Construction and evaluation of DNA vaccine encoding Crimean Congo hemorrhagic fever virus nucleocapsid protein, glycoprotein N-terminal and C-terminal fused with LAMP1.

Crimean-Congo hemorrhagic fever virus (CCHFV) can cause severe hemorrhagic fever in humans and is mainly transmitted by ticks. There is no effective vaccine for Crimean-Congo hemorrhagic fever (CCHF) at present. We developed three DNA vaccines encoding CCHFV nucleocapsid protein (NP), glycoprotein N-terminal (Gn) and C-terminal (Gc) fused with lysosome-associated membrane protein 1 (LAMP1) and assessed their immunogenicity and protective efficacy in a human MHC (HLA-A11/DR1) transgenic mouse model. The mice that were vaccinated three times with pVAX-LAMP1-CCHFV-NP induced balanced Th1 and Th2 responses and could most effectively protect mice from CCHFV transcription and entry-competent virus-like particles (tecVLPs) infection. The mice vaccinated with pVAX-LAMP1-CCHFV-Gc mainly elicited specific anti-Gc and neutralizing antibodies and provided a certain protection from CCHFV tecVLPs infection, but the protective efficacy was less than that of pVAX-LAMP1-CCHFV-NP. The mice vaccinated with pVAX-LAMP1-CCHFV-Gn only elicited specific anti-Gn antibodies and could not provide sufficient protection from CCHFV tecVLPs infection. These results suggest that pVAX-LAMP1-CCHFV-NP would be a potential and powerful candidate vaccine for CCHFV.

Molecular epidemiology and clinical characteristics of drug-resistant Mycobacterium tuberculosis in a tuberculosis referral hospital in China.

BACKGROUND: Despite the large number of drug-resistant tuberculosis (TB) cases in China, few studies have comprehensively analyzed the drug resistance-associated gene mutations and genotypes in relation to the clinical characteristics of M. tuberculosis (Mtb) isolates. METHODOLOGY/PRINCIPAL FINDINGS: We thus analyzed the phenotypic and genotypic drug resistance profiles of 115 Mtb clinical isolates recovered from a tuberculosis referral hospital in Beijing, China. We also performed genotyping by 28 loci MIRU-VNTR analysis. Socio-demographic and clinical data were retrieved from medical records and analyzed. In total, 78 types of mutations (including 42 previously reported and 36 newly identified ones) were identified in 115 Mtb clinical isolates. There was significant correlation between phenotypic and genotypic drug resistance rates for first-line anti-TB drugs (P<0.001). Genotyping revealed 101 MIRU-VNTR types, with 20 isolates (17.4%) being clustered and 95 isolates (82.6%) having unique genotypes. Higher proportion of re-treatment cases was observed among patients with clustered isolates than those with unique MIRU-VNTR genotypes (75.0% vs. 41.1%). Moreover, clinical epidemiological links were identified among patients infected by Mtb strains belonging to the same clusters, suggesting a potential of transmission among patients. CONCLUSIONS/SIGNIFICANCE: Our study provided information on novel potential drug resistance-associated mutations in Mtb. In addition, the genotyping data from our study suggested that enforcement of the implementation of genotyping in diagnostic routines would provide important information for better monitor and control of TB transmission.

Genomic sequence based scanning for drug resistance-associated mutations and evolutionary analysis of multidrug-resistant and extensively drug-resistant Mycobacterium tuberculosis.

OBJECTIVE: To better understand the molecular mechanisms and evolution of drug resistance in Mycobacterium tuberculosis (M. tuberculosis), we performed a genomic sequence based scanning of drug resistance-associated loci for multidrug-resistant (MDR) and extensively drug-resistant (XDR) M. tuberculosis strains. MATERIALS AND METHODS: Forty-five pairs of primers covering known drug resistance-associated loci compiled in the TBDReaMDB database were designed to perform the analysis of drug resistance-associated mutations for 14 M. tuberculosis clinical isolates from TB patients in China. Genetic diversity and evolutionary analysis was done using concatenated nucleotide sequences of drug resistance-associated loci. RESULTS: Forty-four types of mutations were identified in 14 M. tuberculosis clinical isolates. Average nucleotide diversity for drug resistance-associated loci increased in the M. tuberculosis isolates as the drug resistance increased (π = 0, π = 0.00021, and π = 0.00028 for susceptible, MDR, and XDR isolates, respectively). The dN/dS ratios for coding regions of drug resistance-associated genes in MDR and XDR isolates were 2.73 and 1.83, respectively. MDR and XDR isolates were distributed sporadically on different branches in the phylogenetic trees. CONCLUSIONS: Our study provides supporting evidence to demonstrate that the MDR- and XDR-M. tuberculosis strains have evolved independently driven by positive selection.

