[Characteristics on molecular epidemiology of Brucella melitensis in Jiangxi province].

Objective: To understand the molecular characteristics and correlation among isolated strains of Brucella melitensis (BM) so as to improve the strategies on prevention and control of the disease in Jiangxi province. Methods: A total of 25 strains of BM isolated from human in 17 counties of Jiangxi province were analyzed by multiple locus variable-number tandem repeat analysis (MLVA) method. Results: A total of 25 strains of BM were classified into 24 independent genotypes with similarities between 67.00% and 100.00% and Simpson index between 0.000 and 0.773. There were 3 genotypes in MLVA8, including 60.00% (15/25) as 42 genotype, 32.00% (8/25) as 43 genotype, and 8.00% (2/25) as 63 genotype, respectively. There were 7 genotypes in MLVA11 identified, with 116 genotype and 125 genotype the main genotypes, accounting for 56.00% (14/25) of all the identified strains. Conclusions: Genes from all the 25 strains of BM that isolated from human being were with high genetic diversities, and various, genotypes. However, no obvious epidemiological correlation was noticed among these strains, indicating the complexity of the source of infection on Brucella in Jiangxi province.

[The effect of human islet amyloid polypeptide on autophagy in murine INS-1 cells and potential mechanisms].

Objective: The aims of the study were to investigate the effects of human islet amyloid polypeptide (hIAPP) on autophagy in INS-1 cells and its underlying mechanism, and to explore the role of autophagy in hIAPP-induced cytotoxicity and oxidative stress. Methods: INS-1 cells were treated with hIAPP (10 µmol/L) for 24 h in the presence or absence of N-acetyl-L-cysteine (NAC), compound C, 5-aminoimidazole-4-carboxamide-1-ß-D-ribofuranoside (AICAR) and 3-methyladenine (3-MA), respectively. Transmission electron microscopy was used to observe the number of autophagosome in cells. Cell viability was determined by methyl thiazolyl tetrazolium (MTT) test. 2',7'-dichlorofluorescin diacetate (DCFH-DA) assay was used to measure the relative levels of reactive oxygen species (ROS). Western blot was used to detect expression of adenosine monophosphate-activated protein kinase (AMPK) and autophagic markers p62 and microtubule associated protein 1 light chain3 (LC3). Results: Treatment of INS-1 cells with hIAPP resulted in a significant increase in the number of autophagosomes and the expression of LC3-Ⅱ/LC3-Ⅰ (both P<0.05). Meanwhile, treatment of INS-1 cells with hIAPP enhanced the level of ROS to 1.76 times of control cells (P<0.01). Co-treatment with NAC, an antioxidant, inhibited hIAPP-induced ROS generation, and the expression of LC3-Ⅱ/LC3-Ⅰ and p-AMPK in the INS-1 cells (all P<0.05). Pretreatment of INS-1 cells with AMPK inhibitor compound C suppressed hIAPP and AICAR, an activator of AMPK, induced expression of LC3-Ⅱ/LC3-Ⅰ and p-AMPK (all P<0.05). Autophagic inhibitor 3-MA and compound C aggravated the hIAPP-induced cell death and ROS generation in INS-1 cells (All P<0.05). The cytotoxic effects of hIAPP were significantly attenuated by co-treatment with AICAR (P<0.05). Conclusion: Autophagy may act as an adaptive mechanism to alleviate hIAPP-induced oxidative damage and toxicity in INS-1 cells.

[An investigation of prevalence of occupational diseases and treatment implementation in migrant workers in Hunan, China].

Objective: To investigate the prevalence of occupational diseases and treatment implementation in migrant workers in Hunan, China, and to provide a scientific basis for related departments to develop preventive and treatment measures and social security system for migrant workers. Methods: A retrospective investigation was performed in 2015 to collect the information of occupational diseases in migrant workers, and age, type of work, type of occupational disease, and implementation of employment injury insurance for occupation diseases were analyzed. Results: The migrant workers with occupational diseases accounted for 50.43% (11 280/22 368) of all patients with occupational diseases in Hunan, among whom 99.4% (11 212/11 280) were male workers. The mean age of migrant workers with occupational diseases was 55 years. The types of occupational diseases involved 6 categories such as occupational pneumoconiosis and occupational skin diseases, totaling 42 legal occupational diseases; 98.31% of all migrant workers (11 089/11 280) had occupational pneumoconiosis. The main types of work were underground coal miners (62.42%) , heading drivers (29.79%) , and haulage workers (2.20%) in coal mines and non-coal mines. A total of 27.25% migrant workers with occupational diseases (2 072/7 605) enjoyed employment injury insurance, and 20.84% (1 585/7 605) did not receive any medical or life compensations. Conclusion: The occupational diseases in migrant workers in Hunan are mainly pneumoconiosis, and a large proportion of those with occupational diseases do not enjoy implementation of treatment. Coal mines and non-coal mines are the high-risk areas for occupational diseases in migrant workers and should be the focus of prevention and control.

