Objective To perform laboratory detection and molecular traceability analysis on a case of imported kala-azar in Shenzhen to determine the infection strain. Methods Bone marrow puncture fluid and blood samples from a case of kala-azar in Shenzhen were collected for laboratory tests. The patient's bone marrow puncture fluid smears were stained with Giemsa and examined under a microscope. Blood samples were examined for antibodies using the rk39 visceral leishmania rapid diagnostic reagent. Whole blood DNA was extracted, and the ITS-1 sequence was amplified by PCR, sequenced and aligned, and a phylogenetic tree was constructed based on the ITS-1 sequence. Results Microscopic examination of the patient's bone marrow smears revealed a large number of Leishmania amastigotes without flagella, confirming the diagnosis of kala-azar. The patient's blood was tested positive with the rk39 rapid diagnostic reagent, and PCR amplification yielded an ITS-1 gene product sequence that matched the expected size. Sequence alignment with the NCBI database showed 100% sequence similarity with the ITS-1 gene sequence of Leishmania infantum, confirming the infecting strain as Leishmania infantum. Phylogenetic tree construction of the amplified ITS-1 sequence revealed clustering into a clade with Leishmania infantum , and close to KC347299, one of the reference sequence selected. Conclusions The case of kala-azar in Shenzhen was caused by Leishmania infantum. Kala-azar still occurs in China, so the diagnostic technology of medical personnel in non-epidemic areas should be strengthened so that they can actively use new diagnostic technologies to assist in diagnosis, thus improving their prevention and control ability of Leishmania parasites.

Abstract: Objective　To analyze and understand the mutations of drug resistance genes in imported Plasmodium falciparum in Shenzhen, aiming to assess the efficacy of antimalarial drugs and guide effective drug use. Methods A total of 85 samples from individuals with imported Plasmodium falciparum confirmed by fluorescence quantitative polymerase chain reaction (PCR) in Shenzhen from 2022 to 2023 were collected and genomic DNA was extracted. Nested PCR was used to amplify resistance genes, including Plasmodium falciparum Kelch 13 (PfK13), multidrug resistance gene 1 (Pfmdr1), chloroquine resistance transporter (Pfcrt), dihydrofolate reductase (Pfdhfr), and dihydropteroate synthase (Pfdhps) genes. Bidirectional sequencing was conducted, and mutations in these resistance genes were analyzed using MEGA11.06 software. Results　The study found one missense mutation (S549P) and four synonymous mutations in PfK13. For Pfmdr1, 62.69% of the samples showed Y184F mutation, and no N86Y mutation was detected. No mutations at positions 72 and 73 were detected in the Pfcrt gene, while mutations at M74I, N75E, and K76T accounted for 17.46%, 15.87%, and 15.87%, respectively. The wild-type of Pfcrt gene is dominant (82.54%, 52), followed by the triple mutant I74E75T76 (15.87%, 10). The most common mutation type for Pfdhfr is I51R59N108 (91.78%, 67), followed by the wild type (2.74%, 2). More than half (60.32%, 38) of the Pfdhps samples were wild-type, with single mutation K540E being the most common mutation type. S436A, G437A, K540E, A581G, A613S, I431V, G556K, and G579E site mutations were detected. Among the Pfdhfr-Pfdhps combination mutations, I51R59N108-E540 was the most frequent combination mutation (11.48%), with 59.02% of samples showing solitary Pfdhfr mutations. Conclusions In this study, PfK13 mutation rates were low, with no reported resistance mutations. The Y184F mutation emerged as the dominant Pfmdr1 mutation, with no detection of N86Y. For Pfcrt, the wild-type was dominant, followed by the I74E75T76 triple mutation variant. Triple mutant I51R59N108 of Pfdhfr was very common, and our study did not find Pfdhfr Pfdhps completely resistant and super resistant mutants, but there were other quintuple and septuple mutant types. In the future, it is crucial to continue to strengthen the monitoring of malaria parasite resistance genes and to further integrate in vivo efficacy monitoring to effectively guide clinical drug use.

OBJECTIVETo investigate the influence of electroporation on the immunogenicity of the DNA vaccine pVAX- tG250FcGB.

