Establishment of a multiplex PCR for rapid identification of Mycobacterium species / 中华微生物学和免疫学杂志

Objective@#To establish and evaluate a multiplex PCR method for rapid identification of Mycobacterium species in order to provide an approach for rapid diagnosis of pathogens causing tuberculosis.@*Methods@#Four genes including 16S rRNA, Rv0577, RD9 and mtbk_20680 were selected to establish the multiplex PCR method. Specific primers were designed and the reaction system and conditions were optimized. The sensitivity and specificity of the multiplex PCR method were evaluated through identifying Mycobacterium africanum (M.africanum), Mycobacterium bovis (M.bovis), M. bovis BCG, seven common non-tuberculosis Mycobacterium (NTM), seven reference species of common respiratory bacteria and 93 clinical strains of Mycobacterium tuberculosis (MTB) isolated from patients with tuberculosis in Gansu Province of China.@*Results@#The fragments of 16S rRNA, Rv0577, RD9 and mtbk_20680 genes were 543 bp, 786 bp, 369 bp and 231 bp in length, respectively. MTB strains of the Beijing genotype were positive for all of the four genes, while the non-Beijing genotype strains were negative for mtbk_20680. M. africanum, M. bovis and M. bovis BCG strains were negative for 16S rRNA and Rv0577. NTM strains only carried 16S rRNA and none of four genes were detected in the seven species of respiratory bacteria. Among the 93 clinical MTB strains, 80 (86.02%) belonged to the Beijing genotype and the other 13 (13.98%) were non-Beijing genotype strains. The specificity of the multiplex PCR method was 100%.@*Conclusions@#The established multiplex PCR method could accurately distinguish Mycobacterium, Mycobacterium tuberculosis complex (MTBC), NTM, MTB, and Beijing and non-Beijing genotype MTB with high sensitivity and specificity.

Establishment of a multiplex PCR for rapid identification of Mycobacterium species / 中华微生物学和免疫学杂志

Objective To establish and evaluate a multiplex PCR method for rapid identification of Mycobacterium species in order to provide an approach for rapid diagnosis of pathogens causing tuberculo-sis. Methods Four genes including 16S rRNA, Rv0577, RD9 and mtbk_20680 were selected to establish the multiplex PCR method. Specific primers were designed and the reaction system and conditions were opti-mized. The sensitivity and specificity of the multiplex PCR method were evaluated through identifying Myco-bacterium africanum (M. africanum), Mycobacterium bovis (M. bovis), M. bovis BCG, seven common non-tuberculosis Mycobacterium (NTM), seven reference species of common respiratory bacteria and 93 clinical strains of Mycobacterium tuberculosis (MTB) isolated from patients with tuberculosis in Gansu Province of China. Results The fragments of 16S rRNA, Rv0577, RD9 and mtbk_20680 genes were 543 bp, 786 bp, 369 bp and 231 bp in length, respectively. MTB strains of the Beijing genotype were positive for all of the four genes, while the non-Beijing genotype strains were negative for mtbk_20680. M. africanum, M. bovis and M. bovis BCG strains were negative for 16S rRNA and Rv0577. NTM strains only carried 16S rRNA and none of four genes were detected in the seven species of respiratory bacteria. Among the 93 clinical MTB strains, 80 (86. 02％ ) belonged to the Beijing genotype and the other 13 (13. 98％ ) were non-Beijing gen-otype strains. The specificity of the multiplex PCR method was 100％ . Conclusions The established multi-plex PCR method could accurately distinguish Mycobacterium, Mycobacterium tuberculosis complex (MTBC), NTM, MTB, and Beijing and non-Beijing genotype MTB with high sensitivity and specificity.

