[Observation on growth and reproduction of various Leishmania isolates in different culture media].

OBJECTIVE: To compare the growth and reproduction of the promastigotes of Leishmania isolates from various endemic areas of visceral leishmaniasis in China in various culture media, so as to provide experimental evidence for selecting an appropriate medium for the culture of Leishmania. METHODS: A total of 3 × 105 promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates were inoculated into 1 mL NNN medium, 1 mL M199 medium supplemented with 20% fetal bovine serum medium, 1 mL M199 medium supplemented with 20% horse serum medium, and 1 mL brain heart infusion medium containing heme, respectively. All media were placed at 22 ℃ under a sterile condition, and the number of promastigotes was counted continuously for 8 days under a microscope. The growth curve was plotted for the three Leishmania isolates. RESULTS: The promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates all grew and reproduced in the NNN medium, the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium. The number of promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates was all significantly higher in the NNN medium than in the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium at various time points of culture (all P values < 0.05), and the number of promastigotes of the KS-2 isolate was all significantly greater than that of the Cy and JIASHI-5 isolates in the NNN medium, the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium at various time points of culture (all P values < 0.05). In ad dition, the promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates failed to grow and reproduce in the brain heart infusion medium. CONCLUSIONS: The growth and reproduction of the promastigotes of various Leishmania isolates from various endemic areas of visceral leishmaniasis in China vary in the same culture medium, and the growth and reproduction of a Leishmania isolate vary in different culture media. The NNN medium best fits for the culture of Leishmania isolates in the endemic areas of visceral leishmaniasis in China.

Observation on growth and reproduction of various Leishmania isolates in different culture media / 中国血吸虫病防治杂志

Objective To compare the growth and reproduction of the promastigotes of Leishmania isolates from various endemic areas of visceral leishmaniasis in China in various culture media, so as to provide experimental evidence for selecting an appropriate medium for the culture of Leishmania. Methods A total of 3 × 105 promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates were inoculated into 1 mL NNN medium, 1 mL M199 medium supplemented with 20% fetal bovine serum medium, 1 mL M199 medium supplemented with 20% horse serum medium, and 1 mL brain heart infusion medium containing heme, respectively. All media were placed at 22 ℃ under a sterile condition, and the number of promastigotes was counted continuously for 8 days under a microscope. The growth curve was plotted for the three Leishmania isolates. Results The promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates all grew and reproduced in the NNN medium, the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium. The number of promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates was all significantly higher in the NNN medium than in the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium at various time points of culture (all P values < 0.05), and the number of promastigotes of the KS-2 isolate was all significantly greater than that of the Cy and JIASHI-5 isolates in the NNN medium, the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium at various time points of culture (all P values < 0.05). In ad dition, the promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates failed to grow and reproduce in the brain heart infusion medium. Conclusions The growth and reproduction of the promastigotes of various Leishmania isolates from various endemic areas of visceral leishmaniasis in China vary in the same culture medium, and the growth and reproduction of a Leishmania isolate vary in different culture media. The NNN medium best fits for the culture of Leishmania isolates in the endemic areas of visceral leishmaniasis in China.

Comparative proteomics analysis of Leishmania infantum strains isolated from two epidemiological regions of visceral leishmaniasis in China / 中国血吸虫病防治杂志

Objective To analyze the protein abundance differences between two Leishmania infantum strains isolated from different epidemiological types of visceral leishmaniasis in China by comparative proteomics method. Methods Tryptic digests of total proteins were analyzed by using liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by label-free quantitative differential expression analysis. The MS data were analyzed with MaxQuant software (ver 1.3.0.5) against data base. Results This study resulted in the identification of 4 274 proteins across two strains (JIASHI-5 and SC6) . Of these, 1 219 differentially expressed proteins (ratio ＞ 2.0 or ＜ 0.5, P < 0.05) were identified. Considering the proteins differentially or uniquely expressed in the strains, 550 proteins were only found in the JIASHI-5 strain, and 174 proteins were only found in the SC6 strain. Totally 495 differentially proteins were expressed in the two groups, among which 328 proteins were down-regulated and 167 proteins were up-regulated in SC6 strain. Some of the identified differentially expressed proteins were demonstrated and they involved in energy metabolism, stress response, prolonging the lifetime of the infected host cell and survival and proliferation in virulent strains. Conclusion This study reveals a group of differentially expressed proteins and the related biologic function that may lay the foundation for screening and identification of the key Leishmania molecules relative to pathogenicity.

