Optimizing inhalation therapy in the aspect of peak inhalation flow rate in patients with chronic obstructive pulmonary disease or asthma.

BACKGROUND: Pressurized metered dose inhalers (pMDIs) and dry powder inhalers (DPIs) are commonly used drug-delivering devices for patients with chronic airway diseases. Appropriate peak inhalation flow rate (PIFR) and inhaler technique is essential for effective therapy. We aimed at optimizing inhalation therapy through the analysis of PIFRs in patients with chronic obstructive pulmonary disease (COPD) or asthma as well as the effect of technique training using In-Check DIAL® to help patients to achieve their optimal inspiratory flow rates. METHODS: The study continuously enrolled patients who were diagnosed as COPD or asthma from respiratory clinics. PIFRs were described and analyzed between the newly-diagnosed and follow-up patients, and the stable and acute exacerbation patients, respectively. Every participant was trained inhaler technique using In-Check DIAL®. PIFRs before and after training was compared by self-control analysis. RESULTS: Among a total of 209 patients, the average age was 56.9 years. For DPIs users, 10.8% patients had a PIFR < 30 L/min and 44.1% patients had a PIFR ≥ 60 L/min before technique training. After technique training, scarcely patient (1.5%) had a PIFR < 30 L/min, and 60.5% patients had a PIFR ≥ 60 L/min. The patient's average PIFR increased by 5.6L/min after training. The increase in PIFR before and after training was significant (p < 0.001) for most patients, but no significant variation was found in patients with acute exacerbation (p = 0.822). CONCLUSIONS: A considerable number of patients with COPD or asthma were not able to achieve the minimum or optimal PIFR for DPIs. Inhaler training can increase patients' PIFRs and improve their ability to use DPIs. Trail registration The study has registered in chictr.org.cn (ChiCTR1900024707) and been approved by the Ethics Committee of Zhongshan Hospital of Fudan University (B2019-142).

A randomized trial in the investigation of anxiety and depression in patients with coronavirus disease 2019 (COVID-19).

BACKGROUND: In March 2020, the World Health Organization (WHO) declared COVID-19 a public health emergency of international concern. A small proportion of patients infected with COVID-19 go on to develop pneumonia. We speculated that COVID-19 may be likely to result in psychological disorders such as anxiety and depression. In this study, we conducted an investigation of anxiety and depression in patients with COVID-19. METHODS: Sixty-five COVID-19 patients were randomly enrolled into this study. Anxiety and depression among participants were measured through the completion of anonymous Chinese-language Zung self-rating anxiety scale and self-rating depression scale questionnaires. Data were analyzed using independent samples t-tests, Mann-Whitney U-tests, and χ2 tests. RESULTS: The questionnaire results showed that 26.15% and 41.54% of participants suffered from anxiety and depression, respectively, although there was no significantly statistical difference between the proportions of COVID-19 patients with anxiety and depression. Statistically significant differences in employment status, partial pressure of oxygen, and corticosteroid application existed between moderate- and severe COVID-19 patients (P<0.05). In particular, the partial pressure of oxygen was significantly lower in severe COVID-19 patients than in their moderate counter parts (71.31±23.54 vs. 101.06±34.43, U=156, P=0.006). Total lymphocytes was lower in severe group than in moderate group [1.659±0.643 vs. 0.745 (0.645, 0.928), U=109, P=0.000]. Also, a higher proportion of female than male patients had anxiety (χ2=5.388, P=0.02). COVID-19 patients who received antiviral medications also displayed a higher rate of anxiety (χ2=4.481, P=0.034). Total lymphocytes between the non-anxiety and anxiety had statistical difference (U=321, P=0.019). Meanwhile, total lymphocytes between the non-depression and depression also had statistical difference (U=389.5, P=0.01). CONCLUSIONS: Among patients with COVID-19, females and those treated with antiviral medications were more likely to experience anxiety. In addition, our findings reflected the effect of anxiety and depression on immune system.

Epidemiology and issues of NIV-treated AECOPD patients with hypercapnic respiratory failure in Shanghai: A multicentre retrospective survey.