Cross-reactive immune responses to Mycobacterium vaccae, Mycobacterium tuberculosis and Bacillus Calmette-Guerin / 中华微生物学和免疫学杂志

Objective To investigate the cross-reactive immune responses to Mycobacterium vac-cae (M. vaccae), Mycobacterium tuberculosis (M. tuberculosis, H37Rv) and Mycobacterium bovis Bacillus Calmette-Guerin ( BCG) for providing reference for the development of new vaccines with M. vaccae. Meth-ods M. vaccae (ATCC95051), M. tuberculosis (H37Rv) and BCG (China strain) were cultured on L-J solid media and harvested. Total bacterial protein antigens prepared by ultrasonic disruption were used to im-munize BALB/c mice. IgG antibodies in serum samples were detected with enzyme-linked immunosorbent assay ( ELISA) to evaluate humoral immune responses. Cellular immunity was assessed by detecting various cytokines with cytokine release assay ( CRA) . Results The mice that were respectively immunized with the three mycobacterial antigens could produce high titers of antibodies ( IgG) and high levels of IFN-γand IL-2, but low levels of IL-4 and IL-10. Results of the cross reactivity tests showed that ATCC95051, H37Rv and BCG were able to cross-react with the immunized mice, and all of them induced high levels of IFN-γ, IL-2 and IgG antibodies. Conclusions The three Mycobacteria mainly elicited Th1 immune responses. There were cross-reactive immune responses to M. vaccae, M. tuberculosis and BCG, which might provide ref-erence for using M. vaccae in the development of new anti-tuberculous vaccines.

The role of amplitude integrated EEG in evaluation of prognosis in comatose patients with severe traumatic brain injury / 中华行为医学与脑科学杂志

Objective To study the clinical value of amplitude integrated EEG(aEEG),EEG reactivity,EEG patterns,and Glasgow Coma Scale(GCS) scores of predicting the prognosis in comatose patients with severe traumatic brain injury.Methods Sixty-four hospitalized comatose patients with severe traumatic brain injury were evaluated by aEEG,EEG reactivity,EEG patterns and GCS and followed up for one year to observe the prognosis of the patients.Results Accuracy of aEEG,EEG reactivity,EEG patterns and GCS in predicting outcomes of comatose patients with severe traumatic brain injury correctly classified as 73.4％,68.8％,73.4％,64.1％ respectively.The accuracy of GCS in evaluating the prognosis of comatose patients with severe traumatic brain injury was lower than that of the other three methods (P＜0.05).There were positive correlations among aEEG,EEG reactivity,EEG patterns,and GCS (r=0.574-0.843,P＜ 0.05).There were positive correlations between aEEG,EEG reactivity,EEG patterns,GCS and the patients' prognosis(r=0.647,0.609,0.621,0.532,P＜ 0.05).Conclusion As a new electroencephalographic technique,aEEG combined with EEG reactivity,EEG patterns,and GCS can be effectively used to evaluate the prognosis of STBI coma patients,which has a certain clinical value.

Clinical value of surfactant protein-A in exudate pleural effusion / 中南大学学报(医学版)

Objective:To evaluate the clinical value of surfactant protein-A (SP-A) in exudate pleural effusion (EPE).Methods:This clinical study was prospective,observational and cross-sectional.Two hundred and fifteen patients with pleural effusion were divided into the transudate pleural effusions (TPE) group and the EPE group.TPE patients served as the control group.The concentrations of pleural effusions SP-A (SP-Apl) and serum SP-A (SP-Ase) were measured by ELISA,and receiver operator characteristic (ROC) curve and multivarate Cox analysis of SP-A was analysed for its clinical value.Results:SP-Apl concentrations in the EPE group were significantly higher than that in the TPE group [(189.8±43.4) ng/mLvs (22.3±5.1) ng/mL,P＜0.01];SP-Ase concentrations in the EPE group were higher than that in the TPE group [(78.9±11.3) ng/mL vs (25.8±12.4) ng/mL,P＜0.05];SP-Apl concentrations were significantly higher than the concentrations of SP-Ase in the EPE group (P＜0.01).In EPE group,SP-Apl and SP-Ase concentration in the patients with primary lung adenocarcinomas were the highest.The cut off value of SP-Apl concentrations was more than 484.5 ng/mL,yielding a 85.4％ sensitivity and 95.2％ specificity for diagnosing primary lung adenocarcinomas,with an area under the curve (AUC) of 0.943 (95％ CI 0.852 to 0.934,P＜0.01);when SP-Ase concentration was more than 84.2 ng/mL,it yielded a 76.4％ sensitivity and 94.3％ specificity for diagnosing primary lung adenocarcinomas,with an AUC of 0.910 (95％ CI 0.921 to 0.953,P＜0.01).Conclusion:While SP-Apl concentration is more than 484.5 ng/mL and/or SP-Ase concentration is more than 84.2 ng/mL,it may be helpful for the diagnosis of primary lung adenocarcinomas with the usage ofpleural effusion.

