Left ventricular concentric hypertrophy with cardiac magnetic resonance imaging improves risk stratification in patients with Duchenne muscular dystrophy: a prospective cohort study.

BACKGROUND: The development of left ventricular (LV) remodeling has been associated with an increased cardiovascular risk and cardiogenic death, and different patterns of remodeling result in varying levels of prognosis. OBJECTIVE: To investigate the association between different patterns of LV remodeling and clinical outcomes in the preclinical stage of patients with Duchenne muscular dystrophy (DMD). MATERIALS AND METHODS: A total of 148 patients with DMD and 43 sex- and age-matched healthy participants were enrolled. We used the four-quadrant analysis method to investigate LV remodeling based on cardiac magnetic resonance (MR) imaging. Kaplan-Meier curves were generated to illustrate the event-free survival probability stratified by the LV remodeling pattern. Cox regression models were constructed and compared to evaluate the incremental predictive value of the LV remodeling pattern. RESULTS: During the median follow-up period of 2.2 years, all-cause death, cardiomyopathy, and ventricular arrhythmia occurred in 5, 35, and 7 patients, respectively. LV concentric hypertrophy (hazard ratio 2.91, 95% confidence interval 1.47-5.75, P=0.002) was an independent predictor of composite endpoint events. Compared to the model without LV concentric hypertrophy, the model with LV concentric hypertrophy had significant incremental predictive value (chi-square value 33.5 vs. 25.2, P=0.004). CONCLUSION: Age and late gadolinium enhancement positivity were positively correlated with clinical outcomes according to the prediction models. LV concentric hypertrophy was also an independent predictor for risk stratification and provided incremental value for predicting clinical outcomes in the preclinical stage of patients with DMD.

Clinical utilisation of multimodal quantitative magnetic resonance imaging in investigating muscular damage in Duchenne muscular dystrophy: a study on the association between gluteal muscle groups and motor function.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a neuromuscular disease characterised by progressive muscular weakness and atrophy. Currently, studies on DMD muscle function mostly focus on individual muscles; little is known regarding the effect of gluteal muscle group damage on motor function. OBJECTIVE: To explore potential imaging biomarkers of hip and pelvic muscle groups for measuring muscular fat replacement and inflammatory oedema in DMD with multimodal quantitative magnetic resonance imaging (MRI). MATERIALS AND METHODS: One hundred fifty-nine DMD boys and 32 healthy male controls were prospectively included. All subjects underwent MRI examination of the hip and pelvic muscles with T1 mapping, T2 mapping and Dixon sequences. Quantitatively measured parameters included longitudinal relaxation time (T1), transverse relaxation time (T2) and fat fraction. Investigations were all based on hip and pelvic muscle groups covering flexors, extensors, adductors and abductors. The North Star Ambulatory Assessment and stair climbing tests were used to measure motor function in DMD. RESULTS: T1 of the extensors (r = 0.720, P < 0.01), flexors (r = 0.558, P < 0.01) and abductors (r = 0.697, P < 0.001) were positively correlated with the North Star Ambulatory Assessment score. In contrast, T2 of the adductors (r = -0.711, P < 0.01) and fat fraction of the extensors (r = -0.753, P < 0.01) were negatively correlated with the North Star Ambulatory Assessment score. Among them, T1 of the abductors (b = 0.013, t = 2.052, P = 0.042), T2 of the adductors (b = -0.234, t = -2.554, P = 0.012) and fat fraction of the extensors (b = -0.637, t = - 4.096, P < 0.001) significantly affected the North Star Ambulatory Assessment score. Moreover, T1 of the abductors was highly predictive for identifying motor dysfunction in DMD, with an area under the curve of 0.925. CONCLUSION: Magnetic resonance biomarkers of hip and pelvic muscle groups (particularly T1 values of the abductor muscles) have the potential to be used as independent risk factors for motor dysfunction in DMD.