MicroRNA-146a modulates TGF-ß1-induced phenotypic differentiation in human dermal fibroblasts by targeting SMAD4.

During wound healing and tissue repair the dermal fibroblast-to-myofibroblast transdifferentiation plays an important role, transforming growth factor-ß1 (TGF-ß1) is considered to be the main stimuli factor of transdifferentiation. MicroRNAs (miRNAs) have recently emerged as key post-transcriptional regulators of gene expression. The involvement of miRNAs and their roles in TGF-ß1-induced myofibroblast transdifferentiation remains to be determined in detail. The current study found that the expression of miR-146a was upregulated in human dermal fibroblasts cells in response to TGF-ß1 stimulation in dose-dependent manner by quantitative RT-PCR. Bioinformatic analyses predict that signaling effectors mothers against decapentaplegic protein 4 (SMAD4) is a miR-146a target gene. Luciferase assay demonstrated that miR-146a mimics suppressed SMAD4 3'-UTR reporter construct activity. Furthermore, miR-146a overexpression in dermal fibroblast did not decrease target mRNA levels, but significantly reduced target protein expression. In addition, dermal fibroblasts transfected with miR-146a mimics exhibited attenuated TGF-ß1 -induced α-smooth muscle actin (α-SMA) expression compared with the control. This study demonstrated that miR-146a may function as a novel negative regulator to modulate myofibroblast transdifferentiation during TGF-ß1 induction by targeting SMAD4.

Characteristics and treatment outcomes of patients with MDR and XDR tuberculosis in a TB referral hospital in Beijing: a 13-year experience.

BACKGROUND: Information on treatment outcomes among hospitalized patients with multidrug-resistant tuberculosis (MDR-TB) and extensively drug-resistant tuberculosis (XDR-TB) are scarce in China. METHODOLOGY/PRINCIPAL FINDINGS: We conducted this retrospective study to analyze the characteristics and treatment outcomes in MDR- and XDR-TB patients in the 309 Hospital in Beijing, China during 1996-2009. Socio-demographic and clinical data were retrieved from medical records and analyzed. Logistic regression analysis was performed to identify risk factors associated with poor treatment outcomes and Cox proportional hazards regression model was further used to determine risk factors associated with death in TB patients. Among the 3,551 non-repetitive hospitalized TB patients who had drug susceptibility testing (DST) results, 716 (20.2%) had MDR-TB and 51 (1.4%) had XDR-TB. A total of 3,270 patients who had medical records available were used for further analyses. Treatment success rates (cured and treatment completed) were 90.9%, 53.4% and 29.2% for patients with non-MDR-TB, patients with MDR-TB excluding XDR-TB and patients with XDR-TB, respectively. Independent risk factors associated with poor treatment outcomes in MDR-TB patients included being a migrant (adjusted OR = 1.77), smear-positivity at treatment onset (adjusted OR = 1.94) and not receiving 3 or more potentially effective drugs (adjusted OR = 3.87). Independent risk factors associated with poor treatment outcomes in XDR-TB patients were smear-positivity at treatment onset (adjusted OR = 10.42) and not receiving 3 or more potentially effective drugs (adjusted OR = 14.90). The independent risk factors associated with death in TB patients were having chronic obstructive pulmonary disease (adjusted HR = 5.25) and having hypertension (adjusted HR = 4.31). CONCLUSIONS/SIGNIFICANCE: While overall satisfactory treatment success for non-MDR-TB patients was achieved, more intensive efforts should be made to better manage MDR- and XDR-TB cases in order to improve their treatment outcomes and to minimize further emergence of so-called totally drug-resistant TB cases.

Risk factors associated with fluoroquinolone-resistant tuberculosis in a Beijing tuberculosis referral hospital.