OBJECTIVE: To investigate the epidemiology, clinical features, treatment and outcome of Noninvasive ventilation (NIV)-treated acute exacerbation of chronic obstructive pulmonary disease (AECOPD) patients in secondary hospitals of Shanghai. METHOD: Relying on Shanghai alliances for respiratory diseases, a retrospective observational study was performed in 34 secondary hospitals of Shanghai. The AECOPD patients treated with NIV and admitted to the respiratory department or respiratory intensive care unit were recruited between December 1, 2016, and November 30, 2017. RESULTS: There were 555 patients finally recruited in this study. The age was 75.8 ± 9.6 years old and 380 patients (68.5%) were male. 70.5% of all patients had respiratory acidosis (pH <7.35). 55.3% of all patients received nebulised bronchodilator and 77.7% were treated with systemic or inhaled corticosteroids during hospitalisation. 525 patients (94.6%) recovered successfully and the mortality was 3.2%. The hospitalisation was 15.3 ± 6.7 days and hospital expenses were 22 911 ± 13 595 RMB. Inadequate and nonstandard drug treatments were the most important problems during management. CONCLUSION: The NIV can be successfully used for AECOP patients in local hospitals of Shanghai, but accompanied by high costs and long hospital stays. However, the treatments for exacerbation and stable COPD patients are still insufficient.

Assessing risk factors for SARS-CoV-2 infection in patients presenting with symptoms in Shanghai, China: a multicentre, observational cohort study.

Background: The outbreak of COVID-19 has led to international concern. We aimed to establish an effective screening strategy in Shanghai, China, to aid early identification of patients with COVID-19. Methods: We did a multicentre, observational cohort study in fever clinics of 25 hospitals in 16 districts of Shanghai. All patients visiting the clinics within the study period were included. A strategy for COVID-19 screening was presented and then suspected cases were monitored and analysed until they were confirmed as cases or excluded. Logistic regression was used to determine the risk factors of COVID-19. Findings: We enrolled patients visiting fever clinics from Jan 17 to Feb 16, 2020. Among 53 617 patients visiting fever clinics, 1004 (1·9%) were considered as suspected cases, with 188 (0·4% of all patients, 18·7% of suspected cases) eventually diagnosed as confirmed cases. 154 patients with missing data were excluded from the analysis. Exposure history (odds ratio [OR] 4·16, 95% CI 2·74-6·33; p<0·0001), fatigue (OR 1·56, 1·01-2·41; p=0·043), white blood cell count less than 4 × 109 per L (OR 2·44, 1·28-4·64; p=0·0066), lymphocyte count less than 0·8 × 109 per L (OR 1·82, 1·00-3·31; p=0·049), ground glass opacity (OR 1·95, 1·32-2·89; p=0·0009), and having both lungs affected (OR 1·54, 1·04-2·28; p=0·032) were independent risk factors for confirmed COVID-19. Interpretation: The screening strategy was effective for confirming or excluding COVID-19 during the spread of this contagious disease. Relevant independent risk factors identified in this study might be helpful for early recognition of the disease. Funding: National Natural Science Foundation of China.

Initial diagnosis and management of adult community-acquired pneumonia: a 5-day prospective study in Shanghai.