Longitudinal changes in magnetic resonance imaging biomarkers of the gluteal muscle groups and functional ability in Duchenne muscular dystrophy: a 12-month cohort study.

BACKGROUND: Quantitative magnetic resonance imaging (MRI) is considered an objective biomarker of Duchenne muscular dystrophy (DMD), but the longitudinal progression of MRI biomarkers in gluteal muscle groups and their predictive value for future motor function have not been described. OBJECTIVE: To explore MRI biomarkers of the gluteal muscle groups as predictors of motor function decline in DMD by characterizing the progression over 12 months. MATERIALS AND METHODS: A total of 112 participants with DMD were enrolled and underwent MRI examination of the gluteal muscles to determine fat fraction and longitudinal relaxation time (T1). Investigations were based on gluteal muscle groups including flexors, extensors, adductors, and abductors. The North Star Ambulatory Assessment and timed functional tests were performed. All participants returned for follow-up at an average of 12 months and were divided into two subgroups (functional stability/decline groups) based on changes in timed functional tests. Univariable and multivariable logistic regression methods were used to explore the risk factors associated with future motor function decline. RESULTS: For the functional decline group, all T1 values decreased, while fat fraction values increased significantly over 12 months (P<0.05). For the functional stability group, only the fat fraction of the flexors and abductors increased significantly over 12 months (P<0.05). The baseline T1 value was positively correlated with North Star Ambulatory Assessment and negatively correlated with timed functional tests at the 12-month follow-up (P<0.001), while the baseline fat fraction value was negatively correlated with North Star Ambulatory Assessment and positively correlated with timed functional tests at the 12-month follow-up (P<0.001). Multivariate regression showed that increased fat fraction of the abductors was associated with future motor function decline (model 1: odds ratio [OR]=1.104, 95% confidence interval [CI]: 1.026~1.187, P=0.008; model 2: OR=1.085, 95% CI: 1.013~1.161, P=0.019), with an area under the curve of 0.874. CONCLUSION: Fat fraction of the abductors is a powerful predictor of future motor functional decline in DMD patients at 12 months, underscoring the importance of focusing early on this parameter in patients with DMD.

Comparison of Multiple Quantitative Evaluation Indices of Theoretical Knowledge and Clinical Practice Skills and Training of Medical Interns in Cardiovascular Imaging Using Blended Teaching and the Case Resource Network Platform (CRNP).

BACKGROUND This study aimed to compare multiple quantitative evaluation indices of levels of theoretical knowledge and clinical practice skills in training medical interns in cardiovascular imaging based on the use of the blended teaching (BT) online artificial intelligence (AI) case resource network platform (CRNP), including time and frequency indices and effectiveness of the CRNP. MATERIAL AND METHODS The study included 110 medical interns who were divided into the routine teaching (RT) group (n=55) and the blended teaching (BT) group (n=55). The two were assessed using the mini-clinical evaluation exercise (mini-CEX) that assessed clinical skills, attitudes, and behaviors and using an objective written questionnaire. The following four indices were compared between the RT and BT groups: the X-ray score (XS), the computed tomography angiography (CTA) score (CS), the cardiac magnetic resonance imaging (CMRI) score (MS), and the average score (AS). Seven assessment indicators included: the imaging description (ID), the qualitative diagnosis (QD), the differential diagnosis (DD), examination preparation (EP), interview skill (IS), position display (PD), and human care (HC). Indicators of CRNP use included: number of times (TN), average duration (AD), single maximum duration (SMD), and total duration (TD). RESULTS AS significantly correlated with AD (rAD=0.761) and TD (rTD=0.754), and showed moderate correlation with TN (rTN=0.595), but weak correlation with SMD (rSMD=0.404). CONCLUSIONS Levels of theoretical knowledge and clinical practice skills during medical intern training in cardiovascular imaging based on BT using the CRNP teaching technology improved theoretical knowledge and practical skills.

Artificial Intelligence and Myocardial Contrast Enhancement Pattern.