Overexpression of miR-155-5p Inhibits the Proliferation and Migration of IL-13-Induced Human Bronchial Smooth Muscle Cells by Suppressing TGF-β-Activated Kinase 1/MAP3K7-Binding Protein 2

PURPOSE: Molecular mechanisms leading to asthma is still ill-defined. Though the function of microRNAs (miRNAs) in asthma was previously reported, the involvement of miR-155 in important features of this disease remains unknown. The present study was designed to uncover the probable involvement of miR-155-5p in the proliferation and migration of IL-13-induced human bronchial smooth muscle cells (BSMCs) and the intrinsic regulatory mechanism. METHODS: The effects of different concentrations of IL-13 on the proliferation and migration of BSMCs as well as the expression of miR-155-5p and its predicted target transforming growth factor (TGF)-β-activated kinase 1/MAP3K7-binding protein 2 (TAB2) were investigated. The effects of miR-155-5p on the proliferation and migration of interleukin (IL)-13-induced BSMCs was determined in vitro using BSMCs transfected with miR-155 mimic/inhibitor and induced by a high concentration of IL-13. The quantitative real-time polymerase chain reaction (qRTPCR) was employed for determining the expression of miR-155-5p and TAB2. Western blotting was applied to analyze the expression of TAB2 at the protein level. Cell proliferation and migration were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and Transwell assays, respectively. RESULTS: The proliferation and migration of BSMCs were dose-dependently increased with IL-13 treatment. Contrariwise, IL-13 dose-dependently inhibited the expression of miR-155-5p in BSMCs. Mechanistic studies showed that inhibition of miR-155-5p further promoted the stimulatory effects of IL-13, whereas overexpression of miR-155 significantly inhibited these effects. In silico studies and luciferase reporter assays indicated that TAB2 was a negatively regulated miR-155-5p target. CONCLUSIONS: These results suggested that miR-155-5p-inhibit the IL-13-induced proliferation and migration of BSMCs by targeting TAB2 and that the IL-13/miR-155/TAB2 pathway could serve as a therapeutic target for pulmonary diseases, especially asthma.

Associations between Mycobacterium tuberculosis Beijing genotype and drug resistance to four first-line drugs: a survey in China / 医学前沿

Investigations on the genetic diversity of Mycobacterium tuberculosis in China have shown that Beijing genotype strains play a dominant role. To study the association between the M. tuberculosis Beijing genotype and the drug-resistance phenotype, 1286 M. tuberculosis clinical isolates together with epidemiological and clinical information of patients were collected from the center for tuberculosis (TB) prevention and control or TB hospitals in Beijing municipality and nine provinces or autonomous regions in China. Drug resistance testing was conducted on all the isolates to the four first-line anti-TB drugs (isoniazid, rifampicin, streptomycin, and ethambutol). A total of 585 strains were found to be resistant to at least one of the four anti-TB drugs. The Beijing family strains consisted of 499 (53.20%) drug-sensitive strains and 439 (46.80%) drug-resistant strains, whereas the non-Beijing family strains comprised 202 (58.05%) drug-sensitive strains and 146 (41.95%) drug-resistant strains. No significant difference was observed in prevalence (χ= 2.41, P > 0.05) between the drug-resistant and drugsensitive strains among the Beijing family strains. Analysis of monoresistance, multidrug-resistant TB, and geographic distribution of drug resistance did not find any relationships between the M. tuberculosis Beijing genotype and drug-resistance phenotype in China. Results confirmed that the Beijing genotype, the predominant M. tuberculosis genotype in China, was not associated with drug resistance.

