Interferon-Gamma Release Assay is Not Appropriate for the Diagnosis of Active Tuberculosis in High-Burden Tuberculosis Settings: A Retrospective Multicenter Investigation / 中华医学杂志(英文版)

<p><b>BACKGROUND</b>Interferon-gamma release assay (IGRA) has been used in latent tuberculosis (TB) infection and TB diagnosis, but the results from different high TB-endemic countries are different. The aim of this study was to investigate the value of IGRA in the diagnosis of active pulmonary TB (PTB) in China.</p><p><b>METHODS</b>We conducted a large-scale retrospective multicenter investigation to further evaluate the role of IGRA in the diagnosis of active PTB in high TB-epidemic populations and the factors affecting the performance of the assay. All patients who underwent valid T-SPOT.TB assays from December 2012 to November 2015 in six large-scale specialized TB hospitals in China and met the study criteria were retrospectively evaluated. Patients were divided into three groups: Group 1, sputum culture-positive PTB patients, confirmed by positive Mycobacterium tuberculosis sputum culture; Group 2, sputum culture-negative PTB patients; and Group 3, non-TB respiratory diseases. The medical records of all patients were collected. Chi-square tests and Fisher's exact test were used to compare categorical data. Multivariable logistic analyses were performed to evaluate the relationship between the results of T-SPOT in TB patients and other factors.</p><p><b>RESULTS</b>A total of 3082 patients for whom complete information was available were included in the investigation, including 905 sputum culture-positive PTB cases, 914 sputum culture-negative PTB cases, and 1263 non-TB respiratory disease cases. The positive rate of T-SPOT.TB was 93.3% in the culture-positive PTB group and 86.1% in the culture-negative PTB group. In the non-PTB group, the positive rate of T-SPOT.TB was 43.6%. The positive rate of T-SPOT.TB in the culture-positive PTB group was significantly higher than that in the culture-negative PTB group (χ2 = 25.118, P < 0.01), which in turn was significantly higher than that in the non-TB group (χ2 = 566.116, P < 0.01). The overall results were as follows: sensitivity, 89.7%; specificity, 56.37%; positive predictive value, 74.75%; negative predictive value, 79.11%; and accuracy, 76.02%.</p><p><b>CONCLUSIONS</b>High false-positive rates of T-SPOT.TB assays in the non-TB group limit the usefulness as a single test to diagnose active TB in China. We highly recommend that IGRAs not be used for the diagnosis of active TB in high-burden TB settings.</p>

Study on the phenomenon of splashes and sprays from virology / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To investigate the phenomenon of accidental splashes and sprays from manipulation of recombinant virus material and to measure the approximate spilled distance when recombinant virus material inadvertently dropped in the biosafety laboratory.</p><p><b>METHODS</b>first, two groups owning different experience simulated the course of accidental spills and splashes by recombinant adenovirus (rADV) which expressed green fluorescence protein (GFP), the GFP signal were observed in 96 well cell plate after spills appeared; Second, the routine two heights (75 cm and 110 cm) and capacity (1 ml, 1.5 ml, 4 ml and 8 ml) of virus were chose to simulate the experiment of unexpected dropping.</p><p><b>RESULTS</b>First, the positive quantity of the first group owning 5 years' experience is much less than the second group owning 2 years' work experience, the former was 7 positive wells, the latter was 81 positive when they used the pipette to operation. Second, when the unclosed test tubes (1 ml, 1.5 ml, 4 ml and 8 ml recombinant virus) inadvertently dropped, the largest spill distance was 0.92 m, 1.57 m, 2.63 m and2.68 m respectively.</p><p><b>CONCLUSION</b>The better experience is important to make sure safety when we make infectious material; the contaminated distance increased with the amount of recombinant virus material.</p>

Alternations of tau protein and its phosphorylated profiles in the experimental hamsters infected by scrapie agents 263K and 139A / 病毒学报

In human prion diseases, phosphorylated-tau deposition has been described in a rare genetic form, Gerstmann-Straussler-Scheinker disease, but is not considered part of the neuropathological picture of Creutzfeldt-Jakob disease. To investigate the possible changes of tau and phosphorylated tau (Ser396/Ser404) in transmissible spongiform encephalopathies (TSEs), the expressions and transcriptions of above biological factors in the brain tissues of 263K- and 139A-infected hamsters were evaluated by Western blots and Real Time PCR, respectively, followed by quantitative analyses of immunoblot images and relative transcriptional levels compared with normal animals. The contents of total tau increased, but phosphorylated tau at Ser396 and Ser404 decreased, regardless of the types of scrapie agents and clinical incubations. Transcriptions of two tau isoforms were also markedly increased. These findings suggested that dephosphorylation of tau at Ser396/Ser404 was a illness-correlative phenomenon in TSEs. Alterations of tau and phosphorylated tau (Ser396/Ser404) were either intermediate or consequent events in TSE pathogenesis and proposed the potential linkage of these bioactive proteins with the pathogenesis of prion diseases.