Human xenoreactivity is reduced in mice bearing porcine antisense alpha(1,3) galactosyltransferase cDNA.

AIM: To explore the effect of antisense alpha(1,3) galactosyltransferase alpha(1,3) GT cDNA on production of Gal alpha(1,3) Gal (Gal epitope) xenoantigen in vivo. METHODS: Transgenic mice bearing the porcine antisense alpha(1,3) GT cDNA (nt 1alpha-640) were generated by pronuclei microinjection method. The integration of transgene was identified by PCR and Southern-blot analysis. The expression of murine alpha(1,3) GT was characterized by RT-PCR. Morphology of the spleen was examined by histological technique. Gal epitope was detected by immunofluorescent analysis. Binding of human natural xenoantibodies (IgM and IgG) and complement (C3c) to cells from mice was determined by flow cytometric assay. RESULTS: Transgenic mice bearing the porcine antisense alpha(1,3) GT cDNA were born healthy and developed normally. However, necrosis occurred in the spleen of some mice heterozygous for transgene. Cell surface Gal epitope in transgenic heterozygotes was evidently reduced. Substantially less (30 % - 60 %) xenoantibodies in human serum bound to cells from a variety of tissues of transgenic heterozygotes compared with wild-type controls. Consequentially, human complement activation on cells from these mice was reduced by 40 % - 50 %. CONCLUSION: Human xenoreactivity could be effectively reduced by inhibiting the expression of alpha(1,3) galactosyltransferase with an antisense gene.

Transgenic mice ubiquitously expressing human Fas ligand develop a slight form of graft-versus-host-like disease.

AIM: To construct transgenic mice bearing human Fas ligand (FasL/CD95L) cDNA, and further explore the physiological effects of ubiquitous expression of FasL on such animals. METHODS: Transgenic mice were produced by pronuclei microinjection method. Integration and transmission of transgene were identified by nest-PCR and Southern-blot analysis. Level of FasL mRNA was evaluated by semi-quantitative RT-PCR analysis. FasL protein was detected by immunofluorescence analysis. Morphological alterations in tissues were analyzed by histological examination. The percentage of alphabetaT cells in the spleen was determined by flow cytometry analysis. RESULTS: Two independent founder mice bearing human FasL cDNA under the control of CMV promoter were generated healthily. Human FasL was moderately expressed in the majority of tissues examined in F1 heterozygotic mice. Although developing normally, adult transgenic mice exhibited a slight form of graft-versus-host (GVH)-like disease characterized by many morphological abnormalities occurring locally in the spleen, testis, lung and liver. In addition, the percentage of alphabetaT cells in the spleen was respectively decreased approximately by 32 % and 24 % in two independent transgenic lines, relative to wild-type mice. CONCLUSION: Ubiquitous expression of Fas ligand can lead to slight GVH-like disease

Adenovirus-mediated expression of pig alpha(1, 3) galactosyltransferase reconstructs Gal alpha(1, 3) gal epitope on the surface of human tumor cells.

Gal alpha(1, 3) Gal (gal epitope) is a carbohydrate epitope and synthesized in large amount by alpha(1, 3) galactosyltransferase [alpha(1, 3) GT] enzyme on the cells of lower mammalian animals such as pigs and mice. Human has no gal epitope due to the inactivation of alpha(1, 3) GT gene but produces a large amount of antibodies (anti-Gal) which recognize Gal alpha(1, 3) Gal structures specifically. In this study, a replication-deficient recombinant adenoviral vector Ad5sGT containing pig alpha(1, 3) GT cDNA was constructed and characterized. Adenoviral vector-mediated transfer of pig alpha(1, 3) GT gene into human tumor cells such as malignant melanoma A375, stomach cancer SGC-7901, and lung cancer SPC-A-1 was reported for the first time. Results showed that Gal epitope did not increase the sensitivity of human tumor cells to human complement-mediated lysis, although human complement activation and the binding of human IgG and IgM natural antibodies to human tumor cells were enhanced significantly after Ad5sGT transduction. Appearance of gal epitope on the human tumor cells changed the expression of cell surface carbohydrates reacting with Ulex europaeus I (UEA I) lectins, Vicia villosa agglutinin (VVA), Arachis hypogaea agglutinin (PNA), and Glycine max agglutinin (SBA) to different degrees. In addition, no effect of gal epitope on the growth in vitro of human tumor cells was observed in MTT assay.