METHODSThe DNA vaccine pVAX-tG250FcGB was constructed by inserting the coding gene of tG250 fusion genes into the expression vector pVAX. The DNA vaccine was delivered in BALB/c mouse by electroporation or intramuscular injection, and the induced antigen specific immune responses were compared.

RESULTSThe vaccine delivered by electroporation and intramuscular injection both induced immune responses in BALB/c mouse, but electroporation produced an obviously stronger effect than intramuscular injection.

CONCLUSIONElectroporation-mediated DNA vaccine delivery can produce strong immune response in mice and is an effective means for studying the immunogenic effect of DNA vaccine pVAX-tG250FcGB.

Animals ; Antibody Formation ; Antibody Specificity ; Antigens, Neoplasm ; genetics ; immunology ; Electroporation ; Gene Fusion ; Granulocyte-Macrophage Colony-Stimulating Factor ; genetics ; immunology ; HEK293 Cells ; Humans ; Injections, Intramuscular ; Male ; Mice ; Mice, Inbred BALB C ; Plasmids ; Random Allocation ; Recombinant Fusion Proteins ; genetics ; immunology ; Transfection ; Vaccines, DNA ; genetics ; immunology

OBJECTIVETo obtain 1-4 IgG-like domains of mouse vascular endothelial growth factor receptor 2 (VEGFR2) fusion protein (mVEGFR2D1-4/GST) and identify its antiginicity and biological activity.

METHODSThe gene of mVEGFR2D1-4 was amplified by RT-PCR from 14-days embryos of Balb/c mice. The PCR product was cloned into pET-42a prokaryotic expression vector to construct the recombinant plasmid pET-42a-mVEGFR2D1-4, which was transformed into E. coli BL21 (DE3) strain for mVEGFR2D1-4/GST expression. The fusion protein was identified by SDS-PAGE and Western blotting, and the antigenicity of the protein purified by affinity chromatography was characterized by ELISA. The VEGF blocking effect of the purified protein in human umbilical vein endothelial cells (HUVECs) were evaluated in in vitro cell cultures.

RESULTSThe mVEGFR2D1-4 gene was obtained, which had an identical sequence to that retrieved in GenBank. The prokaryotic expression vector for mVEGFR2D1-4 was successfully constructed as confirmed by enzyme digestion and DNA sequencing. Both Western blotting and ELISA demonstrated the antigenicity of the purified mVEGFR2D1-4 fusion protein, which obviously blocked the effect of VEGF in promoting HUVEC proliferation in vitro.

CONCLUSIONThe mVEGFR2D1-4/GST fusion protein obtained shows a strong antigenicity and biological activity to facilitate further study of active anti-tumor immunotherapy targeting VEGFR2.

Animals ; Cell Proliferation ; Escherichia coli ; genetics ; metabolism ; Female ; Gene Expression ; Genetic Vectors ; Human Umbilical Vein Endothelial Cells ; Humans ; Mice ; Mice, Inbred BALB C ; Plasmids ; Recombinant Fusion Proteins ; genetics ; immunology ; isolation & purification ; Reverse Transcriptase Polymerase Chain Reaction ; Vascular Endothelial Growth Factor Receptor-2 ; genetics ; immunology ; isolation & purification

Objective To investigate the influence of electroporation on the immunogenicity of the DNA vaccine pVAX-tG250FcGB. Methods The DNA vaccine pVAX-tG250FcGB was constructed by inserting the coding gene of tG250 fusion genes into the expression vector pVAX. The DNA vaccine was delivered in BALB/c mouse by electroporation or intramuscular injection, and the induced antigen specific immune responses were compared. Results The vaccine delivered by electroporation and intramuscular injection both induced immune responses in BALB/c mouse, but electroporation produced an obviously stronger effect than intramuscular injection. Conclusion Electroporation-mediated DNA vaccine delivery can produce strong immune response in mice and is an effective means for studying the immunogenic effect of DNA vaccine pVAX-tG250FcGB.

Objective To investigate the influence of electroporation on the immunogenicity of the DNA vaccine pVAX-tG250FcGB. Methods The DNA vaccine pVAX-tG250FcGB was constructed by inserting the coding gene of tG250 fusion genes into the expression vector pVAX. The DNA vaccine was delivered in BALB/c mouse by electroporation or intramuscular injection, and the induced antigen specific immune responses were compared. Results The vaccine delivered by electroporation and intramuscular injection both induced immune responses in BALB/c mouse, but electroporation produced an obviously stronger effect than intramuscular injection. Conclusion Electroporation-mediated DNA vaccine delivery can produce strong immune response in mice and is an effective means for studying the immunogenic effect of DNA vaccine pVAX-tG250FcGB.