Molecular interaction between PrP protein and the signal protein 14-3-3 beta / 病毒学报

The molecular interaction between PrP and 14-3-3 beta and the possible interactional domain between two proteins were studied by co-immunoprecipitation, pull down and FRET assays. The results showed that PrP protein could interact with 14-3-3 beta in vitro and in vivo. The domain which responded for the interaction was located at C-terminal of PrP (amino acid residues 106 to 126). This study of the interaction between PrP and 14-3-3 protein further provided the insight into the potential role of 14-3-3 in the biological function of PrP and the pathogenesis of prion disease.

Preparations of the specific antibodies against exon 2 and exon 3 of human tau protein / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To prepare the specific antibodies against exon 2 and exon 3 of human tau protein.</p><p><b>METHODS</b>Sequences encoding exon 2 and exon 3 of human tau protein were amplified from human peripheral blood DNA and cloned into a prokaryotic expression vector pGEX-2T. Fusion proteins GST-E2 and GST-E3 were expressed and purified from E. coli system. The antisera were elicited by immunization of the purified fusion proteins to rabbits and mice. The specific antibodies were purified by Protein G/A and CNBr-activated sepharose 4B coupled with GST protein. The specificity and sensitivity of the purified antibodies were evaluated by Western blotting and ELISA.</p><p><b>RESULTS</b>Recombinant fusion proteins GST-E2 and GST-E3 were expressed and purified from E. coli, which showed Mr. 29 x 10(3). Various antisera were collected from the immunized experimental animals. Reliable immunoreactive specificity and titers of the purified antibodies against exon 2 and exon 3 of human tau protein were confirmed by Western blotting and ELISA after serial purification processes.</p><p><b>CONCLUSION</b>Four specific antibodies against exon 2 and exon 3 of human tau protein have been successfully prepared, which provides basis for analyzing the role of tau in neurodegenerative diseases.</p>

Expression of cytosolic PrP and analysis of its cytotoxic activities / 病毒学报

In order to study the physicochemical characteristics of cytosolic PrP (CytoPrP) and evaluate its possible influence on cell viability, a recombinant plasmid expressing human CytoPrP eukaryoticly was constructed and transfected into human neuroblastoma cell line SH-SY5Y transiently. Proteinase-resistant activities of CytoPrP were evaluated by a proteinase K (PK) digestion and cytotoxic effects of CytoPrP were tested by MTT assay and Trypan Blue cell-counting. The presence of CytoPrP in cytoplasm after transfection was controlled by the presence of protease inhibitor. Compared with wild-type PrP, CytoPrP possessed relatively stronger PK-resistant activities. Obvious cytotoxic effects were observed in the cells after inducement of CytoPrP in cytoplasm by protease inhibitor, showing a dose-dependent manner. The results provide useful scientific evidences for further studies of potential role of CytoPrP in pathological mechanism of prion disease.

Establishment of PrP(Sc) conversion based on serial PMCA in vitro / 病毒学报

In order to establish an amplification system in vitro with which the PrP(Sc) is able to convert PrP(C) into proteinase K-resistant isoform infinitely and whether this system is more efficient than conventional protein misfolding cyclic amplification (PMCA), scrapie strain 263K-infected hamster's brain homogenate and homologous normal brain homogenate were prepared, respectively. A new methodology, namely serial PMCA, was utilized to reveal the continuous propagation ability of PrP(Sc). Totally 8 rounds of serial PMCA were proceeded and each round contained 48 cycles of alternative sonication and incubation. Simultaneously 144 cycles of conventional PMCA was used as a control. The results showed the PrP(Sc) from hamsters' brain tissues of scrapie agent 263K could replicate efficiently and infinitely with serial PMCA compared with finite propagation of PrP(Sc) with conventional PMCA system. The study of PrP(Sc) continuous propagation in brain homogenate with serial PMCA may further provide insight into the unsettled mechanism of prions misfolding and replication and apply to detect trace amount of PrP(Sc).