BACKGROUND: Despite the release of a national guideline in 2016, the actual practices with respect to adult community-acquired pneumonia (CAP) remain unknown in China. We aimed to investigate CAP patient management practices in Shanghai to identify potential problems and provide evidence for policy making. METHODS: A short-period, 5-day prospective cross-sectional study was performed with sampled pulmonologists from 36 hospitals, encompassing all the administrative districts of Shanghai, during January 8-12, 2018. The medical information was recorded and analyzed for the patients with the diagnosis of CAP who were cared for by 46 pulmonologists during the study period. RESULTS: Overall, 435 patients were included in the final analysis, and 94.3% had a low risk of death in terms of CRB-65 criteria (C: disturbance of consciousness, R: respiratory rate, B: blood pressure, 65: age). When diagnosed with CAP, 70.1% of patients were not evaluated using the CURB-65 score (CRB-65 + U: urea nitrogen), but most patients (95.4%) were evaluated using CRB-65. Time to achieve clinical stability was longer in patients with hypoxemia than in those without hypoxemia (8.42±6.36 vs. 5.53±4.12 days, P=0.004). Overall, 84.4% of patients with a CRB-65 score of 0 were administered antibiotics intravenously, and 19.4% were still hospitalized after excluding hypoxemia and comorbidities. The average duration of antibiotic treatment was 10.4±4.9 days. Overall, 72.6% of patients received antibiotics covering atypical pathogens whose time to clinical stability was significantly shortened compared with those without coverage, but the antibiotic duration was similar and not correspondingly shortened. CONCLUSIONS: CRB-65 seems to be more practical than CURB-65 for the initial evaluation of CAP in the context of local practice, and oxygenation assessment should be included in the evaluation of severity. Overtreatment may be relatively common in patients at low risk of death, including unreasonable hospitalization, intravenous administration, and antibiotic duration.

Effect of PIFR-based optimised inhalation therapy in patients recovering from acute exacerbation of chronic obstructive pulmonary disease: protocol of a prospective, multicentre, superiority, randomised controlled trial.

INTRODUCTION: Acute exacerbation (AE) is a major cause of disease progression and death in patients with chronic obstructive pulmonary disease (COPD), accounting for majority of medical expenditures. Correct inhalation therapy is effective in preventing AE attacks. However, inappropriate usage of dry powder inhaler, partially due to the unrecovered peak inhalation flow rate (PIFR) after acute exacerbation of COPD (AECOPD), results in increased risk of early treatment failure. Therefore, we designed a multicentre, randomised clinical trial to determine whether PIFR-based optimised inhalation therapy and training on inhaler usage at discharge could effectively reduce early treatment failure events. METHODS AND ANALYSIS: A total of 416 hospitalised patients just recovering from AECOPD will be recruited and equally randomised into the PIFR group and the control group at a 1:1 ratio. The PIFR group will receive additive support before discharge, including choice of PIFR-guided inhaler and education on its usage. PIFR is measured by InCheck DIAL. In comparison, the control group will receive inhalers based on judgement of the respiratory physician. The primary outcome of the study is 30-day treatment failure rate. Other endpoints include PIFR, error rate of inhalation device use, satisfaction with inhalation devices, 30-day mortality, 90-day mortality, symptoms and quality of life of patients, and COPD-related treatment costs. ETHICS AND DISSEMINATION: The trial has been approved by the Ethics Committee of Zhongshan Hospital of Fudan University (B2019-142). Participants will be screened and enrolled from hospitalised patients with AECOPD by clinicians, with no public advertisement for recruitment. After the trial has completed, the results will be reported to the public through conference presentations and peer-reviewed journals. TRIAL REGISTRATION NUMBER: NCT04000958.

Increased catabolism of nucleic acid in nonalcoholic fatty liver disease patients of different ages.

Uric acid is the end product of nucleic acid catabolism for humanity. Serum uric acid level has been suggested to be associated with nonalcoholic fatty liver disease (NAFLD). This study aims to examine the association between serum uric acid levels and NAFLD in subjects of different ages. A cross-sectional study was performed among the patients of different ages with NAFLD (abnormal group) or without NAFLD (normal group) in Lianyungang, Jiangsu, China. The levels of serum uric acid, total cholesterol, triglyceride, and low density lipoprotein in the abnormal group were significantly higher than those in the normal group, while the level of high density lipoprotein in the abnormal group was significantly lower than that in the normal group (p<0.05). The serum uric acid prevalence, total cholesterol, triglyceride, high density lipoprotein, and low density lipoprotein in the abnormal group was significantly higher than those in the normal group (p<0.05). The serum uric acid, total cholesterol, triglyceride, and low density lipoprotein levels and prevalence rates in elderly patients were higher than younger and middle aged patients. Serum uric acid level is highly associated with NAFLD in patients of different ages.

Preparation and antibacterial activity of oligosaccharides derived from dandelion.

