Antibodies, Anticardiolipin ; blood ; Autoantibodies ; blood ; Child ; Child, Preschool ; Female ; Humans ; Infant ; Male ; Mucocutaneous Lymph Node Syndrome ; immunology ; beta 2-Glycoprotein I ; immunology

Background Efficient and lowcost way to isolate keratocytes is helpful for research on cornea.Either relatively expensive or inefficient is the shortage of those means now applied,while raising the keratocytes through passage will change the phenotype of them quickly.Our aim is to approach the way getting keratocytes effectively utilizing modified two step enzymatic digestion by type I collagenase. Objective To evaluate the effect of isolating the bovine keratocytes utilizing two step enzymatic digestion and observe the morphological changes of the keratocytes during cultivation in vitro. Methods Keratocytes were isolated from bovine corneas using 0.5 mg/mL and 1 mg/mL type I collagenase digestion.The harvesting rate and viability rate of the primary keratocytes were evaluated.During the primary cultivation in vitro,the morphological changes of the keratocytes and their F-action distribution were observed.Results(1)The extracellular matrix of the bovine corneas were almost dissolved by the two step enzymatic digestion,followed the keratocytes completely isolated from the solid matrix.The amount of the harvested keratocytes from each cornea was(2.11±0.15)X106 on average while the viability rate was(91.69±3.73)% and the inoculation rate Was(81.20±1.25)%.(2)The primary keratocytes attached and spreaded out with dendritic and stellate morphology.After 3 days cultured,the branches of the keratocytes were contacting and formed networks.The F-actin detected by phalloidin binding showed a limited cortical localization. Conclusion (1)The method of two step enzymatic digestion can make the extracellular matrix of bovine cornea stroma completely degraded with the advantages in high efficiency of harvesting keratocytes and high cell viability and relatively simple manipulation. (2) The primary bovine keratocytes have dendritic morphology and with limited F-action distribute in cellular cortex.

BACKGROUND: Cytochrome C oxidase(CCO) is the terminal enzyme in respiration chain of mitochondrion, and it plays a key role in aerobic metabolism and energy production during the process of oxidative phosphorylation. Recently, it is found that energy production of mitochondrion is closely related to the cellular apoptosis, and the changes of CCO activity is closely related to the neuronal impairment after cerebral ischemia and anoxia.OBJECTIVE: To investigate the protective mechanisn of compound pinggan xifeng decoction on the neuronal impairment in cerebral hemorrhage (CH) according to the mitochondrial energy metabolism and cellular apoptosis in neurons.DESIGN: A randomized and controlled trial based on experimental animals.SETTING: Institute of Integrated Traditional Chinese Medicine and Western Medicine, Xiangya Hospital, and Center of Telemedicine.MATERIALS: The experiment was completed in the animal laboratory(key laboratory of province) of Institute of Combination of Traditional Chinese Medicine and Western Medicine from November 2 to 9 in 2003. A total of 80 healthy male SD rats were selected from Experimental Animal Center of Xiangya School of Medicine, Public Health Ministry.METHODS: CH rat models were induced with collagenase Ⅶ, CCO activity was assayed with histochemistry combined with semi-quantification of gray scale, and the cellular apoptosis was evaluated with Tunel method.MAIN OUTCOME MEASURES: CCO activity of CH rats in lateral hippocampal CA1;lateral cellular apoptosis of CH rats.RESULTS: After 12-hour model establishment, CCO activity in CH group was decreased dramatically compared with that in sham operation group (P＜ 0.01), which was 52.12 ±3.75 and 26.98 ±6.32 respectively in lateral hippocampal CA1. And the cellular apoptosis in CH group was increased notably compared with that in sham operation group(P ＜ 0.01),which was(13.56 ± 1.72)/sight and(4. 32 ± 1.04)/sight respectively.Then the two had deteriorated afterwards. After the treatment with pinggan xifeng decoction, CCO activity can be maintained, and the cellular apoptosis was reduced.CONCLUSION: Neuronal injury was closely related to the decrease of CCO activity and the cellular apoptosis in CH. Pinggan xifeng decoction could maintain CO activity of mitochondrion, improve the cellular aerobic netabolism, and reduce the cellular apoptosis, which might be one of the protective mechanisms for secondary neuronal injury in CH.