Adenovirus-mediated expression of antisense RNA transcripts complementary to pig alpha(1,3) galactosyltransferase mRNA inhibits expression of Gal alpha(1,3) Gal epitope.

AIM: To examine the effects of the expression of antisense RNA transcripts complementary to the pig alpha(1,3) galactosyltransferase [alpha(1,3)GT]mRNA on the expression of Gal alpha(1,3) Gal structure (gal epitope) in cultured cell lines. METHODS: Human adenoviral vectors were used to mediate the expression of antisense RNA. The expression levels of H blood group antigens and gal epitopes were analyzed by flow cytometry using FITC-UEA-I and FITC-GS-IB4 lectins, respectively. RESULTS: Recombinant adenoviruses, Ad5anti-sGT600 and Ad5-anti-sGT1100, which express antisense RNA complementary to different regions of the pig alpha(1,3) GT mRNA, were constructed and used to infect cell line of NIH3T3. The results showed about 30% reduction in the expression level of gal epitopes on the surface of NIH3T3 cells. In addition, co-expression of human secretor type alpha(1,2) fucosyltransferase [alpha(1,2)FT]cDNA and antisense RNA complementary to the pig alpha(1,3) GT mRNA led to a further reduction in the gal epitope level. CONCLUSION: Recombinant adenoviruses, Ad5anti-sGT600 and Ad5anti-sGT1100, are effective to down-regulate the gal epitope expression.

Adenovirus-mediated expression of human secretor type alpha(1,2) fucosyltransferase reduces level of Gal alpha(1,3)Gal epitope.

AIM: To test the potential of human secretor type alpha(1, 2) fucosyltransferase [Se alpha(1,2)FT] to downregulate the expression of Gal alpha(1,3)Gal epitope (gal epitope) in cultured cell lines. METHODS: Expression of Se alpha(1,2) FT was mediated by human adenoviral vector. Flow cytometric analysis was used to compare the expression level of H blood group antigen or gal epitope. MTT was employed to assess the susceptibility of mouse NIH3T3 cells to human natural antibody and complement mediated lysis. RESULTS: A recombinant replication-deficient adenovirus (rAdv) containing human Se alpha(1,2)FT cDNA (Ad5hSeFT) was designed and successfully constructed. Flow cytometric analysis showed that after mock infection, Ad5null infection, and Ad5hSeFT infection, the mean fluorescence intensity (MFI) values for the binding of Ulex europaeus I (UEA-I) lectin to NIH3T3 cells were 2.3 +/- 0.6, 2.1 +/- 1.0, and 36.5 +/- 5.9, respectively; MFI values for the binding of Griffonia simplicifolia isolectin B4 (GS-IB4) lectin to NIH3T3 cells were 167 +/- 23, 170 +/- 19, and 100 +/- 14, respectively; MFI values for the binding of human natural IgG and IgM antibodies to NIH3T3 cells were 31 +/- 3, 32 +/- 4, and 22 +/- 4, respectively. CONCLUSION: H blood group antigen was detected on NIH3T3 cells after Ad5hSeFT infection and resulted in more than 40% reduction in the level of gal epitope on the cell surface. This reduction increased the resistance of NIH3T3 cells to lysis by normal human serum.

Experimental and clinical studies on one-stage arterialization of the venous channels for revascularization of severely ischemic limbs.

A new approach to perfuse arterial blood through venous channels for revascularization of severely ischemic limbs is reported. The procedure studied and used consists of creating an arteriovenous fistula between the normal arterial trunk proximal to the occlusion and the deep venous trunk of the diseased limb, constricting the venous trunk proximal to the anastomosis to one third of its lumen diameter, ligating the communicating and small tributary veins distal to the constriction in the operative field. The results of the experimental and clinical studies have shown that the treated ischemic limb was quickly revascularized without undesirable influence on cardiac function. This new approach has been used in the treatment of severe ischemia involving total 212 limbs in 156 patients, and the results appeared more satisfactory than those treated with staged arteriovenous reversal.