In this study, we prepared oligosaccharides from dandelion (Taraxacum officinale) by hydrolysis with hydrogen peroxide (H2O2) and investigated their antibacterial activity. The optimum hydrolysis conditions, as determined using the response surface methodology, were as follows: reaction time, 5.12h; reaction temperature, 65.53 °C and H2O2 concentration, 3.16%. Under these conditions, the maximum yield of the oligosaccharides reached 25.43%. The sugar content in the sample was 96.8%, and the average degree of polymerisation was approximately 9. The oligosaccharides showed high antibacterial activity against Escherichia coli, Bacillus subtilis and Staphylococcus aureus, indicating that dandelion-derived oligosaccharides have the potential to be used as antibacterial agents.

Up-regulation of cyclin D1 expression in asthma serum-sensitized human airway smooth muscle promotes proliferation via protein kinase C alpha.

Abnormal hypertrophy and hyperplasia of airway smooth muscle cells play an important role in airway remodeling in chronic asthma. The authors' previous studies have indicated that protein kinase C alpha (PKC alpha) is involved in the proliferation of passively sensitized human airway smooth muscle cells (HASMCs). However, the underlying mechanisms remain unknown. Here, the authors examined the possible role of the alpha isoform of PKC in the control of cyclin D1 expression, using HASMCs passively sensitized on human atopic asthmatic serum as a model system. Cultured HASMCs were passively sensitized with serum from atopic asthmatic patients. Cell proliferation was measured by [(3)H]thymidine incorporation and an MTT assay. Cell cycle status was analyzed by flow cytometry. The mRNA and protein expression profiles of cyclin D1 were measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blotting, respectively. Furthermore, the authors assessed the role of cyclin D1 in PKC alpha-induced HASMC proliferation by transfection with a recombinant cyclin D1 antisense construct. The activation of PKC alpha with phorbol myristate acetate (PMA), a PKC activator, up-regulated cyclin D1 expression and increased the proliferation of passively sensitized HASMCs. This effect was significantly decreased by specific inhibition of PKC alpha with Go6976. In addition, the authors showed that transfection with antisense cyclin D1 abolished PMA-induced G1/S progression and HASMC proliferation. These results demonstrate that PKC alpha promotes the proliferation of HASMCs sensitized with atopic asthmatic serum via up-regulation of cyclin D1 expression.

Role of protein kinase C alpha and cyclin D1 in the proliferation of airway smooth muscle in asthmatic rats.

BACKGROUND: Airway smooth muscle (ASM) is suspected to be a determining factor in the structural change of asthma. However, the role of protein kinase C alpha (PKCalpha) and cyclin D1 involved in the dysfunction of ASM leading to asthmatic symptoms is not clear. In this study, the central role of PKCalpha and cyclin D1 in ASM proliferation in asthmatic rats was explored. METHODS: Thirty-six pathogen-free male Brown Norway (BN) rats were randomly divided into 2 groups: control groups (group N1, N2 and N3) and asthmatic groups (group A1, A2, and A3). Groups A1, A2 and A3 were challenged with ovalbumin (OA) for 2 weeks, 4 weeks and 8 weeks respectively. Control animals were exposed to an aerosolized sterile phosphate buffered saline (PBS). The ASM mass and nucleus numbers were studied to estimate the degree of airway remodeling by the hematoxylin-eosin staining method. PKCalpha and cyclin D1 expression in the ASM cells was detected by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemistry. The relation between PKCalpha and cyclin D1 was assessed by linear regression analysis. PKC agonist phorbol 12-myristate 13-acetate (PMA), PKC inhibitor Ro31-8220 and an antisense oligonucleotide against cyclin D1 (ASOND) were used to treat ASM cells (ASMCs) obtained from the 2 weeks asthmatic rats. The cyclin D1 protein expression level was detected by Western blotting. RESULTS: Compared with the control group, the PKCalpha and cyclin D1 mRNA levels were increased in the asthmatic group. Similar to RT-PCR results, immunohistochemistry analysis for PKCalpha and cyclin D1 expression revealed an increased production in ASMCs after allergen treatment for 2, 4 and 8 weeks compared with the respective control groups. No difference in expression of PKCalpha and cyclin D1 in ASM were found in the 2, 4 or 8 weeks asthmatic rats. There were significant positive correlations between PKCalpha and cyclin D1 expression, both transcriptionally (r = 0.944, P < 0.01) and translationally (r = 0.826, P < 0.01), in ASM. The content of cyclin D1 in asthmatic ASMCs increased after being stimulated by PMA, and decreased when induced by Ro31-8220. ASOND targeting for cyclin D1 lowered the expression of cyclin D1 induced by PMA. CONCLUSIONS: Increased expression of PKCalpha and cyclin D1 in ASM along with smooth muscle structure changes might implicate PKCalpha and cyclin D1 participation in the proliferation of ASM and contribute to the pathogenesis of asthma after repeated allergen exposure in rats. The results suggested that cyclin D1 might be downstream of PKC signal transduction pathway.