Mycena subpiligera, a new taxon in sect. Fragilipedes that can strongly enhance the germination efficiency of Gastrodia elata seeds, was discovered in subtropical areas of China. As revealed by a morphological comparison with related Mycena species as well as maximum likelihood (ML) and Bayesian phylogenetic analyses based on sequences of the internal transcribed spacer (ITS) and the large subunit (LSU) regions of nuclear ribosomal RNA, the new taxon can be distinguished from phenotypically similar and phylogenetically related species. Optimal cultural conditions for M. subpiligera basidiomata are reported, and the germination rate of the new species is compared with that of M. citrinomarginata.

OBJECTIVETo compare the serological characters of an outbreak of hepatitis E and evaluate sensitivity and specificity of anti-HEV E2-IgM.

METHODSThe sera collected from the employees of an outbreak unit were detected for anti-HEV E2-IgM and IgG, and the serum samples from a neighboring department were used as control. The results detected with anti-HEV E2-IgM, IgG and Genelab anti-HEV IgM, IgG in some samples were compared.

RESULTSThe positive rate of anti-HEV E2-IgM in the control group was 0.11%. The results between the positive and the negative samples can be distinguished easily. The specificity of anti-HEV E2-IgM is about 99.89%. The positive rate of anti-HEV E2-IgM in outbreak stricken population was 8.66%, significantly higher than that in the control group (P < 0.001). The results from HEV patients' serial samples in the outbreak unit showed that the anti-HEV E2-IgM titer was high 30-60 days after the infected and then declined clearly. The positivity seemed unrelated to neither sex nor age. Among the 115 positive to anti-HEV E2-IgM, 27 were negative to Genelab anti-HEV IgG, the fact indicated a rather high risk of misdiagnosis of about 23.48%. In the 179 randomized samples of the control group, the positive rate of Genelabs anti-HEV IgG was about 11.17%. In 110 samples for the positive anti-HEV E2-IgM, the positive ratio of Genelabs anti-HEV IgG was about 76.36%, and that of Genelabs anti-HEV IgM only 69.09%. There were 16 samples negative for both Genelabs anti-HEV IgG and IgM. The ratio of the difference between the Genelabs anti-HEV IgG and IgM was about 25.45%.

CONCLUSIONThe specificity of anti-HEV E2-IgM was about 99%, and false positive rate was low. The sensitivity of anti-HEV E2-IgM in acute hepatitis E infection was 25%-30% higher than that of Genelabs anti-HEV IgM,IgG. The infected persons in the outbreak unit can be preferably distinguished from the non-infected persons by anti-HEV E2-IgM. Anti-HEV E2-IgM can image the characters of the outbreak of HEV and played a great role in the control of outbreak and in the early diagnosis for hepatitis E.

Antibodies, Viral ; blood ; China ; epidemiology ; Disease Outbreaks ; Enzyme-Linked Immunosorbent Assay ; Hepatitis E ; blood ; epidemiology ; virology ; Hepatitis E virus ; immunology ; Humans ; Immunoglobulin G ; blood ; Immunoglobulin M ; blood

OBJECTIVETo understand the risks and associated factors of HIV transmission by sharing syringes among HIV-positive drug users.

METHODThe survey was conducted among HIV-positive injecting drug users (IDUs-HIV+) who received HIV counseling, testing and treatment in Changsha city Infectious Disease Hospital and Hengyang city No.3 People's Hospital from July 2012 to May 2013 to understand their socio-demographic characteristics, HIV prevalence and syringe sharing. A total of 503 IDUs-HIV+ were involved in and provided the contact list of 2 460 drug users who had the syringe sharing experience over one month with IDUs-HIV+. 420 IDUs-HIV+ among 503 were defined as infection sources due to sharing syringe with at least one drug user. Among them, 234 HIV-negative persons were in control group, and 186 HIV-positive were in cased group. A total of 1 220 drug users were followed up among 2 460 and defined as vulnerable population. The HIV transmission rate was calculated based on the HIV prevalence among vulnerable population. Based on the result of HIV transmission to vulnerable population from 420 infection sources, case-control study and the multivariate logistic regression analysis were adopted to explore the associated factors of HIV transmission among IDUs-HIV+.