PURPOSE OF REVIEW: Machine learning (ML) and deep learning (DL) are two important categories of AI algorithms. Nowadays, AI technology has been gradually applied to cardiac magnetic resonance imaging (CMRI), covering the fields of myocardial contrast enhancement (MCE) pattern and automatic ventricular segmentation. This paper mainly discusses the relationship between machine learning and deep learning based on AI and pattern of MCE in CMRI. RECENT FINDINGS: It found that some histogram and GLCM parameters in ML algorithm had significant statistical differences in diagnosis of cardiomyopathy and differentiation of fibrosis and normal myocardial tissue. In the DL algorithm, there was no significant difference between CNN and observers in measuring myocardial fibrosis. The rapid development of texture parameter analysis methods would promote the medical imaging based on AI into a new era. Histogram and GLCM parameters are the research hotspot of unsupervised learning of MCE images. CNN has a great advantage in automatically identifying and quantifying myocardial fibrosis reflected by LGE images.

Evaluation of Early Gadolinium Enhancement (EGE) and Cardiac Functional Parameters in Cine-Magnetic Resonance Imaging (MRI) on Artificial Intelligence in Patients with Acute Myocarditis: A Case-Controlled Observational Study.

BACKGROUND The diagnosis of myocarditis is challenging, and the treatment is generally delayed due to misdiagnosis or missed diagnosis. Endomyocardial biopsy (EMB) is not a specific or sensitive method. A case-controlled observational study was conducted to evaluate early gadolinium enhancement (EGE) and left ventricular functional parameters on Artificial Intelligence in cine-MRI in patients with acute myocarditis. MATERIAL AND METHODS We selected 21 patients with pathologically proven acute myocarditis. We analyzed the EGE findings (total/serial number and location of positive-segments using the 17-segment model according to the American Heart Association) and clinical characteristics (symptoms, arrhythmias in ECG, coronary angiography, and EMB). All patients were divided into positive EGE and negative EGE groups to analyze left ventricular functional parameters (LVEF, FS, LVEDD, LVEDV, LVESV, LVMM, LVSV, CO, and CI) on Artificial Intelligence. RESULTS We enrolled 21 patients (11 males) with a mean age of 32.6±9.8 years (range, 16 to 51 years). Abnormalities on EGE were found in 2/3 of patients, involving 41 segments among multiple locations on the myocardium. The differences in LVEF (40.2±10.2% vs. 51.3±3.6%), LVESV (69.0±16.1ml vs. 52.5±10.6ml) and LVSV (42.6±11.4 vs. 52.8±2.8 ml) on Artificial Intelligence was statistically significant between the positive EGE and negative EGE groups (p<0.05). CONCLUSIONS Our results suggest a significant role of EGE on the basis of Lake Louise criteria in evaluating patients with clinical suspicion of acute myocarditis. Parameters, including LVEF, LVESV, and LVSV, on Artificial Intelligence, may be useful independent predictors for capillary leakage and microcirculatory disturbance in myocarditis.

Inactivated Sendai virus suppresses murine melanoma growth by inducing host immune responses and down-regulating ß-catenin expression.

