The early and effective diagnosis is crucial to efficacious treatment of pulmonary tuberculosis, especially for smear negative cases and MDR-TB, and is one of the priorities of global tuberculosis control and prevention. The traditional methods which have been used for decades of years, show many well-known drawbacks, in terms of fast speed and time consuming. Recently, some new promising diagnostic methods emerged, which have been expected to optimize the diagnosis of tuberculosis, especially for MDR-TB.

Objective To investigate the incidence of drug resistance among new and retreatment TB patients from special hospital.Methods Totally 500 smear positive TB patients from June 2013 to December 2014 in Beijing Chest Hospital were enrolled.Phenotypic susceptibilities to cultured isolates were analyzed in 15 anti tuberculosis drugs by MGIT 960,include Isoniazid (INH),Rifampicin (RFP),Streptomycin (STR),Ethambuto (EMB),Kanamycin (Km),Amikacin (Am),Capreomycin (Cm),Ofloxacin (Ofx),Levofloxacin (Lfx),Moxifloxacin (Mfx),Paza-aminosalicylate (PAS),Protionamide (Pto),Linezolid (Lzd),Ethionamide (Eto),Pyrazinamide (PZA).Results A total of 500 TB cases were enrolled.71 samples among these were NTM infection and 12 samples were contaminted.The rest of 417 of cases infected with Mycobacteriuma,and the rate of drug resistance was 47.2％ (192/417) and the MDR rate was 28.2％ (120/417).The retreatment was significantly higher than that of the new in any drug resistance rate and MDR rate (P =0.000).100 of the retreatment isolates and 50 of the new isolates with Mycobacteriuma were selected to do the drug susceptibility test in 11 subsequent anti tuberculosis drugs (include:PZA,Am,Km,Cm,Ofx,Lfx,Mfx,PAS,Pto,Lzd,Eto).Five cases were contaminated,and in the rest of the cases,48 was the new and 97was the retreatment.The rate of the drug resistance to PZA,Am,Km,Cm,Ofx,Lfx,Mfx for the retreatment were significantly higher than the new (P ＜ 0.05).The rate of drug resistance to PAS,Pto,Lzd and Eto for the new and reteartment were no markedly differential in statistics.Conclusion This study further confirmed the rate of drug resistance in retreatment cases is higher,and the management of tuberculosis patients should be further strengthened.

BACKGROUND:At present, the development of reprogramming technology provides a wide prospect for stem cellresearch. Through the ectopic co-expression of reprogramming factors, the somatic cells can be reprogrammed to a pluripotent state, termed as induced pluripotent stem cells, which can avoid the ethical controversy faced in the research and application of embryonic stem cells. Also, we can generate patient-specific and disease-specific induced pluripotent stem cells, which significantly decrease immuno-rejection. However, reprogramming technology faces some chal enges, such as low efficiency and safety.
OBJECTIVE:Based on the characteristics of induced pluripotent stem cells and the principles of reprogramming, to detail the progress in reprogramming technology from five aspects, including cellresources, carriers, transcription factors, microRNA and signal transduction pathway. 
METHODS:A computer-based online retrieval was performed to search papers published form January 1990 to April 2013 in VIP periodical ful-text database, Wanfang periodical ful-text database, CNKI periodical ful-text database, PubMed database and Springer database with key words of“reprogramming, induced pluripotent stem cell, signal transduction pathway, epigenetics, microRNA, transcription factor, vector, somatic cell, smal molecule compound, safety”both in Chinese and English. After excluding objective-independent papers, 67 papers were included for further analysis.
RESULTS AND CONCLUSION:By exploring different cellresources, different carriers, various combination of transcription factors, microRNAs or inhibition of the signal transduction pathways, the reprogramming efficiency and safety have been improved greatly. However, currently, induced pluripotent stem cells stil could not meet the requirement of clinical application. To achieve the clinical application of induced pluripotent stem cells, it is urgent to explore the mechanism of reprogramming, and to optimize the programming strategy.