RESULTSAs the sources of HIV transmission, 420 IDUs-HIV+ had an average duration of (4.5 ± 1.2) years for drug use. As a susceptible population, 1 220 drug users sharing syringes with the 420 IDUs-HIV+ had an average duration of (1.1 ± 0.5) years for drug use. There were 238 HIV-positive persons among 1 220 vulnerable drug users, with a transmission rate of 0.57. In the case-control study, the proportion of male subjects was 87.1% (162/186) in the case group, which was higher than that in the control group (77.8%, 182/234). The proportion of subjects who received support after knowing their HIV infection status was 51.1% (95/186) in the case group, which was lower than that in the control group (79.5%, 186/234). The proportion of subjects sharing syringes every time of using drugs was 47.8% (89/186) in the case group, which was higher than that in the control group (36.8%, 86/234). The proportion of subjects having AIDS awareness was 21.0% (39/186) in the case group, which was lower than that in the control group (64.5%, 151/234); the proportion of subjects having close contact with HIV-positive persons for more than 106 days was 60.2% (112/186) in the case group, which was higher than that in the control group (31.6%, 74/234). The proportion of subjects maintaining the original drug use method after being infected with HIV was 50.5% (94/186) in the case group, which was higher than that in the control group (16.7%, 39/234) (all P values < 0.05). The multivariate logistic regression analysis was carried out to analyse high correlate factors of HIV transmission by sources of transmission, and the AIDS awareness, duration of contact between sources of transmission and vulnerable population, access to support following confirmed HIV infection were protective factors, OR (95% CI) values were 0.155 (0.104-0.262), 0.170 (0.106-0.253), and 0.306 (0.189-0.450), respectively; while the frequency of syringe sharing and continuous drug use after being infected with HIV were risk factors, and the OR (95% CI) values were 3.06 (1.77-5.29), and 3.54 (2.16-5.80), respectively.

CONCLUSIONHIV transmission by IDUs-HIV+ might be contained by raising AIDS awareness, providing comprehensive psychological support, conducting needle exchange and methadone maintenance treatment and reducing syringe sharing.

Aged ; Case-Control Studies ; Drug Users ; HIV Infections ; Humans ; Male ; Methadone ; Needle Sharing ; Prevalence ; Risk Factors ; Substance Abuse, Intravenous

Interleukin-5 (IL-5) accompanies the development of airway inflammation and hyperresponsiveness through the activation of eosinophils. Therefore, interference of IL-5 expression in lung tissue seems to be an accepted approach in asthma therapy. In this study, we designed a small interfering RNA (siRNA) to inhibit the expression of IL-5. The siRNAs against IL-5 were constructed in a lentivirus expressing system, and 1.5x10(6) IFU (inclusion-forming unit) lentiviruses were administered intratracheally to ovalbumin (OVA)-sensitized murine asthmatic models. Our results show that lentivirus-delivered siRNA against IL-5 efficiently inhibited the IL-5 messenger ribonucleic acid (mRNA) expression and significantly attenuated the inflammation in lung tissue. Significant decrease of eosinophils and inflammatory cells were found in peripheral blood, bronchoalveolar lavage fluid (BALF), and lung tissue. In addition, significant inhibition of airway hyperresponsiveness (AHR) was found in the mice treated with siRNA against IL-5. These observations demonstrate that siRNA delivered by means of the lentivirus system is possibly an efficacious therapeutic approach for asthma.
Animals ; Asthma ; immunology ; prevention & control ; Bronchial Hyperreactivity ; immunology ; prevention & control ; Disease Models, Animal ; Genetic Therapy ; methods ; Humans ; Interleukin-5 ; genetics ; immunology ; Male ; Mice ; Mice, Inbred BALB C ; Pneumonia ; immunology ; prevention & control ; RNA ; therapeutic use ; Treatment Outcome