PrP protein can bind to protein kinase CK2 both in native and recombinant forms in vitro / 病毒学报

To explore the possible molecular interaction between CK2 and PrP, the full length sequences of human CK2alpha and CK2beta genes were amplified with RT-PCR using the mRNA from cell line SH-SY5Y as the template, and then the fusion proteins HIS-CK2alpha and GST-HIS-CK2beta were expressed in E. coli. The interaction between CK2 and PrP was evaluated with immunoprecipitation and pull-down assays. The results demonstrated that recombinant PrP bound specifically with CK2alpha, but not with CK2beta. The native CK2 and PrP in the hamster brains interacted each other, forming protein complexes. The domain responsible for interacting with CK2alpha was located at the C-terminal segment of PrP (residues 90-231). This study proposed reliable experimental data for the molecular interaction between PrP and CK2alpha, both in recombinant and native categories. These results supply scientific clues for further assessing the potential biological significance of the interaction of PrP with CK2 and possible role of CK2 in the pathogenesis of prion diseases.

The construction and identification of the PRNP vectors with ubiquitin or the lysosome-targeting signal / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To evaluate PrP expression characteristic of PRNP nucleic acid vaccine vector with ubiquitin or the lysosome-targeting signal.</p><p><b>METHODS</b>The gene of ubiquitin and lysosome-targeting signal were ligated to PRNP and pcDNA3.1 vector that is, pcDNA3.1-UPrP and pcDNA3.1-PrPL were constructed. The expression characteristics of PrP with two signals were evaluated by Western Blot and the localization was observed by indirect immune fluorescence.</p><p><b>RESULTS</b>The protein expressed by pcDNA3.1-UPrP and pcDNA3.1-PrPL with ubiquitin and lysosome-targeting signal can be recognized by prion-specific antibody. The protein has three glycosylation molecules form as native PrP.PrP with ubiquitin was degraded gradually with time extension,whereas quantity of PrP with lysosome signal reduced in 48 h after transfection. The protein with two location signals can direct fusion proteins to cytoplasm.</p><p><b>CONCLUSION</b>The PRNP vectors with ubiquitin or the lysosome-targeting signal were constructed and expressed in eukaryocyte successfully. There will be one of good foundation on PRNP nucleic acid vaccine.</p>

Investigating the risk of two model virus in the laboratory / 中华实验和临床病毒学杂志

<p><b>OBJECTIVE</b>To study the survival time of recombination rival in environment and inactivation ability of different disinfectant and ultraviolet radiation against virus.</p><p><b>METHODS</b>NC membranes absorbed the recombinant adenovirus (rADV) or herpes simplex virus (rHSV) with green fluorescence protein (GFP) were laid, or immersed in various concentration of different disinfectants such as ethanol, sodium hypochlorite, lysol and geramine and then taked out them every 15 min, or exposed under ultraviolet radiation, then the NC membranes were adsorbed 1 h in cell, 37 degrees C 5% CO2 48 h. The results were observed under the fluorescence microscope.</p><p><b>RESULTS</b>(1) the average survival time of rHSV under environment is less than 60 min, rADV is almost up to 2 h. (2) The infection ability of rHSV and rADV was inactived 15 min by both ethanol (100%, 70% and 50%) and sodium hypochlorite (5%, 2.5% and 1.25%). (3) Two virus can be killed by 0.1% bromogeramine. (4) Both 5% and 2.5% lysol, but rADV can not lost the infection on Vero Cell until 75 min by 1.25% Lysol. (5) The rHSV was inactivated under ultraviolet radiation, but rADV was not.</p><p><b>CONCLUSION</b>The survival time of is different from both envelope rival and the no-envelope viral under nature environment and the inactivate ability of disinfectant also is different between two model virus; Disinfectant should be choose according to virus type.</p>

Clinical and Eletrophysiology Analysis of Infantile Spinal Muscular Atrophy / 实用儿科临床杂志

Objective To explore the feature of electrical physiology and the key aspect of diagnosis in early stage of spinal muscular atrophy in infants.Method The back clinical data of the musle change of electrical physiology within 25 cases were analyzed.Results The children were ill in 1 year old,with the symptom that four limbs present symmetry and paralysing slowly,low limbs being more serious than upper limbs,the close and the distant of the four limbs.The levels of creatine kinase(CK) and lactate dehydrogenase(LDH) in serum were normal and the case unconcentrated was not seen rarely.The show of electromyogram(EMG) was that three limbs presenting wide-range losing nerve electric potential in peace state,when straingently the deadline was long and the motor unit potential(MUP) was high,when strain highly the eletric potential was decreased;the speed of nerve conduction was common.Muscle inspection was typical nervine myatrophy.Conclusion The clinical feature,the changes of muscle electrical physiology and the muscle inspection are valuable for diagnosis so as to afford reliable evidence for further gene diagnosis.J Appl Clin Pediatr,2006,21(3):167