Objective To investigate the efficacy of sophocarpine in rats with alcoholic liver disease and its effects on the expression of tumor necrosis factor (TNF)-α,interleukin (IL)-6 and transforming growth factor (TGF)-β1.Methods A total of 48 male Sprague-Dawley adult rats were evenly divided into healthy control group,model group,prevention group and treatment group.The rats in the healthy control group were gavaged with 0.9％NaCl every day for 12 weeks.The rats in the model group,prevention group and treatment group were gavaged with alcohol for 12 weeks to establish the model.The prevention group was injected with 20 mg · kg1 · d1 sophocarpine for 12 weeks.Since the fifth week,the treatment group was continuously injected with 20 mg · kg1 · d-1 sophocarpine for eight weeks.The histological changes were evaluated.The serum levels of alanine aminotransferase (ALT), aspartate aminotransferase ( AST ), alkaline phosphatase (AKP),triglyceride (TG) and total cholesterol (TC) were examined.And the expression of TNF-α,IL-6 and TGF-β1 in liver tissue at mRNA and protein level were detected with immunohistochemistry and real-time polymerase chain reaction (PCR) assay.Comparison among groups was perform with single factor analysis of variance,pairwise comparisons with least significant difference method (LSD method),ranked data with Kruskal-Wallis H-test and multiple pairwise comparison with Nemenyi test.Results Compared with model group,hepatic steatosis and inflammatory cell infiltration were significantly improved in the treatment group and prevention group.The levels of ALT (41.40 U/L± 10.53 U/L and 40.75 U/L±6.94 U/L vs 58.37 U/I±5.35 U/L),AST(121.60 U/L±16.24 U/L and 109.50 U/L±9.23 U/L vs 156.63 U/L±32.47 U/L),AKP(114.88 U/L±40.37 U/L and 112.60 U/L±44.34 U/L vs 161.75 U/L±28.95 U/L),TG (4.19 mmol/L±0.99 mmol/L and 2.69 mmol/L± 1.35 mmol/L vs 4.50 mmol/L±0.99 mmol/L) and TC (1.48 mmol/L±0.28 mmol/L and 1.43 mmol/L±0.19 mmol/L vs 1.67 mmol/L±0.20 mmol/L) significantly decreased and the difference was statistically significant ( all P＜0.05).The expression of TNF-α,IL-6 and TGF-β1 at mRNA and protein level in liver tissue of model group were significantly higher than those of healthy control group,prevention group and treatment group.After treated with sophocarpine,the expression of TNF-α(mRNA:1.36 ± 0.08,1.16 ± 0.05 ; protein:3.38 ％ ± 0.82 ％,1.74 ％ ± 0.65 ％ ),IL-6 (mRNA:1.51 ± 0.05,1.39 ± 0.02; protein:5.89％ ± 0.96％,4.26％ ± 0.53％) and TGF-β1 (mRNA:1.39±0.04,1.37±0.02; protein:4.27％ ±0.97％,2.11％ ±0.83％) of treatment group and prevention group at mRNA and protein level significantly lower than those of model group (mRNA:1.81±0.16,1.95 ±0.13,1.84±0.22; protein:5.82％ ± 1.21％,7.63％ ±1.03％,5.33％± 1.12％) and the difference was statistically significant (all P＜0.01).Conclusion Sophocarpine significantly alleviates alcohol induced liver injury in rats,improves liver steatosis and inflammatory reaction degree,which may be related with the downregulation of TNF-α,TGF-β1 and IL-6 expression in liver tissue of ALD rats.