Recent studies have found that ABO blood group antigen is also closely related to the onset and development of many diseases. More and more attention is being paid to the decrease of A/B blood group antigen caused by some tumors. This study was purpose to investigate the correlation between DNA methylation of the ABO gene promoter CpG island and leukemia. The relative contents of ABH antigen on the surface of RBC from kinds of blood disease patients and healthy individuals were detected by using flow cytometry and confocal laser scanning microscopy. The DNA sequences and CpG methylation of ABO gene promoter in patients with hematopathy and healthy individuals, as well as the -102 site methylation of ABO gene promoter in patients with hematopathy and healthy individuals were detected by PCR and MSP-PCR respectively. The results showed that RBC from leukemia patients displayed different degree of A/B antigen decrease. The sequences of ABO gene promotor of patients with hematopathy were not different from healthy individuals indicating high conservation of promoter sequences. Comparison of sequences between patients with hematopathy and healthy individual indicated that CpG islands on ABO gene promoter either from blood disease patients or from healthy individual had no methylated site in AA patients, but C residues at position -102, -101, -100, -99 and -97 on the promoter of ABO gene in AML, CML, ALL and some MDS patients were methylated. It is concluded that methylation of CpG islands in promoter of ABO gene may result in AB antigen decrease in patients with leukemia. The methylation sites -102, -101, -100, -99 and -97 may be specific for leukemia. The methylation of site -102 can be used as a molecular marker in differential diagnosis for leukemias.
ABO Blood-Group System ; genetics ; Base Sequence ; CpG Islands ; genetics ; DNA Methylation ; Humans ; Leukemia ; genetics ; Molecular Sequence Data ; Promoter Regions, Genetic ; Sequence Analysis, DNA

This study was aimed to prepare a reconstructed B. Fragilis-derived recombinant α-galactosidase developed for human B to O blood group conversion. Based on the construction of recombinant E. Coli (DE3) which can express α-galactosidase, the inducing time and inducer concentration were optimized for high expression of α-galactosidase. Then, the expression products in supernatant were purified by cation and anion exchange column chromatography. The purified α-galactosidase was used to treat B group red blood cells in phosphate buffer (pH 6.8) for 2 hours to prepare O group red blood cells. The results showed that the optimal inducing conditions for α-galactosidase expression were IPTG 0.1 mmol/L, 37°C and 2 hours. The specific enzyme activity of purified protein increased from 0.42 U/mg to 2.1 U/mg as compared with pre-purification. And, the conditions of B to O blood group conversion were 26°C, pH 6.8 (neutral pH condition) and 2 hours. Moreover, 225 µg of the enzyme could converse 1 ml B red blood cells to O completely. It is concluded that the technology of expression and purification of recombinant α-galactosidase has been established, and the purified protein can converse B red blood cells to O completely, which means that an effective enzyme conversing B red blood cells to O has been obtained.
ABO Blood-Group System ; immunology ; Bacteroides fragilis ; enzymology ; Cloning, Molecular ; Escherichia coli ; metabolism ; Humans ; Recombinant Proteins ; biosynthesis ; alpha-Galactosidase ; biosynthesis

αGal, a xenotransplantations antigen (XTA), can lead to hyper acute reaction (HAR) in xenotransplantation. α-Galactosidase from B. fragilis is a novel galactosidase belong to CAZy GH110 which can clear the terminal αGal from branched and linear oligosaccharides. This study was purposed to investigate the removal effect of a novel α-galactosidase on α-Gal XTA on surface of red blood cells. The αGal XTA from the red blood cells of cattle, pig, dog and rabbit was digested by using recombinant α-galactosidase; the α-Gal antigens on surface of cells was detected by flow cytometry. The results showed that the XTA was disappeared completely or mainly. It is concluded that the novel α-galactosidase is a potential enzyme to remove the XTA on the surface of xenotransplants and can be used to overcome the HAR in xenotransplantation.
Animals ; Antigens, Heterophile ; immunology ; Cattle ; Dogs ; Epitopes ; Erythrocytes ; immunology ; Macaca mulatta ; Mice ; Mice, Inbred BALB C ; Rabbits ; Swine ; Transplantation, Heterologous ; alpha-Galactosidase ; immunology