[Recombination and identification of sense and antisence CyclinD1 eukaryotic expression vectors and the effects of the vectors on the proliferation of airway smooth muscle cell in asthmatic rats].

This study is to investigate the expression of CyclinD1 in asthmatic rats and construct expression plasmids of sense and antisense CyclinD1 gene and transfect them to asthmatic airway smooth muscle cell to study the effects of CyclinD1 on the proliferation of airway smooth muscle cells in asthmatic rats. CyclinD1 cDNA was obtained by RT-PCR of total RNA extracted from the airway smooth muscle in asthmatic rats. The sequence was inserted into eukaryotic expression vector pcDNA3.1 (+) to recombinate the sense and antisense pcDNA3.1-CyclinD1 eukaryotic expression vector. The two recombinations and vector were then separately transfected into airway smooth muscle cell in asthmatic rats by using liposome. The expression level of CyclinD1 was certificated by Western blotting analysis. The proliferations of ASMCs isolated from asthmatic rats were examined with cell cycle analysis, MTT colorimetric assay and proliferating cell nuclear antigen (PCNA) immunocytochemical staining. Results showed (1) Compared with control group, the content of CyclinD1 was significantly increased; (2) It was comformed by restriction endonucleasa digestion and DNA sequence analysis that the expression plasmid of sense and antisense CyclinD1 were successfully recombinated. There was significant change of CyclinD1 expression between vector and sense CyclinD1 transfected cells, and the expression level of CyclinD1 in ASMC transfected with antisense CyclinD1 was lower than that in vector transfected cells (P <0.01); (3) In the asthmatic groups, compared with the vecter group, the percentage of S + G2M phase, absorbance A value of MTT and the expression rate of PCNA protein in ASMC transfected with pcDNA3. 1-CyclinD1 vector significantly increased. The values decreased remarkably in the pcDNA3,1-as CyclinD1 group. Statistical analysis revealed that there were significant differences in these indicators of cell proliferation in three groups (P <0.01). In the normal groups, statistical analysis revealed that there were significant differences in the percentage of S + G2M phase, a value of MTT and the expression rate of PCNA protein in three groups (P <0.01). Sense CyclinD1 eukaryotic expression vectors could have a positive effect on the proliferation of ASMC, however the antisence one have a negative effect, which implicated that CyclinD1 might contribute to the process of airway smooth muscle cell proliferation.

[Inhibition of NF-kappaB through IkappaBalpha transfection affects invasion of human lung cancer cell line A549].