OBJECTIVE: This paper aims to investigate the anti-tumor mechanism of inactivated Sendai virus (Hemagglutinating virus of Japan envelope, HVJ-E) for murine melanoma (B16F10). METHODS: The murine dendritic cells (DCs) were treated with HVJ-E, and then the cytokines secreted from DCs and costimulation-related molecules on DCs were measured. Meanwhile, the expression of ß-catenin in HVJ-E treated murine melanoma cells was detected. In addition, HVJ-E was intratumorally injected into the melanoma on C57BL/6 mice, and the immune cells, CTL response and tumor volume were analyzed. RESULTS: HVJ-E injected into B16F10 melanoma obviously inhibited the growth of the tumor and prolonged the survival time of the tumor-bearing mice. Profiles of cytokines secreted by dendritic cells (DCs) after HVJ-E stimulation showed that the number of cytokines released was significantly higher than that elicited by PBS (1P<0.05). The co-stimulation-related molecules on DCs were comparable to those stimulated by LPS. Immunohistochemical examinations demonstrated the repression of ß-catenin in B16F10 melanoma cells after HVJ-E treatment. Meanwhile, real-time reverse transcription PCR revealed that HVJ-E induced a remarkable infiltration of CD11c positive cells, chemokine ligand 10 (CXCL10) molecules, interleukin-2 (IL-2) molecule, CD4(+) and CD8(+) T cells into HVJ-E injected tumors. Furthermore, the mRNA expression level of ß-catenin in the HVJ-E injected tumors was also down-regulated. In addition, B16F10-specific CTLs were induced significantly after HVJ-E was injected into the tumor-bearing mice. CONCLUSION: This is the first report to show the effective inhibition of melanoma tumors by HVJ-E alone and the mechanism through which it induces antitumor immune responses and regulates important signal pathways for melanoma invasion. Therefore, HVJ-E shows its prospect as a novel therapeutic for melanoma therapy.

[TNFR-Fc prevents lipopolysaccharide-induced acute lung injury in mice via oxidative stress inhibition].

OBJECTIVE: Acute lung injury (ALI) was associated with excessive inflammation caused by high TNF-α concentration. We hypothesized that anti-TNF-α therapy would have beneficial effects in experimental ALI in mice via oxidative stress inhibition. METHODS: BALB/c mice (8 - 10 week old) were randomly assigned to an LPS group, a TNFR-Fc + LPS group and a control group. LPS (5 mg/kg) was intratracheally administered to the mice. For the TNFR-Fc + LPS group, TNFR-Fc was given intraperitoneally once at a dose of 0.4 mg/kg before LPS treatment, at 0 and 2 h after LPS treatment, the mice were sacrificed. Wet to dry ratio of the lung and the protein concentration in BALF were measured. Lung histology was evaluated for lung injury. TNF-α concentration in serum, MDA and total anti-oxidative stability were assayed. Finally, the transcription level of iNOS, Nox1, Nox2, Nox4, XO and SOD were evaluated by qRT-PCR. RESULTS: TNFR-Fc treatment significantly decreased the serum TNF-α concentration (P < 0.001), reduced the score of lung histology and decreased the protein concentration in BALF (P < 0.001), but did not change the wet to dry ratio of the lung. The ALI mice treated with TNFR-Fc had a lower transcription level of Nox1, Nox2, Nox4 and XO (all P < 0.001). The total anti-oxidative stability of mice treated with TNFR-Fc was preserved better than those treated with LPS alone (P < 0.05). CONCLUSION: Treatment with TNFR-Fc significantly reduced LPS-induced ALI. The transcription level of Nox and XO was down-regulated by anti-TNF-α therapy, followed by oxidative stress attenuation.

Frontiers of COVID-19-related myocarditis as assessed by cardiovascular magnetic resonance.

Coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. In some patients, COVID-19 is complicated with myocarditis. Early detection of myocardial injury and timely intervention can significantly improve the clinical outcomes of COVID-19 patients. Although endomyocardial biopsy (EMB) is currently recognized as the 'gold standard' for the diagnosis of myocarditis, there are large sampling errors, many complications and a lack of unified diagnostic criteria. In addition, the clinical methods of treating acute and chronic COVID-19-related myocarditis are different. Cardiac magnetic resonance (CMR) can evaluate the morphology of the heart, left and right ventricular functions, myocardial perfusion, capillary leakage and myocardial interstitial fibrosis to provide a noninvasive and radiation-free diagnostic basis for the clinical detection, efficacy and risk assessment, and follow-up observation of COVID-19-related myocarditis. However, for the diagnosis of COVID-19-related myocarditis, the Lake Louise Consensus Criteria may not be fully applicable. COVID-19-related myocarditis is different from myocarditis related to other viral infections in terms of signal intensity and lesion location as assessed by CMR, which is used to visualize myocardial damage, locate lesions and quantify pathological changes based on various sequences. Therefore, the standardized application of CMR to timely and accurately evaluate heart injury in COVID-19-related myocarditis and develop rational treatment strategies could be quite effective in improving the prognosis of patients and preventing potential late-onset effects in convalescent patients with COVID-19.