Observation on growth and reproduction of various Leishmania isolates in different culture media / 中国血吸虫病防治杂志

Objective To compare the growth and reproduction of the promastigotes of Leishmania isolates from various endemic areas of visceral leishmaniasis in China in various culture media, so as to provide experimental evidence for selecting an appropriate medium for the culture of Leishmania. Methods A total of 3 × 105 promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates were inoculated into 1 mL NNN medium, 1 mL M199 medium supplemented with 20% fetal bovine serum medium, 1 mL M199 medium supplemented with 20% horse serum medium, and 1 mL brain heart infusion medium containing heme, respectively. All media were placed at 22 ℃ under a sterile condition, and the number of promastigotes was counted continuously for 8 days under a microscope. The growth curve was plotted for the three Leishmania isolates. Results The promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates all grew and reproduced in the NNN medium, the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium. The number of promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates was all significantly higher in the NNN medium than in the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium at various time points of culture (all P values < 0.05), and the number of promastigotes of the KS-2 isolate was all significantly greater than that of the Cy and JIASHI-5 isolates in the NNN medium, the M199 medium supplemented with 20% fetal bovine serum medium, and the M199 medium supplemented with 20% horse serum medium at various time points of culture (all P values < 0.05). In ad dition, the promastigotes of KS-2, Cy and JIASHI-5 Leishmania isolates failed to grow and reproduce in the brain heart infusion medium. Conclusions The growth and reproduction of the promastigotes of various Leishmania isolates from various endemic areas of visceral leishmaniasis in China vary in the same culture medium, and the growth and reproduction of a Leishmania isolate vary in different culture media. The NNN medium best fits for the culture of Leishmania isolates in the endemic areas of visceral leishmaniasis in China.

[Comparative proteomics analysis of Leishmania infantum strains isolated from two epidemiological regions of visceral leishmaniasis in China].

OBJECTIVE: To analyze the protein abundance differences between two Leishmania infantum strains isolated from different epidemiological types of visceral leishmaniasis in China by comparative proteomics method. METHODS: Tryptic digests of total proteins were analyzed by using liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by label-free quantitative differential expression analysis. The MS data were analyzed with MaxQuant software (ver 1.3.0.5) against data base. RESULTS: This study resulted in the identification of 4 274 proteins across two strains (JIASHI-5 and SC6) . Of these, 1 219 differentially expressed proteins (ratio ＞ 2.0 or ＜ 0.5, P < 0.05) were identified. Considering the proteins differentially or uniquely expressed in the strains, 550 proteins were only found in the JIASHI-5 strain, and 174 proteins were only found in the SC6 strain. Totally 495 differentially proteins were expressed in the two groups, among which 328 proteins were down-regulated and 167 proteins were up-regulated in SC6 strain. Some of the identified differentially expressed proteins were demonstrated and they involved in energy metabolism, stress response, prolonging the lifetime of the infected host cell and survival and proliferation in virulent strains. CONCLUSIONS: This study reveals a group of differentially expressed proteins and the related biologic function that may lay the foundation for screening and identification of the key Leishmania molecules relative to pathogenicity.

Comparative proteomics analysis of Leishmania infantum strains isolated from two epidemiological regions of visceral leishmaniasis in China / 中国血吸虫病防治杂志

Objective To analyze the protein abundance differences between two Leishmania infantum strains isolated from different epidemiological types of visceral leishmaniasis in China by comparative proteomics method. Methods Tryptic digests of total proteins were analyzed by using liquid chromatography-tandem mass spectrometry (LC-MS/MS), followed by label-free quantitative differential expression analysis. The MS data were analyzed with MaxQuant software (ver 1.3.0.5) against data base. Results This study resulted in the identification of 4 274 proteins across two strains (JIASHI-5 and SC6) . Of these, 1 219 differentially expressed proteins (ratio ＞ 2.0 or ＜ 0.5, P < 0.05) were identified. Considering the proteins differentially or uniquely expressed in the strains, 550 proteins were only found in the JIASHI-5 strain, and 174 proteins were only found in the SC6 strain. Totally 495 differentially proteins were expressed in the two groups, among which 328 proteins were down-regulated and 167 proteins were up-regulated in SC6 strain. Some of the identified differentially expressed proteins were demonstrated and they involved in energy metabolism, stress response, prolonging the lifetime of the infected host cell and survival and proliferation in virulent strains. Conclusion This study reveals a group of differentially expressed proteins and the related biologic function that may lay the foundation for screening and identification of the key Leishmania molecules relative to pathogenicity.

[Interpretation of Diagnostic Criteria for Kala-azar].

