ALD pedigree were collected and PCR productsequencingwereperformedtoresearch into the change of ALD gene. Results Diagnosis of ALD was determined by the clinical manifestations and brain MRI. The homozygote mutation GGG(Gly)→AGG(Arg) at codon 266 in exon 1 was found in the ALD patient and the heterozygote mutation at the same loci was found in his mother,butwasnotfoundin other members of the family and 2 normal subjects. Conclusions The ALD patient′s mother is the first person taking this point mutation and the mutation causes severe clinical manifestations. The gene research could be regarded as the molecular base of the antenatal diagnosis.

Objective To evaluate the sensitivity and specificity of various assays in diagnosing Cushing′s syndrome. Methods The plasma cortisol, urinary free cortisol (UFC), circadian rhythm in cortisol secretion and dexamethasone suppression test were assessed in 173 patients clinically diagnosed Cushing′s syndrome. The data were compared with the postoperative pathologic diagnosis. Results The normal diurnal rhythm of cortisol secretion was lost in 92.9% patients with Cushing′s syndrome. The loss of normal diurnal rhythm of cortisol secretion of 2 time points occurred in 85.1% (8:00, 16:00) and 91.8% (8:00, 24:00), and that of 3 time points (8:00, 16:00, 24:00) in 94.7% of the cases. The excretion of UFC was increased in 91.7% of patients with Cushing′s syndrome. Low-dosedexamethasonedidnotsuppressthe excessive secretion of glucocorticoid in 79.7% (1 mg) and 84.3% (2 mg) patients with Cushing′s syndrome. The basal level of plasma cortisol was raised in 75.6% patients. The sensitivity of 8 mg dexamethasone suppression test was 50%-70% as the standard was set at 50% suppression, and specificity was more than 95%. Conclusion The most sensitive tests for Cushing′s syndrome are the loss of normal circadian rhythm of cortisol secretion and increased UFC. The method of 3 time points is more sensitive than that of 2 time points in the assessment of circadian rhythm. The 8 mg dexamethasone suppression test is the most useful method in differentiating Cushing′s disease from adrenal adenoma.

Objective To investigate the epidemiological status of visceral leishmaniasis in Hamangou coal mine area of Korla City of Xinjiang Uygur Autonomous Region.Methods Based on a hint of possible existence of patients, a retrospective survey was carried out house by house to find cases with suspected signs/symptoms of the disease.Meanwhile, a survey on current status was conducted, including physical examination(liver and spleen palpation) to those less than 15 years-old, leishmanin skin test and rk39 immunochromatographic strip test for part of the residents.Bone marrow smears were examined for the cases with clinical signs/symptoms and positive rk39 strip test.Sandflies were collected using routine methods in and around the area, identified, and dissected to find infection of promastigotes.Results Leishmanin skin test was performed in 185 people with a positive rate of 21.1%(39/185), 39 out of 140 local residents who have lived there for more than 6 years showed positive(27.9%) , while all residents who have lived less than 6 years and children under 5 years old were negative.Of the 81 children under 15 years old with a negative skin test, one showed positive for rk39 strip test, and leishmania body was found in the bone marrow smear of this case, so confirmed as visceral leishmaniasis.12 sandfies were identified as Phlebotomus alexandri, and natural infection with promastigotes was found in one sandly.Conclusion The investigation confirms that visceral leishmaniasis is endemic in the Hamangou coal mine area.