BACKGROUND & OBJECTIVE: Recent studies have shown that activation of nuclear factor-kappaB(NF-kappaB) can regulate the invasion and metastasis of cancer cells. The present study was to investigate inhibition of NF-kappaB activity on invasion of human lung cancer cell line A549 and the possible mechanism. METHODS: The recombinant plasmid pcDNA3.1(+)/IkappaBalpha, expressing the alpha isoform (IkappaBalpha) of the NF-kappaB inhibitor, was constructed. A549 cells were cultured in vitro and divided into non-transfection group, pcDNA3.1(+) transfected group and pcDNA3.1(+)/IkappaBalpha transfected group. The expression of IkappaBalpha was detected by reverse transcription-polymerase chain reaction (RT-PCR) and Western blotting. The activity of NF-kappaB was determined by electrophoretic mobility shift assay (EMSA). Invasion of A549 cells was assessed by transwell chamber assay. The expression of MMP-2 and MMP-9 was detected by RT-PCR and gelatin zymography. RESULTS: Plasmid pcDNA3.1(+)/IkappaBalpha was successfully constructed and expressed in A549 cells. The activity of NF-kappaB, the number of invasive cells, the activity of MMP-2 and MMP-9 of A549 cells in pcDNA3.1(+)/IkappaBalpha transfected group were significantly lower than those in non-transfection group and pcDNA3.1(+) transfected group (all P<0.05). CONCLUSION: Transfection of IkappaBalpha can inhibit NF-kappaB activity, thus inhibit cell invasion of A549, which may be through the down-regulation of MMP-2 and MMP-9 expressions.

PKC promotes proliferation of airway smooth muscle cells by regulating cyclinD1 expression in asthmatic rats.

AIM: To determine whether protein kinase C (PKC) has any effect on the expression of cyclinD1, a key regulator of growth control and G1/S transition, and to investigate the underlying molecular mechanisms of PKC involving the remodeling of the asthmatic airway smooth muscle (ASM). METHODS: The treatment of synchronized ASM cells from asthmatic rats with PKC-specific agonist phorbol 12-myristate 13-acetate (PMA) and antagonist 2-{1-[3-(amidinothio) propyl]-1Hindol-3-yl}-3-(1-methylindol-3-yl) maleimide methanesulfonate salt (Ro31-8220) was followed by the proliferation assay. PKCalpha and cyclinD1 expressions in ASM cells (ASMC) were detected by RT-PCR and Western blotting. The relation between PKCalpha and cyclinD1 was assessed by linear regression analysis. The effect of the construct recombinant plasmid pcDNA3.1-antisense cyclinD1 (pcDNA3.1-ascyclinD1) on the proliferation of ASMC was found to be induced by PMA. RESULTS: The data showed phorbol ester-dependent PKCalpha promoted the proliferation of ASMC. The closely-positive correlation existed between the expression of PKCalpha and cyclinD1 at the transcriptional (r=0.821, P<0.01) and translational (r=0.940, P<0.01) levels. pcDNA3.1-ascyclinD1 could inhibit the proliferation of ASMC. pcDNA3.1-ascyclinD1 almost completely attenuated the PMA-induced proliferation effect as Ro31-8220+pcDNA3.1. CONCLUSION: The proliferation of ASMC by PKC might by regulated by the cyclinD1 expression in asthmatic rats.

[PKCalpha-ERK1/2 cascade is involved in up-regulation of cyclinD1 and P21(cip1) in human airway smooth muscle cells sensitized by sera from atopic asthmatics].