Role of desmosine in prediction of treatment response in chronic obstructive pulmonary disease / 实用医学杂志

Objective To explore the association of serum and sputum desmosine with treatment response in patients with chronic obstructive pulmonary disease(COPD). Methods Serum and induced sputum desmosine were measured with enzyme linked immunosorbent assay in 65 patients with newly diagnosed COPD and 26 healthy people. The associations of desmosine with pulmonary function ,modified Medical Research Council dyspnea scale(mMRC),and St. George's Respiratory Questionnaire score(SGRQ)were analyzed before and after treat-ment with inhaled corticosteroid/long acting β2-agonist. The relationship between desmosine and treatment re-sponse in COPD were explored. Results Level of sputum desmosine was higher in patients with COPD than in healthy controls(1061.2 ± 933.9 ng/mL vs. 443.5 ± 501.7 ng/mL;t=2.277,P=0.027). Sputum desmosine level was negatively related with forced expiratory volume in 1 second(FEV1)(r=-0.357,P=0.001)and forced vital capacity(FVC)(r =-0.479,P = 0.02). Serum desmosine level was correlated with pulmonary function,MRC, and SGRQ(P>0.05 for all comparisons). 3 months after treatment,neither serum nor sputum desmosine declined significantly(P>0.05). FVC,MRC,and the total scores and activity scores on the SGRQ improved more markedly in patients with lower expression of sputum desmosine than in those with higher expression(P < 0.05 for all com-parisons). Conclusions Level of sputum desmosine is inversely correlated with pulmonary function in stable COPD. Patients with lower expression of sputum desmosine have more significant improvement in symptoms.

Protein kinase C-nuclear factor-erythroid 2-related factor 2 regulating the expression of heme oxygenase-1 in rat airway epithelial cells / 中南大学学报(医学版)

Objective:To observe the effect of the signaling pathway of protein kinase C (PKC)-nuclear factor-erythroid 2-related factor 2 (Nrf2) on the expression of heme oxygenase -1 (HO-1) induced by cigarette smoke extract in rat airway epithelial cells.Methods:The airway epithelial cells of 25 male SD rats were randomly divided into a control group, a CSE3h group, a RO318220 group (PKC inhibitor), a Nrf2 siRNA group and a Nrf2 siRNA+RO318220 group, 5 rats in each group. hTe control group was incubated with DMEM/F12 alone. hTe CSE3h group was treated with 10% CSE for 3 h. hTe RO318220 group was pretreated with 3 μmol/L RO318220 for 0.5 h and subsequently treated with 10% CSE for 3 h. hTe Nrf2 siRNA group was pretreated with Nrf2 siRNA, and then treated with 10% CSE for 3 h. hTe Nrf2 siRNA+RO318220 group was pretreated with Nrf2 siRNA and 3 μmol/L RO318220 for 0.5 h, and then treated with 10% CSE for 3 h. hTe protein levels of Nrf2 in the nucleus and cytoplasm, and HO-1 and PKC in the whole cells were semi-quantified by Western blot. The protein expression of HO-1 was measured by immunocytochemistry. HO-1 mRNA expression was detected by RT-PCR. Immunolfuorescence staining was used to observe the nuclear translocatin of Nrf2. Results: CSE markedly induced Nrf2 nuclear translocation in the rat airway epithelial cells, and RO318220 pretreatment blocked the CSE induced Nrf2 nuclear translocation. Immunocytochemistry showed that HO-1 protein expression was strongly positive in the CSE3h group, weakly positive in the other 4 groups. hTe expression of PKC protein, HO-1 mRNA and protein signiifcantly increased in the CSE3h group, and HO-1 activity markedly improved in the CSE group (P0.05). Conclusion: CSE induces the nuclear translocation of Nrf2 by PKC signaling pathway, thus upregulating the HO-1 expression in the rat airway epithelial cells.

The clinical effects of cowpox vaccination in inflammatory skin extract in the treatment ofdiabetic peripheral neuropathy / 中国基层医药

Objective To study the effectiveness of vaccinia vaccination rabbit inflammatory skin extracts on blood homocysteine(Hcy) and insulin-like growth factor-1(IGF-1) in patients with diabetic peripheral neuropathy. Methods 100 patients with diabetic peripheral neuropathy were randomly divided into the two groups,the observa-tion group(n=50 cases) and the control group(n=50 cases).The observation group was treated through vaccinia vaccination rabbit inflammatory skin extracts, while the control group was treated through mecobalamin.They were treated for 15 days.Median nerve,peroneal motor nerve conduction velocity (MCV) and sensory nerve conduction velocity ( SCV) were determined before and after treatment.Blood IGF-1 and Hcy were detected.Results TSS were significantly lowered after treatment (t=9.772,13.624,all P<0.01).Compared with the control group,the observa-tion group was significantly decreased (t=3.925,P<0.05).After treatment,the median nerve,peroneal nerve MCV and SCV were significantly increased (t=5.103,3.019,4.998,2.928,5.128,3.112,5.286,3.118,all P<0.05). Compared with the control group,theobservation group was observed to increase more significantly(t=2.826,2.743, 3.268,3.096,all P<0.05).After treatment,serum IGF-1 were significantly elevated,and compared with the control group after treatment,post-treatment observation group increased more significantly(t=4.179,P<0.05).After treat-ment plasma Hcy were significantly lower than that of the control group after treatment,the observation group after treatment significantly decreased(t=3.165,P<0.05).Conclusion Vaccinia vaccination rabbit inflammatory skin extracts can improve blood Hcy and IGF-1 in patients with diabetic peripheral neuropathy.