Objective To investigate and analyze the impact of gender difference on outcome and prognosis of ST-segment elevation myocardial infarction (STEMI) in patients treated with primary percutaneous coronary intervention (PCI).Methods This was a prospective and multicentered observation study.All the patients with acute STEMI admitted to the hospitals from June 1st 2009 to June 1st 2010 were continuously recruited.In this study,a unified questionnaire was applied and the 382 patients satisfied the criteria.A unified follow-up questionnaire was used on patients who were discharged from the hospital.Results On average,the female patients were 8 years older than the males.The median “symptom-to-balloon time” was 312.5 minutes in females and 270.0 minutes in males,and it was significantly different (P=0.007).During hospitalization,a higher proportion of female patients developed heart failure,angina and bleeding.No gender differences were found on the in-hospital mortality rates and medical therapy recommended by the guideline.The female patients were more prone to multi-vessel disease than males (P=0.002).Success rates of primary PCI did not show any gender differences.One-month mortality and other cardiovascular events also did not show gender difference when the patients were followed for one month after being discharged.The rates of heart failure and re-hospitalization due to cardiac incidents among female patients were obviously higher than the males,three months after being discharged (P=0.007,respectively).However,the three-month and long-term cardiac mortality did not show differences related to gender.Female patients were associated with higher all-cause mortality than that in males,but there was no statistically significant difference (female 4.2％ vs.male 1.6％;P=0.056).Data from multi-factor regression analysis showed that being female was not an independent predictor related to in-hospital mortality or during the follow-up period.Conclusion Being female was not an independent predictor of in-hospital mortality or during follow-up period among patients who were treated with primary PCI.Worse long-term outcome seen in female patients was likely to be explained by older age or longer pre-hospital delayed time.

Objective To detect infection of Angiostrongylus cantonensis and examine effection of treatment to prepare monoclonal antibodies(McAbs). Methods Six-week-old BALB/c mice were imrnunized by the intraperitoneal injection of e/s antigens of Angiostrongylus cantonensis. Fusion of splecn cells from immunized mice with prepared SP2/0-Ag14 myeloma cells was performed in RPMI 1640. Fused cells were suspended in RPMI 1640 containing 1% HAT and 20% fetal calf serum and dispensed into 96-well cell culture plates. The supernatants of clones were screened by ELISA with sera of patients of angiostrongyliasis.Distribution of cohere antigen of 12D5 and 21B7 monoclonal antibodies was analyzed with immunohistochemistry. Two McAbs ( 12D5 and 21B7) were applied to detect the circulating antigen (CAg) in the sera of rats infected with A. cantonensis and angiostrongyliasis patients respectively by double antibody sandwich ELISA.Results 12D5 McAb was identified as IgG1 and 21 B7 McAb was IgM. Western blot result showed two McAbs could used to identified 55 × 103 protein of adult worms of A. cantonensis. Cohere antigen of 12D5 and 21B7 monoclonal antibodies were distributed on intestine surface of A. cantonensis. The detection rates of CAg in the sera of infected rats 100% (48/48), the detection rates of CAg in the sera of angiostrongyliasis patients was 100% (32/32). No cross-reaction to sera of patients with other infection of parasites, such as clonochiasis, fasiolopsiasis, ancylostomiasis, trichinosis, anisakiasis as well as schsitosomiasis, and health srea did not reacted with 12D5 and 21B7 McAbs,and detaction rate of antibody of angiostrongyliasis patients only reached 75% (24/32) with antigen of A. cantonensis. Conclusion Cohere antigen of 12D5 and 21B7monoclonal antibodies were antigens of enteric epithelium. Sandwich ELISA with 12D5 and 21B7 McAbs showed high specificity act as detecting CAg of A. cantonensis in sera of infection animal and patients. It is apparent that Sandwich ELISA with 12D5 and 21 B7 is not only rapid and simple without requirement of special instrument, but also rather sensitive and specific for the detection of current infection with A. cantonensis.

To identify the gene differentially expressed in female Anopheles anthropophagus and to analyze its gene sequence, this gene amplified by PCR was identified by real-time PCR and its cDNA was then amplified with rapid amplification of cDNA ends (RACE) technology. It was found that the expression ratio of the female differentially expressed gene in female and male mosquitoes was 267.49 according to the formula F=2~(-⊿⊿CT).The size of mRNA of the gene was 364 bp, and the amino acid sequence deduced from the open reading frame (ORF) was found to be similar to the sequence of tectin protein of Culex quinquefasciatus and proteins of other species. The mRNA sequence of this gene was submitted to NCBI with a accession number of FJ907236. This gene may provide a foundation for further studies on the biological functions of mosquitoes.