Identification of the major photodegradant in metronidazole by LC-PDA-MS and its reveal in compendial methods.

Metronidazole in aqueous solution is sensitive to light and UV irradiation, leading to the formation of N-(2-hydroxyethyl)-5-methyl-l,2,4-oxadiazole-3-carboxamide. This is revealed here by liquid chromatography with tandem photo diode array detection and mass spectrometry (LC-PDA-MS) and further verified by comparison with the corresponding reference substance and proton nuclear magnetic resonance (1H-NMR). However, in current compendial tests for related substances/organic impurities of metronidazole, the above photolytic degradant could not be detected. Thus, when photodegradation of metronidazole occurs, it could not be demonstrated. In our study, an improved LC method was developed and validated, which includes a detection at a wavelength of 230 nm and optimization of mobile phase composition thereby a better separation was obtained.

Study on the preventive effect of intravenous esomeprazole in the management of nonvarices upper gastrointestinal bleeding.

ABSTRACT: This retrospective study investigated the preventive effect of intravenous esomeprazole (IVEO) in the prevention of nonvarices upper gastrointestinal bleeding (NUGIB).This study enrolled 130 patients with NUGIB and all of them underwent successful endoscopic hemostasis, of which 65 cases received routine management and IVEO (Group A) and the other 65 cases received routine management alone (Group B). The primary outcome (recurrent bleeding rate within 72-hour, 7-day, and 30-day), and secondary outcomes ((all-cause mortality, bleeding-related mortality, blood transfused, hospital stay (day), and incidence of adverse events)) were compared between 2 groups.Patients in the group A showed lower recurrent bleeding rate within 72-hour(P < .05), 7-day (P < .05), and 30-day (P < .05), than that of patients in the group B. However, no significant differences were identified in all-cause mortality(P = .26), bleeding-related mortality (P = .57), blood transfused (P = .33), and hospital stay (P = .74) between 2 groups. In addition, both groups had similar safety profile.This study found that routine management and IVEO was superior to the routine management alone for preventing the recurrent bleeding rate after successful endoscopic hemostasis in patients with NUGIB.

The 4D B-spline method of calculating left ventricular functional parameters of cardiac MRI to evaluate myocardial injury of the apical segment in patients with myocarditis: a case-controlled observational study.

BACKGROUND: Myocarditis does not have typical clinical manifestations and thus is difficult to accurately diagnose by virtue of infection history, and electrocardiogram (EKG) and peripheral blood abnormalities. Endomyocardial biopsy is the gold standard for diagnosis of myocarditis, but is invasive, high risk, and has an observational blind area. Cardiac magnetic resonance imaging (CMRI) is multiparameter and multidirectional with high spatial resolution and high contrast of soft tissue. However, the optimal method of calculating left ventricular (LV) function in patients with apical-segment-injured myocarditis is unresolved. We compared and analyzed the differences between two different methods (Simpson and 4D B-spline surface model (known as the 4D method)) of measuring LV function by CMRI in patients with myocarditis in the 17th segment of the left ventricle. METHODS: The basic clinical data of two groups (myocarditis and non-myocarditis) were statistically analyzed, and differences in the LV function parameters by the two imaging methods were compared in the myocarditis group. Receiver-operating characteristic curves of single parameters and combined parameters based on the Simpson and 4D methods were drawn and the area under the curve, diagnostic threshold, maximum sensitivity interval, and maximum specificity interval were calculated. RESULTS: In the myocarditis and non-myocarditis groups the respective number of patients was 22 and 17, the percentage of males was 54.55% and 47.06%, and the average age was 32.20±11.59 and 43.06±11.62 years. The difference in LV ejection fraction (LVEF) (P=0.033) and LV end systolic volume (LVESV) (P=0.030) in the myocarditis group was statistically significant. The respective AUCs based on the Simpson and 4D methods were LVEF 0.602 vs. 0.778, LVESV 0.556 vs. 0.751, LVEF-and-LVESV 0.634 vs. 0.775. Based on the 4D method, the diagnostic thresholds of LVEF and LVESV were 34.965 (sensitivity 0.882, specificity 0.591) and 69.090 (sensitivity 0.727, specificity 0.706), the maximum sensitivity intervals of LVEF and LVESV were (24.610, 27.450) and (35.355, 37.200), and the maximum specificity intervals of LVEF and LVESV were (60.530, 65.625) and (91.625, 95.835), respectively. CONCLUSIONS: Compared with the Simpson method, the 4D method might be more effective for CMRI diagnosis of apical-segment-injured myocarditis. When the Simpson method is used, LVEF combined with LVESV is recommended for comprehensive evaluation to improve diagnostic efficiency. When the 4D method is used, LVEF might be the preferred parameter for evaluation of LV function.