OBJECTIVE: To explore the role of PKCalpha-ERK1/2 cascade in PMA induced up-regulation of cyclinD1 and P21(cip1) in human airway smooth muscle cells (HASMCs) sensitized by sera from atopic asthmatics. METHODS: HASMCs in cultures were passively sensitized by 10% serum from asthmatic patients and were randomly divided into five groups: the control group, PMA treated group, PMA and PKCalpha mismatched Oligodeoxynucleotides (PKCalpha-mmODN) treated group, PMA and PKCalpha antisense Oligodeoxynucleotides (PKCalpha-asODN) treated group, PMA and U0126 (MAP Kinase Kinase inhibitor)treated group. The expression of p-PKCalpha, ERK1/2, p-ERK1/2, cyclinD1 and P21(cip1) protein were determined by western blotting. The proliferation of HASMC was examined by cell cycle analysis and MTT colorimetric assay. RESULTS: Compared with the control group, the expression of p-PKCalpha and ERK1/2, p-ERK1/2 protein increased, the expression of cyclinD1, P21(cip1) protein increased correspondingly (the A value % control was 2.10 +/- 0.29, 1.67 +/- 0.19, 2.20 +/- 0.27, 1.99 +/- 0.22 and 3.11 +/- 0.29 respectively; q value was 9.87, 7.06, 10.57, 11.10 and 20.33 respectively; all P < 0.05) in PMA treated group, and cells proliferation [the percentage of cells in S phase was (30.3 +/- 2.4)%, A(490) value was 0.80 +/- 0.06] enhanced significantly compared with those [the percentage of cells in S phase was (13.9 +/- 2.6)%, A(490) value was 0.41 +/- 0.04] of the control group (q = 6.07, 12.63; all P < 0.05). In PMA and PKCalpha-asODN treated group, the level of p-PKCalpha decreased, the expression of ERK1/2, p-ERK1/2 and the expression of cyclinD1, P21(cip1) decreased correspondingly (the A value % control was 1.23 +/- 0.19, 1.34 +/- 0.18, 1.52 +/- 0.20, 1.45 +/- 0.18 and 1.49 +/- 0.18 respectively; q value was 7.49, 3.58, 5.97, 6.06 and 15.65 respectively; all P < 0.05), and cells proliferation reduced significantly [the percentage of cells in S phase was (21.2 +/- 2.8)%, A(490) value was 0.51 +/- 0.04; q = 6.07, 12.63; all P < 0.05], as compared with those of the PMA treated group. In PMA and U0126 treated group, the level of p-PKCalpha had no significant change (A value was1.99 +/- 0.18, q = 0.94, P > 0.05), but the levels of ERK1/2, p-ERK1/2 decreased, the expression of cyclinD1, P21(cip1) reduced (the A value % control was 0.95 +/- 0.21, 1.15 +/- 0.19, 1.37 +/- 0.15 and 1.96 +/- 0.21 respectively; q value was 7.79, 9.16, 6.92 and 11.16 respectively; all P < 0.05), and cells proliferation reduced significantly [the percentage of cells in S phase was (22.0 +/- 3.2)%, A(490) value was 0.49 +/- 0.03; q = 5.51, 13.45; all P < 0.05], as compared with those of the PMA treated group. CONCLUSION: ERK1/2 is one of the downstream regulators of PKCalpha, and PKCalpha-ERK1/2 cascade is involved in PMA induced up-regulation of cyclinD1 and P21(cip1) and proliferation in HASMC sensitized by sera from atopic asthmatics.

[Structure-based design and biosynthesis of collagen proteins].

Collagen is the most abundant protein in human body and a periodic helix, i. e. , triple helix, fibrous protein, which provides the scaffold structures for the cell adhesion and macromolecule aggregation, etc. With the development of gene engineering and biomaterial technologies, and the incessant studies on the technique to obtain the proteins with special functions, the collagen protein has been one of the third generation biomaterials that attract more attention than others. In this paper, we reviewed the recent structure-based design and biosynthesis of collagen.

[Nuclear factor-kappaB activity and its correlation with cell proliferation in non-small cell lung cancer tissues].