Effect of adenosine A2A receptor antagonist ZM241385 on amygdala-kindled seizures and progression of amygdala kindling / 华中科技大学学报（医学）（英德文版）

The purpose of this study was to evaluate the effect of adenosine A2A receptor antagonist ZM241385 on amygdala-kindled seizures and its roles in epileptogenesis. Electrodes were implanted into the right amygdala of male adult Wistar rats. Kindling was accomplished by using stimulus strength of 500 μA applied daily to the amygdala until 10 consecutive stage 5 seizues were induced. Then effect of ZM241385 was studied in fully kindled rats after intracerebroventricular administration of the drug. In addition, the effect on kindling progression was evaluated through ZM241385 injection before daily stimulation. In all experiments, behavioral changes in the rats in response to ZM241385 were monitored closely. The results showed that, in fully amygdala-kindled rats, ZM241385 (0.001-0.1 nmol/L) decreased afterdischage duration (ADD), motor seizure duration (MSD), stage 5 duration (S5D) and seizure duration (SD), but only the effect on ADD was dose-dependent. The doses of 0.001-0.1 nmol/L had no influence on stage 4 latency (S4L) and seizure stage (SS). The dosages of 0.0001 and 1 nmol/L of ZM241385 did not exert any effect on all seizure parameters. In contrast to the results in fully amygdala-kindled rats, ZM241385 (0.001-0.1 nmol/L) had minimal or no effects on the progression of amygdala-kindled seizures. We are led to the conclusion that although ZM241385 had no influence on the progression of amygdala-kindled seizures, it had potent anti-convulsant profile and little adverse effects at the dosage of 0.001-0.1 nmol/L, suggesting that the agent is effective against the amygdala-kindled seizures.

A new method for the identification of the "Beijing family" strain of Mycobacterium tuberculosis / 中华微生物学和免疫学杂志

Objective To examine the foasibility of a new method for Mycobacterium tuberculosis (M. tuberculosis)"Beijing family"strain identifjcation——RD105 deletion test. Methods Two methods,Spoligotyping and RD105 deletion test,were used for M. tuberculosis"Beijing family"strain identification,respectively. The difference of the two identification methods was compared. Results Three hundred and forty-two clinical isolates from four areas(Beijing,Fujian,Xinjiang and Jilin)were assayed in this study.Among the total samples,261 isolates belonged to"Beijing family"accounting for 76.32%,while the other 81 isolates belonged to"non-Beijing family"accounting for 23.68%. The coincidence rate for these two methods was 100%. Conclusion The simple and rapid new method——RD105 deletion test can be used to identify"Beijing family"instead of the traditional method——Spoligotyping.

Extraction of type I collagen and preparation of a collagen-gelatin scaffold / 中国矫形外科杂志

[Objective]To develop an ideal method for extracting type I collagen from cortical bone and to prepare a collagen-gelatin scaffold.[Method]The cortical bone was disintegrated into bone matrix powder in a high speed mill and was subsequently dehydrated in alcohol,decalcificated in hydrochloric acid and defatted in chloroform:methanol(1:1,v/v).The osscins were extracted using improved pepsin digestion method after the bone matrix powder was dissolved,centrifuged,dialyzed and lyophilized.Type I collagen was then characterized by SDS-PAGE and amino-acid composition analysis.The biomaterial was made of type I collagen and gelatin using freeze-drying method,and the alignment regularities of microscopic channels and their course directions were observed under the scanning electronic microscope.The size of the micropores and the factor of porosity were also measured.[Result] The collagen extracted was confirmed to be type I collagen by SDS-PAGE and amino-acid composition analysis.All the scaffolds looked like circular cylinder,the microscopic channels were arranged in parallel manners,and the pore sizes of the channels were uniform.[Conclusion]Ossein extracted from cortical bone is a real type I collagen that can be applied in the construction of collagen products.