Objective To explore the pathological features and the causative particles of severe acute respiratory syndrome (SARS) for providing evidences of SARS prevention and clinical treatment. Methods A dead case of SARS of China was studied by light microscopy, electron microscopy, histochemical and immunohistochemical stain. Results The major pathological changes of lung in the SARS case were acute pulmonary interstitial exudative and leakage inflammation, with predominant lymphocyte infiltration. The hyaloid membranes were formed in 20%～30% pulmonary alveoli. The diffuse pulmonary epithelial injury was observed, and virus like inclusions were found in about 30% of total alveolar epithelia by histochemical stain, but chlamydia like inclusions were found occasionally. Meanwhile, the extra pulmonary organs, such as lymph nodes and spleen, showed extensive haemorrhagic necrosis inflammation, accompanied macrophage/histocyte reactive proliferation with erythrocytophage. The double adrenal glands also presented focal haemorrhagic necrosis inflammation. Under the electron microscopy observation, virus like particles with 100 ～150 nm diameter and halo or garland envelopes were found in more than 30 % alveolar epithelial cells, endothelial cells in lung tissues, and also in a part of cardiomyocytes, lymphocytes and macrophages in lymph nodes. The virus like particles were mainly located in cytoplasm and dilated reticular endoplasm. In contrast, chlamydia like particles were commonly visualized in multiple extra lung organs such as liver, but very few in the lung. Immunohistochemistry showed the positive reactions in the lung tissues with the serum IgG and/or IgM from the dead case himself and other SARS convalescent stage cases from Guangdong province of China. Conclusion In the severe SARS case, predominant acute interstitial exudative and leakage inflammation, often with the formation of hyaloid membranes in pulmonaryalveoli, and the haemorrhagic necrosis inflammation of immune organs might be pathological features of SARS. According to the structures, diameter and location of the virus like particles found in this case, combined with the pathological changes, we should consider that those virus like particles might be a new kind of coronavirus, and this kind of virus might be the main causative agent of SARS. However, the chlamydia like particles frequently observed in extra lung organs also suggested the potential new kind of coronavirus might be coexist and synergicallly cause SARS. Our findings in this study provide several evidences for SARS clinical therapy such as application of corticosteroid and enhancement of immune ability and combination of anti virus and anti chlamydium drugs.

Objective To study the pathological changes in organs remote from the lung in SARS patient. Methods The pathological changes in extra lung organs and potential coronavirus infection were studied by using light and electron microscopic examinations as well as special virus inclusion stains in the tissues obtained from an autopsy of a patient who died of SARS. Results Besides the lesions in the lung, pathological changes were found also in the central nervous system (CNS), including the cerebrum, cerebellum, thalamus, pons, and medulla oblongata, such as widening of the Virchow Robin′s space, infiltration of a few lymphocytes and macrophages in the parenchyma, vasodilatation and congestion. However, no significant neuron degeneration or necrosis was identified. Vasodilatation in the lamina propria of mucosa and submucosa of the digestive tract with some lymphocytes infiltration, and epithelial nuclear vacuolation, and occasional apoptosis were observed in the mucosal epithelial and glandular cells, as well as focal hemorrhage in segments of the small intestine. Mesenchymal edema and infiltration of a few lymphocytes in the pancreas were noted. Very mild lymphocyte infiltration, but no viral inclusions, was found in the convoluted seminiferous tubules of the testis. The patient who died of SARS was proved to have arteriosclerosis of the coronary arteries, and coronavirul particles were identified in the blood vessels under electron microscopic examination, however no coronavirul particles were found in the brain or the testis of the patient. Conclusion There were mild hypoxic changes in the tissue of CNS in the patient with severe SARS without invasion of the virus. It was confirmed that there were coronavirul particles in the blood of the patient at the acute stage of SARS. Since the patient who succumbed to the disease had a history of coronary arteriosclerosis, it was inferred that cardiovascular disease might be a contributory factor of mortality in this patient with severe SARS.

Objective To explore the target cells of SARS coronavirus infection in vivo and to provide the evidence of multi organ injuries produced by SARS coronavirus infection. Methods Three biotin labeling oligonucleotide probes were synthesized according to the published gene sequence of SARS coronavirus. The location, distributtion and quantity of SARS coronavirus in 2 autopsy cases of SARS were studied by in situ hybridization and electron microscopic examination. Results SARS coronavirus particles were identified in multiple organs. In lungs, SARS coronaviruses were located predominantly in the cytoplasm of bronchiolar and alveolar epithelial cells, in a part of macrophages and endothelial cells as well as a few infiltrated lymphocytes. In situ hybridization showed that in target cells SARS coronavirus distribution presented a cytoplasmic or inclusive pattern, and the mean number of positive cells in the pulmonary tissue was 80?25 per 200? field. Electron microscopic examination showed that the coronaviral particles were 100～150 nm in diameter, with low density electron cores with halo or garland envelopes. About 15% of renal tubular epithelial cells harbored SARS coronavirus, and a few parenchymal cells and sinusoid capillary endothelial cells of adrenal glands were hybridization positive. In the gastro intestinal tract, SARS coronaviruses were seen in the cytoplasm of mucosal and crypt epithelial cells, mostly in 2/3 of superficial mocosa. Under both electron microscopy and in situ hybridization observation, SARS coronaviruses were found focally distributed in some cardiomyocytes. The SARS coronavirus positive particles were also noted in macrophages/histocytes, sinusoid endothelial cells, as well as a few lymphocytes in thoracic and celiac lymph nodes. In addition, coronavirus particles were also seen in a few testicular epithelial cells and Leydig's cells. Conclusion SARS coronavirus could attack multiple target cells, implicating that SARS might cause multi organ damages, with lungs as the predominant organ of injury.