OBJECTIVE: To investigate the nuclear factor (NF)-kappaB activity in human non-small cell lung cancer (NSCLC) tissues and to explore the correlation between NF-kappaB activity and cell proliferation, between NF-kappaB activity and cell spontaneous apoptosis. METHODS: Thirty samples of non-small cell lung cancer tissues and 15 normal lung tissues were collected from May to October in 2006. NF-kappaB activation was determined by electrophoretic mobility shift assay (EMSA). CyclinD1 level was examined by RT-PCR and Western blot. Proliferation cell nuclear antigen (PCNA) protein was examined by immunohistochemical analysis. Spontaneous cell apoptosis was determined by the TUNEL method. RESULTS: There was significant difference (F=78.96, P<0.01) in NF-kappaB activity among normal lung tissue group (24,826+/-3724), squamous-cell carcinoma tissue group (28,028+/-4204), and adenocarcinoma tissue group (35,425+/-5317). The NF-kappaB activity in the squamous-cell carcinoma group and the adenocarcinom tissue group was higher than that in the normal lung tissue group (all P<0.01); and the NF-kappaB activity is in the adenocarcinoma tissue group was higher compared with that in the squamous-cell carcinoma group (P<0.05). There was significant difference (F=62.43, P<0.01) in the cyclinD1 mRNA level among normal lung tissue group (2.04+/-0.24), the squamous-cell carcinoma group (2.91+/-0.37), and the adenocarcinoma group (4.13+/-0.36). There was significant difference (F=89.24, P<0.01) in cyclinD1 protein level among normal lung tissue group (0.31+/-0.06), the squamous-cell carcinoma group (0.43+/-0.07), and the adenocarcinoma group (0.58+/-0.08). There was significant difference (F=45.61, P<0.01) in PCNA protein level among the normal lung tissue group (0.32+/-0.09), the squamous-cell carcinoma group (0.42+/-0.10), and the adenocarcinima group (0.54+/-0.16). There was no significant difference (F=1.86, P>0.05) in apoptosis index among the normal lung tissue group (2.58%+/-0.39%), the squamous-cell carcinoma group (2.27%+/-0.34%), and the adenocarcinoma group (2.92%+/-0.59%). The NF-kappaB activity was positively correlated with cyclin D1 mRNA level, cyclin D1 protein level, and PCNA protein level in the squamous-cell carcinoma group (r=0.51, P<0.05, r=0.54, P<0.05, r=0.60, P<0.05), respectively; the NF-kappaB activity was also positively correlated with cyclinD1mRNA, cyclinD1protein level, and PCNA protein level in the adenocarcinoma group (r=0.60, P<0.05; r=0.64, P<0.05; r=0.68, P<0.05), respectively. The NF-kappaB activity in the squamous-cell group and the adenocarcinoma group was not related to cell apoptosis index. CONCLUSION: NF-kappaB activity increased in NSCLC tissues. Abnormal NF-kappaB activation may be associated with cell proliferation, but do not affect spontaneous cell apoptosis in NSCLC tissues.

[Effects of recombinant adeno-virus vector carrying interleukin-5 antisense on the expression of interleukin-5 mRNA and protein in CD4+ T lymphocytes of asthmatic rats].

OBJECTIVE: To construct recombinant adeno-associated virus vector carrying antisense interleukin-5 (IL-5) gene (rAAV-asIL-5), and to explore the effects of this virus transfection on IL-5 mRNA and protein in CD(4)(+) T lymphocytes of asthmatic rats. METHODS: The eukaryotic antisense IL-5 expressing vector plasmid of recombinant adeno-associated virus (pasIL-5/rAAV) was constructed by gene recombination technique. The rAAV-asIL-5 particles were produced by co-transfection of pasIL-5/rAAV, pXX2, and pXX6 in package cell 293 through phosphate calcium deposit, and the titers of rAAV-asIL-5 were measured by Southern blot. The rAAV-asIL-5 particles were transfected into CD(4)(+) T lymphocytes obtained by gradient of Ficoll and immunomagnetic beads from the peripheral blood of asthmatic rats. Then IL-5 mRNA in T lymphocytes and IL-5 protein in supernatant of cell culture were determined with semi-quantitative RT-PCR and ELISA respectively. RESULTS: (1) The rAAV-asIL-5 was constructed and identified, and the titer of rAAV-asIL-5 was 1.3 x 10(11) virus particles/ml. (2) The relative ratio A of absorbance (IL-5/beta-actin) of rAAV group was 1.0515 +/- 0.1477, which was significantly lower than that of the control group (1.4271 +/- 0.1655) (n = 6, P < 0.01). (3) The protein level of IL-5 in supernatant of culture of rAAV group was (12.0840 +/- 1.4769) ng/L, significantly lower than that of the control group [(15.3590 +/- 1.2685) ng/L, n = 6, P < 0.01]. CONCLUSION: Construction of rAAV-asIL-5 was successful, and transfection of this virus was capable of inhibiting the expression of IL-5 mRNA and protein in CD(4)(+) T lymphocytes of asthmatic rats. The results of this study provide experimental data for further study of gene therapy for asthma.