Floral-like enhancement might reflect an active liver tuberculous lesion to avoid systemic hematogenous dissemination by surgery: A case report.

After primary dissemination of Mycobacterium tuberculosis bacillus infection that is localized in liver, disease progression and changes to immune function in the body occur. Various forms of tuberculosis, including granuloma, caseous necrosis, liquefactive necrosis, fibrosis, and calcification, occur that could be presented at different stages, and imaging examination shows findings that are consistent with these stages. Not all liver tuberculosis patients are suitable for liver resection, and preoperative imaging examination and pathological immunohistochemical results could be used to determine whether tuberculosis was active, avoid unnecessary liver resection, and prevent the postoperative spread of tuberculosis. Here, we reported a case of miliary tuberculosis, pelvic tuberculosis, and tuberculous abscess of the thigh muscle in a 51-year-old man after liver lesion resection. The liver lesion was confirmed to be tuberculosis by surgical pathology, which is rare and has not been previously reported. The purpose of this case report is to remind radiologists of the importance of the floral-like enhancement and to estimate whether liver tuberculosis is active. This will help to guide clinicians to determine the timing of surgery, avoid unnecessary liver resection, and avoid hematogenous transmission.

LGE-MRI in the Assessment of Left-ventricular Remodelling in Myocarditis.

BACKGROUND: The exact morbidity of myocarditis is unknown, as the treatment is generally delayed in virtue of misdiagnosis or missed diagnosis. AIM: The aim of this study was to identify prognostic factors of left-ventricular remodeling on CMRI performed in patients with pathological proven myocarditis. METHODS: Sixty-two cases with various presentations of myocarditis (39 cases with heart failure; 23 cases with arrhythmias) were selected. All patients, who underwent coronary angiography, endomyocardial biopsy, were divided into positive-remodeling and negative-remodelling groups to analyse LGE and cardiac cine parameters at presentation and subsequent to 3 months. RESULTS: Comparison of two subgroups in CMRI is as follows: positive LGE (65.6% vs. 86.7%; p<0.05), LVEF (41.3±14.8% vs. 37.6±10.1%; p=0.62), (25.7±2.0% vs. 24.0±2.5%; p=0.81), (44.5±3.9mm vs. 46.3±5.4mm; p=0.76), (129.1±8.5ml vs. 135.3±12.2ml; p=0.26), (74.8±7.3ml vs. 79.1±10.0ml; p=0.55), (52.0±5.7g vs. 49.6±6.5g; p=0.71), (34.9±3.5ml vs. 32.4±6.2ml; p=0.68), (3.8±0.7L/min vs. 3.1±0.5L/min; p=0.64), (2.9±0.6L/min*m2 vs. 2.7±0.5L/min*m2; p=0.79). CONCLUSION: LGE-MRI is rewarding as an independent predictor in left-ventricular positive and negative remodelling of myocarditis.