Kala-azar was once transmitted in the northern area of the Yangtze River in China, including 16 provinces (cities or autonomous regions). Through the great continuing prevention and control effort, this disease has been effectively controlled in the most of endemic areas. However, because the epidemic factors of the disease are complex, this disease still transmits or sporadically occurs in the western part of China, including 60 counties of Xinjiang, Gansu, Sichuan, Inner Mongolia, Shanxi, and Shaanxi provinces (autonomous regions). Following the Management Measures for Health Criteria, the Diagnostic Criteria for Kala-azar (WS 258-2006) was compiled by the ex-Ministry of Health of the People's Republic of China and it was issued in April 7, 2006 and implemented in December 1, 2006. The Criteria consists of six parts, including the application range, terms and definitions, diagnostic principle, diagnostic standard, and differential diagnosis. Two informative appendices (epidemiology and differential diagnosis) and two normative appendices (immune-detection and etiological examination) are attached. The Criteria provides the technical reference for diagnosis of kala-azar in medical institutions and disease control institutions. Combined with the current epidemic situation of kala-azar in China, this paper interprets the main contents of the Diagnostic Criteria for Kala-azar (WS 258-2006), so as to promote its learning and implementing.

[Cloning, expression and serological evaluation of H3 protein from Echinococcus granulosus].

OBJECTIVE: To clone and express the basement membrane specific heparan sulfate proteoglycan core protein (H3), and to evaluate its effect in detection of human cystic echinococcosis (CE). METHODS: The H3 gene immunoscreened from the cDNA library was cloned into pGEX-4T vector. The recombinant plasmid pGEX-3X-AgB8/3 was transformed into Escherichia coli BL21 strains and induced by isopropyl-ß-D-thiogalactopyranoside (IPTG). Then the expressed recombinant protein was purified by affinity chromatography and its effect in the detection of CE was evaluated by ELISA. Meanwhile, the effects of H3 and two antigens that the research group prepared before (purified HCF and rAgB8/2) in CE detection were compared. RESULTS: The plasmid pGEX-4T-H3 was successfully constructed and H3 was successfully expressed in prokaryotic cells. The sensitivities of the recombinant H3, purified HCF and rAgB8/2 in CE detection were 84.0% (68/81), 90.1% (73/81) and 77.8% (63/81) respectively, and there was no statistical difference among them (χ2 = 4.58, P > 0.05). The cross reactions of recombinant H3 with the sera of the patients with CE, cysticercosis and schistosomiasis were 63.3% (19/30), 16.7% (5/30) and 5.0% (1/20) respectively, and the cross reaction was 0 with the sera of healthy people. The specificities of recombinant H3, purified HCF, and rAgB8/2 were 80.8% (105/130), 71.5% (93/130) and 82.3% (107/130) respectively, and there were no statistical difference among them (χ2 = 5.71, P > 0.05). CONCLUSIONS: The recombinant H3 is a potential diagnostic antigen for CE detecting.

99Tcm-MIBI adenosine myocardial perfusion imaging in patients with slow coronary flow / 中华核医学杂志

Objective To investigate the relationship between myocardial ischemia and slow coronary flow phenomenon with 99Tcm-methoxyisobutylisonitrile (MIBI) adenosine myocardial perfusion SPECT imaging. Methods Forty-four patients were divided to three groups according to the result of coronary angiography(CAG). There were GAG-positive(P-GAG) (n=12),slow coronary flow (CSF) (n =22),and normal coronary flow (NCF) (n = 10). Results of adenosine myocardial perfusion imaging were compared among these three groups. Semi-quantitative visual scoring method was used to evaluate the myocardial perfusion:0 = normal,1 = mild decrease,2 = moderate decrease,3 = severe decrease,4 = defect. Statistical analysis was performed using variance analysis,t-test and x2-test. Results No significance was observed at age ( t =0.27,0. 54 and 0. 59),sex (x2 = 0. 92),hypertension,hyperlipemia and diabetes (x2 = 1.23,all P ＞ 0.05 ) among the three groups. A significantly higher frames of the coronary thrombolysis in myocardial infarction (TIMI) flow was noted in CSF than in NCF groups (33.7 ±5.5 vs 17.6 ±3.9,t = 9. 58,P ＜0. 001 ). The positive adenosine myocardial perfusion imaging rate were significant among these three groups with 100% (12/12) in P-CAG group,77.3% (17/22) in CSF group,and 20% (2/10) in NCF group. When using semi-quantitative visual scoring method,significantly higher average ischemia segments were noted in CSF group than in NCF group ( 1.06 ± 0.77 and 0. 91 ± 0.80,t = - 2. 02,P ＜ 0. 05 ),but was less than that in P-CAG group (2.41 ±0.79,t =4. 54,P ＜0.001 ). The degree of ischemia of CSF group was higher than that in NCF group ( 8.01 ± 6.06,and 2.73 ± 2.60,t = - 2.07,P ＜ 0.05 ) and was less than that in P-CAG group (14. 07 ±12. 77 ,t=1.44,P＞0. 05). Conclusion Slow coronary flow phenomenon can be detected by adenosine myocardial perfusion image to offer the evidence of diagnosis and treatment for the chest pain patients with negative coronary angiography results.