BACKGROUND AND OBJECTIVE: The rapid increase in the number of multi-drug-resistant tuberculosis (MDR-TB) cases worldwide emphasizes the importance of rational use of key important life-saving second-line anti-TB drugs such as fluoroquinolones. In order to provide information for better case management, so as to minimize the further spread of extensively drug-resistant TB, a retrospective study was performed to assess the risk factors associated with fluoroquinolone resistance among TB patients attending the 309 Hospital in Beijing, China. METHODS: Drug susceptibility testing results and clinical data for hospitalized TB patients for the period 2000-2010 were analysed. Univariate and multivariate analyses were used to determine the risk factors associated with fluoroquinolone-resistant TB. RESULTS: From July 2000 to July 2010, ofloxacin resistance was observed in 306 of 3546 (8.6%) hospitalized TB patients who were tested. The independent risk factors associated with fluoroquinolone-resistant TB were being single (adjusted OR 1.65), being a migrant living in Beijing (adjusted OR 2.15), being a migrant from another area (adjusted OR 5.07), being a patient who was retreated (adjusted OR 2.84), exposure to fluoroquinolones (adjusted OR 2.73), having COPD (adjusted OR 3.53), having COPD with known exposure to fluoroquinolones (adjusted OR 2.47), having MDR-TB (adjusted OR 1.67), and having poly-resistant TB (adjusted OR 2.34). CONCLUSIONS: These findings suggest that in high-risk populations continuous surveillance of fluoroquinolone resistance should be maintained. In addition, programs should be implemented for the management of patients with MDR-TB and more complicated TB, so as to minimize the further spread of fluoroquinolone resistance and extensively drug-resistant TB.

[Comparison of four imaging examinations of shoulder joint injury in forensic expertise].

OBJECTIVE: To compare the value of four imaging examinations, including the X-ray, CT, MRI and gas-iodine double contrast CT analyses, in the forensic expertise of shoulder joint injury. METHODS: Imaging data of shoulder joint injury, by the X-ray, CT, MRI and gas-iodine double contrast CT were retrieved and analyzed. RESULTS: The correct diagnosis rates of fracture and soft tissue injury by X-ray, CT and MRI were 52.8%, 72.0% and 63.2%, as well as 0.0%, 32.9% and 82.5%, respectively. The correct diagnosis rate of soft tissue injury by gas-iodine double contrast CT was 100%. CONCLUSION: X-ray is a useful screening method, CT is better for diagnosis of fracture, and MRI is fit for diagnosis of soft tissue injury. Gas-iodine double contrast CT can reflect not only the soft tissue injury but also its severity. Thus, combined application of X-ray, CT, MRI and gas-iodine double contrast CT can provide important imaging information for forensic expertise in shoulder joint injury.

[Forensic clinical analysis on 25 cases of traumatic cerebral infarction].

OBJECTIVE: To investigate characteristics of forensic clinical identification on traumatic cerebral infarction(TCI). METHODS: Twenty-five cases of TCI were analyzed retrospectively, including the general situation, location of infarction and clinical feature. RESULTS: TCI occurred primarily in children and elderly. All the cases had definite cerebral trauma which was located mainly in the regions of basal ganglia-internal capsule, frontal, temporal and parietal cerebral cortex. CONCLUSION: The consequence of TCI has direct correlation with location of cerebral infarction. More attention should be paid to this issue in forensic practice.

[Effects of triptolide on hypothalamic-pituitary-adrenal axis of rats].

OBJECTIVE: To observe the effects of triptolide on the hypothalamic-pituitary-adrenal axis (HPAA) of rats in light of morphological and functional changes. METHODS: Thirty Sprague-Dawley (SD) male rats were randomized into 3 groups and given 2% propylene glycol, mixture of propylene glycol and prednisone acetate or compounds of propylene glycol and triptolide by gavage, respectively, for consecutive 7 weeks. Determination in the 3 groups was conducted concerning the contents of blood plasma cortisol (COR), adrenocorticotropic hormone (ACTH) and corticotropin-releasing hormone (CRH) besides measurement of the rats' body weight, coefficient of the adrenal gland and observation of the histopathological changes in fascicular zone of adrenal cortex. Immunohistochemical staining technique was used to detect the expression of ACTH in pituitary in the 3 groups. RESULTS: (1) The content of COR in the groups of triptolide and prednisone acetate appeared lower and serum ACTH showed no significant difference, but CRH in the group of triptolide was augmented as compared with the control group (P < 0.05). (2) The rats' weight in the groups of triptolide and prednisone acetate was declined, and yet, the coefficient of the adrenal gland remained no significant change in comparison with the controls. HE staining and electron microscopy examination revealed thinned and constricted zona fasciculata in adrenal gland in the rats of triptolide and prednisone acetate, with hypofunction. ACTH expression in the group of triptolide was higher than that of the control group (P < 0.05). CONCLUSION: Morphologically and functionally, the findings suggest that long-term use of triptolide may result in atrophied cortex and hypofunction of the adrenal gland, leading to augmented production and secretion of CRH and ACTH from respective hypothalamic and pituitary.