Objective To explore the impact of blockade of SDF-1/CXCR4 signaling pathway by T140 on degradation of typeⅡ collagen in human articular cartilage and to define the mechanism of action of T140. Methods 144 pieces of cartilage (Mankin score of 0 or 1) were obtained from osteoarthritis patients undergoing total knee replacement ( OA cartilage group) and 144 pieces of cartilage (Mankin score of 0 or 1) were obtained from patients receiving traumatic amputation (normal cartilage group). Each group was treated with SDF-1 of 100 ng/mL, then divided into three subgroups A, B, and C. The cartilage tissue in each group was cultured in the nutrient solution containing of T140, MAB310, or SDF-1 (1 000 nmol/L) for 48 or 96 hours. RT-PCR was used to detect expression of typeⅡcollagen mRNA in the cartilage tissue. Results Level of type Ⅱcollagen mRNA was markedly higher in subgroup A than in subgroup B and subgroup C at the same group and the same time (P <0.05). The expression level of type Ⅱcollagen mRNA at the same time and in the same subgroup of OA cartilage group were lower than that in normal cartilage group (P < 0.05). Conclusions SDF-1 induces degradation of typeⅡcollagen in human articular cartilage thruogh the SDF-1/CXCR4 signaling pathway. T140 can block the SDF-1/CXCR4 signaling pathway and reduce the degradation of type II collagen.

Objective To detect the activity of α1 antitrypsin(AAT) with initial velocity of enzymatic reaction in order to detect the activity of samples in the process of separating and purifying plasma protein ,chromogenic substrate assay was optimized.Methods The effect of trypsin concentration and reaction time on enzymatic reaction was acquired by the kinetic monitoring mode of the microplate reader .Initial velocity was calculated to confirm the largest concentration of trypsin which was saturated by substrate .AAT was incubated with trypsin and absorbance produced by enzymatic reaction of remaining trypsin and substrate could reflect the activity of AAT .A standard curve was established with △D fitting with the activity of AAT standard.The activity of related samples was detected and the precision and accuracy of the method were evaluated . Results Trypsin concentration was 0.0625 mg/ml.Within 20 minutes, enzymatic reaction was with initial velocity .The range of the standard curve was 200-1200 IU/ml.Correlation coefficient was more than 0.99.The activity of Cohn Ⅳ, samples of pre-processing and elution were (720.59 ±18.63), (601.84 ±19.18),and (568.09 ±24.83)IU/ml, respec-tively.The relative standard deviation was less than 10%. Sample recovery rate was 90%-110%.Conclusion The optimized chromogenic substrate assay greatly improves accuracy and precision .The method can be used for the detec-tion of AAT activity of samples in laboratories and workshops .

Objective To establish an assay for detecting α2-macroglobulin activity in Cohn fraction Ⅳ.Methodsα2-M reacted with trypsin to form α2-M-trypsin complex.After the chromogenic substrate Na-benzoyl-DL-arginine 4-nitroanilide hydrochloride ( BAPNA) was added, absorption at 410 nm was detected with the microplate reader .α2-M activity in Cohn fractionⅣwas quantitatively detected according to the established standard curve of plasma α2-M activity. Result Several critical parameters in this assay were optimized .A standard curve of plasma α2-M activity was established . According to this standard curve ,α2-M activity in Cohn fraction Ⅳsample was detected to be 1.578 PU/ml.Conclusion Using normal human plasma as the reference material , theα2-M activity in Cohn fractionⅣcan be detected through chro-mogenic substrate assay.This study provides a simple method to detect α2-M activity during the purification process of α2-M from Cohn fraction Ⅳ.

AIM: To investigate the influence of GM-CSF on human vascular endothelial cells induced to form new blood vessels and the role of VEGF.METHODS: HUVECs were cultured by Matrigel to set up a stable angiogenesis system with the stimulating factors.The rhGM-CSF concentration-dependent and time-dependent effects and the role of VEGF165 were detected.CD34 was measured by immunochemical staining and numbers of vessel formation was calculated under microscopic observation.RESULTS: After treatment with rhGM-CSF at various concentrations and at different time points,the numbers of vessel formation increased in a dose-dependent and time-dependent manner.In the presence of VEGF165,the numbers of vessel formation increased evidently.CONCLUSION: HUVECs were induced to develop tubular structure in vitro cultured with Matrigel.GM-CSF promotes human vascular endothelial cells to form vessel-like structure in vitro in a dose-dependent and time-dependent manner.VEGF also in vitro promotes human vascular endothelial cells to form new vessel-like structure.