A high throughout assay for human papillomavirus genotypes with fluorescence polarization / 中华医学杂志(英文版)

<p><b>OBJECTIVE</b>To develop a simple, cheap, quick, accurate and practical method for a high throughout genotypes assay of human papillomavirus (HPV) DNA.</p><p><b>METHODS</b>Crude DNA was extracted by a simplified proteinase K digesting method. HPV common conservative primers: GP5+/6+ system was used to amplify HPV DNA in 127 samples of condylomata acuminatum (CA) and cervical scrapes by PCR, then the PCR product was assayed using a template directing terminator incorporation (TDI) and genotypes were detected with fluorescence polarization (FP). Major HPVs type-specific probes (HPV6, 11, 16, 18, 31, 33, 35 and 58) designed by us were hybridized with the specific PCR products and a special fluorescent ddNTP terminator was directly added to the end of the probe under direction of specific PCR products. The results were measured with FP and compared with the results of the DNA sequence.</p><p><b>RESULTS</b>Compared with the results of DNA sequencing, the results detected with fluorescence polarization were all correct. The proposed method could detect more than one type of HPV infection, but DNA sequencing method could not. The positive rate of HPV was 100% in 78 CA biopsies. Among them, there were 14 HPV double infections [HPV6B and 11 (9 cases), HPV11 and 16 (4), HPV11 and 18 (1)], 5 HPV triple infections [HPV6B, 11 and 16 (4), HPV11, 16 and 18 (1)], and one HPV quadruple infection (HPV6B, 11, 16 and 18). The positive rate of HPV was 77% in the 49 cervical scrapes. Six HPV double infections [HPV6B and 11 (2), HPV11 and 16 (1), HPV6B and 16 (1), HPV16 and 18 (1), HPV18 and 58 (1)], 3 HPV triple infections [HPV6B, 11 and 16 (2), HPV11, 16 and 18 (1)] and one HPV quadruple infection (HPV6B, 11, 16 and 18) were detected in cervical cancer scrapes.</p><p><b>CONCLUSIONS</b>The proposed method allowed a high throughout, special, simple, rapid, automatic and economical detection of HPV-DNA genotyping without a use of labeling probes. It can detect multiple HPV genotype infection and will be and useful tool in HPV genotype screening.</p>

The Quality Control and Targeting Studies of Anti-hepatoma Monoclonal Antibodies / 中国肿瘤生物治疗杂志

The cell suspensions prepared from surgically resected hepatoma specimens were used to immunize BALB/c mice and, with the hybridoma technique, a battery of high affinity monoclonal anti-hepatoma antibodies were obtained which were designated HAbl8, Fll, E5 and A10 separately. Immunohistochimical staining showed that the above 4 antibodies possessed good selective reactivity with hepatoma tissue. After radioiodination of Fll, E5, A10, HAbl8 IgG and It's F(ab')_(2) fragments, the labelled reagents were employed for radioimmunoimaging in hepatoma-bearing nude mice and the in vivo detection appeared promising, with tumor/non-tumor ratios being 6.88, 5.14, 5.67, 5.15 and 14.47 respectively. The in vivo localization ablities of the antibodies seemed better compared to other similar findings published elswhere (Dunk AA, 1987). Also, ~(131) I -HAbl8 I gG and its radiolabelled fragments were utilized for radioimmunotherapy in hepatoma-bearing nude mice, with complete response rate and partial response rate being 42%(5/12) and 50% (6/12)respectively. When the HAbl8 conjugate with radioiodine was introduced for the in vivo imaging in hepatoma patients, a positivity rate of 86.5% (45/52)was witnessed, with the smallest tumor foci detected being only 0.5cm in diameter. In the in vivo targeting therapy with the immunoconjugate, a partial response rate of 69.6% (16/23) was obtained. In summary, the antibodies reported here have lended a novel regime for the present comprehensive therapy protocol of hepatoma.