[The pathological observation of acute intoxication by Alangium Chinese in mice].

OBJECTIVE: To investigate the pathological change of mice organ intoxicated by Alangium Chinese and its poisoning mechanism. METHODS: Mice were intoxicated by gavage with extract of Alangium Chinese. Then the histopathologic examination was made for evaluating the pathological changes in the organs of the poisoned mice by HE staining. RESULTS: The main pathological changes included alveolar hemorrhage, pulmonary interstitial hemorrhage, sinus hepaticus expansion and congestion, hepatocyte edema, subarachnoid hemorrhage, congestion and hemorrhage of other organs. CONCLUSION: The main target organs or tissue of Alangium Chinese are the lungs, liver and vascular smooth muscle. There is correlation between the toxic effect and the dosage.

[The value of TNFalpha testing in evaluation of death due to drug anaphylactic shock].

OBJECTIVE: To analyze the changes of the serum TNFalpha level in cadavers of drug anaphylactic shock(DAS) and to explore the significance of the TNFalpha testing in evaluation of death caused by DAS. METHODS: The level of serum TNFalpha from cardiac blood was determined by double antibody-sandwich ELISA method. RESULTS: The level of TNFalpha in the experimental group was (131.6+/-9.4) pg/mL, while the level in the infectious diseases control group and the normal control group were (87.3+/-6.4) pg/mL and (17.2+/-4.5) pg/mL, respectively. There was statistically significant difference among the groups (P<0.05). CONCLUSION: The serum TNFalpha level may be used as one of the indexes for evaluation of death caused by highly suspected drug anaphylactic shock.

[Apoptosis in myocardial cells after macleaya cordata total alkaloids poisoning in rats].

OBJECTIVE: To observe pathological changes and apoptosis in rats myocardial cells after Macleaya cordata total alkaloids poisoning, and to provide some references for Macleaya cordata total alkaloids poisoning detection. METHODS: An experimental model of Macleaya cordata total alkaloids poisoning was established, and the technology of TUNEL staining was used.The results were analyzed by computer image analysis competitive system. RESULTS: Quantities of apoptosis in myocardial cells in poisoning groups were much more than those in the control groups at different tages (P<0.01). In addition the quantities of apoptosis were different after different poisoning duration. CONCLUSION: Although clinical symptoms was not obvious and could not be detected by poison analysis. Pathological changes induced by Macleaya cordata total alkaloids could be found through the apoptosis detection.

[The expression of Bcl-2 protein on acute myocardial ischemia/reperfusion and the significance in forensic pathology].

OBJECTIVE: To search the objective morphologic evidence for the forensic pathological diagnosis on the acute myocardial ischemia/reperfusion. METHODS: The expression of Bcl-2 protein in the early stage of acute myocardial ischemia/reperfusion(I/R) in rats were studyed with immunohistochemistry. RESULTS: There was no expression of Bcl-2 protein in control group. The expression of Bcl-2 protein began to increase in myocytes 0.5 h after I/R in the ischemic areas and the expression of Bcl-2 protein in inner layer was stronger than in outer layer. The expression of Bcl-2 protein appears firstly in the inner layer of myocardium and extended gradually toward the outer layer 1 h after I/R. It was found that the expression of Bcl-2 protein in outer layer was stronger than in inner layer, then the expression of Bcl-2 protein became weaken gradually. CONCLUSION: Bcl-2 gene may be an important regulative gene in the process of apoptosis and the changes observed in this paper might be useful for the forensic pathological diagnosis on the early myocardial ischemia.

[A study on the expression of C-FOS protein after experimental rat brain concussion].

OBJECTIVE: To study the relationship between expression of C-FOS protein and brain concussion and find a sensitive marker of diagnosis of the brain concussion. METHODS: Fifty-five rats were randomly divided into brain concussion groups and control group. The expression of C-FOS protein was microscopically observed by immunohistochemical method. RESULTS: There were negative expression of C-FOS protein in control group. In brain concussion group, however, positive expression of C-FOS protein in some neurons was seen at 15 min after brain concussion, and reach to the peak at 6 h after brain concussion, then decreased gradually. CONCLUSION: These findings suggest that detection of C-FOS protein could be an index of diagnosis of brain concussion and a sensitive marker of timing of injury after brain concussion.