Molecular epidemiological and phylogenetic analyses of canine parvovirus in domestic dogs and cats in Beijing, 2010-2013.

Fifty-five samples (15.62%) collected from dogs and cats were identified as canine parvovirus (CPV) infection in Beijing during 2010-2013. The nucleotide identities and aa similarities were 98.2-100% and 97.7-100%, respectively, when compared with the reference isolates. Also, several synonymous and non-synonymous mutations were also recorded for the first time. New CPV-2a was dominant, accounting for 90.90% of the samples. Two of the 16 samples collected from cats were identified as new CPV-2a (12.5%), showing nucleotide identities of 100% with those from dogs. Twelve samples (15.78%) collected from completely immunized dogs were found to be new CPV-2a, which means CPV-2 vaccines may not provide sufficient protection for the epidemic strains.

[Gefitinib inhibits α-smooth muscle actin expression in mice with bleomycin-induced lung fibrosis].

OBJECTIVE: To evaluate the effect of epidermal growth factor receptor tyrosine kinase inhibitor, gefitinib, on the expression of α-smooth muscle actin (α-SMA) in mice with lung fibrosis induced by bleomycin. METHODS: Thirty male BALB/c mice were randomly divided into control, bleomycin, and bleomycin plus gefitinib groups. The mice in the control group were subjected to intratracheal administration of normal saline, those in bleomycin group received bleomycin (3 mg/kg) intratracheally, and those in bleomycin plus gefitinib group received oral gefitinib (20 mg/kg administering) plus intratracheal bleomycin administration. All the mice were sacrificed 14 days after the treatments, and the left lung was examined pathologically with HE staining and Masson staining and also immunohistochemically for assay of the total EGFR, phosphorylated EGFR and α-SMA. The right lungs were sampled for RT-PCR to detect the mRNA levels of α-SMA. RESULTS: Gefitinib administration lessened lung fibrosis induced by bleomycin and significantly reduced lung collagen accumulation. The phosphorylation of EGFR in the pulmonary mesenchymal cells and epithelial cells and the expression levels of α-SMA mRNA and protein were inhibited by gefitinib treatment in mice with intratracheal administration of bleomycin (P<0.05). CONCLUSION: Gefitinib offers protection against lung fibrosis induced by bleomycin in mice probably by inhibiting the downstream signals of EGFR and by downregulating the expression of α-SMA.

[Construction and identification of recombinant avian adeno-associated virus expressing GFP reporter gene].

To generate recombinant avian adeno-associated virus (rAAAV) for gene transfer studies in avian cells, the recombinant plasmid containing the whole genome of AAAV was digested with restriction enzymes to remove the Rep and Cap genes, resulting in AAAV transfer vector pAITR. GFP-expressing cassette was amplified by PCR and inserted into the AAAV transfer vector. The Rep-Cap gene of AAAV amplified by high fidelity PCR was subcloned into eukaryotic expression vector pcDNA3, resulting in an AAAV helper vector pcDNA-ARC. The Rep and Cap genes amplified by high fidelity PCR were subcloned separately into the co-expression vector pVITRO2-mcs, resulting in another AAAV helper vector pVITRO2-ARC. Using calcium phosphate precipitation method, rAAAV-GFP was generated by co-transfecting AAV-293 cells with a cocktail of pAITR-GFP, pcDNA-ARC or pVITRO2-ARC, and adenovirus helper vector pHelper. The three structural proteins VP1, VP2 and VP3 of correct molecular masses were detected by SDS-PAGE and the GFP reporter gene was detected by PCR in purified rAAAV-GFP virions. Chicken embryonic fibroblast (CEF) cells and CEL cell line were transduced with the recombinant virus, the GFP-positive cells were easily observed under fluorescent microscope, expression of which lasted for at least two weeks. These data demonstrate that an efficient helper virus-free packaging system has been established for generating recombinant AAAV particles for gene transfer studies in avian cells and for development of recombinant vaccines against avian diseases.