Effect of TLR7 gene expression mediating NF-κB signaling pathway on the pathogenesis of bronchial asthma in mice and the intervention role of IFN-γ.

OBJECTIVE: To explore the mechanism of TLR7 mediating NF-κB signaling pathway on the pathogenesis of bronchial asthma in mice and the intervention effect of IFN-γ in the process. MATERIALS AND METHODS: The experimental animals were 70 C57BL/6J female mice of clean grade, which were divided into 7 groups according to different treatment protocols, including Normal group, Asthma group, Model+1-MT group, Model+IFN-γ group, Model+TLR7 agonist group, TLR7 deficient group, and Model+TLR7 deficient group. Hematoxylin-eosin (HE) staining was used to observe the pathological changes of lung tissues. The positive expression rates of TLR7, p-IKKα and NF-κBp65 were detected by immunohistochemistry. bronchoalveolar lavage fluid (BALF) cells were classified and counted. The contents of interleukin (IL)-4, IL-10, IL-12 and interferon (IFN)-γ in BALF supernatant were detected by enzyme-linked immunosorbent assay (ELISA). Following isolation, culture and plasmid construction of airway smooth muscle cells (ASMCS) from normal mice and asthmatic mice, cells were transfected and divided into the Control group, pcDNA-TLR7 NC group, siRNA-TLR7 NC group, pcDNA-TLR7 group, siRNA-TLR7 group, Asatone group, Triptolide group, and pcDNA-TLR7 +Asatone group. The expression of TLR7, IDO, p-IKKα and NF-κBp65 was detected by real-time polymerase chain reaction (RT-PCR) and Western blot, respectively. 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT) was used to detect the proliferation of ASMCS. The cell cycle and apoptosis of ASMCS were detected by flow cytometry. RESULTS: HE staining showed successful modeling of asthma. Immunohistochemical test showed that the positive expression rate of TLR7 in the Asthma group was significantly decreased, and that of IKKα and NF-κBp65 was significantly increased, with significantly increased IL-4, IL-10, IL-12 and IFN-γ levels (all p<0.05). Model+1-MT group and Model+TLR7 deficient group had a large number of inflammatory cell infiltration, increased IL-4, IL-10, IL-12 and IFN-γ levels, decreased expression levels of TLR7 and IDO, and increased expression of p-IKKα and NF-κBp65 (all p<0.05); while the opposites results were detected in Model+IFN-γ group and Model+TLR7 agonist group (all p<0.05). Cell transfection experiments revealed that pcDNA-TLR7 group and Triptolide group had increased TLR7 expression while decreased p-IKKα and NF-κBp65, decreased proliferation level, and increased cell apoptosis (all p<0.05); while the contrary results were found in siRNA-TLR7 group and Asatone group (all p<0.05); yet without significant difference in pcDNA-TLR7+Asatone group (all p>0.05). CONCLUSIONS: Upregulation of TLR7 can inhibit the activation of NF-κB signaling pathway, reduces airway inflammation, inhibits ASMCS proliferation and thus promotes cell apoptosis in asthmatic mice. Besides, IFN-γ can exert a protective role in suppressing the progression of inflammation in asthma.

Azithromycin influences airway remodeling in asthma via the PI3K/Akt/MTOR/HIF-1α/VEGF pathway.

Asthma is a respiratory disease that affects people of all walks of life, and is a hotspot of continuous research, with significant manpower and resources invested in its study. Airway remodeling is an important associated pathological change, and a mark of the irreversible damage produced by asthma. It involves compositional and functional changes in the cells of the airway walls, leading to reversible structural changes, and complicating treatment. Airway remodeling is mediated by different inflammatory pathways which have been targeted for treatment, with good results. However, given its complexity, systematic study of the pathogenesis of airway remodeling is still needed, and additional targeted therapies are necessary. Macrolide drugs, such as erythromycin, azithromycin, and clarithromycin, have antibacterial effects and also influence the cytokine secretion of macrophages and T-lymphocytes. They have direct effects on a variety of cytokines, inhibiting inflammation and reducing airway reactivity. In this study, we investigated the protective effect of azithromycin on airway remodeling through the phosphoinositol-3 kinase/Akt/mechanistic target of rapamycin kinase/hypoxia-inducible factor 1α (HIF-1α)/vascular endothelial growth factor (VEGF) pathway. We observed that a long course of azithromycin could significantly reduce airway reactivity and ovalbulmin-induced pathological alterations in asthmatic mice. Gene expression analysis confirmed that HIF-1α and VEGF were significantly down-regulated following a long course of azithromycin administration.

MiR-26a protects type II alveolar epithelial cells against mitochondrial apoptosis.

OBJECTIVE: This study aims to investigate the miR-26a effects on H2O2-induced apoptosis of Type II alveolar epithelial cells (AEC-II) and the potential mechanism. MATERIALS AND METHODS: AEC-II cells were treated with 0.5 mmol/L H2O2 to mimic cellular model of acute lung injury. Transmitting electron microscopy (TEM) was employed to observe the change of morphological structures. After infecting with miR-26a mimics, flow cytometry was performed to detect cell apoptosis. Western blot was also done to explore mitochondrial apoptosis-related markers: Caspase-3, B-cell lymphoma-2 (Bcl-2) and Bax. AEC-II cells treated with 0.5 mmol/L H2O2 exhibited significant cell apoptosis. Overexpression using miR-26a mimics partially reversed the effects of H2O2-induced apoptosis in AEC-II cells, evidenced by flow cytometry results. RESULTS: Further Western blot results revealed increased levels of Caspase-3 and Bax, and the decreased Bcl-2 level after infecting with miR-26a mimics, indicating miR-26a has protective effects against mitochondrial apoptosis in AEC-II cells. CONCLUSIONS: MiR-26a protected AEC-II cells against apoptosis via mitochondrial pathway. Thus, miR-26a promises to be a potential therapy in treatment of Acute Respiratory Distress Syndrome (ARDS).

Close encounters with DNA.

Over the past ten years, the all-atom molecular dynamics method has grown in the scale of both systems and processes amenable to it and in its ability to make quantitative predictions about the behavior of experimental systems. The field of computational DNA research is no exception, witnessing a dramatic increase in the size of systems simulated with atomic resolution, the duration of individual simulations and the realism of the simulation outcomes. In this topical review, we describe the hallmark physical properties of DNA from the perspective of all-atom simulations. We demonstrate the amazing ability of such simulations to reveal the microscopic physical origins of experimentally observed phenomena. We also discuss the frustrating limitations associated with imperfections of present atomic force fields and inadequate sampling. The review is focused on the following four physical properties of DNA: effective electric charge, response to an external mechanical force, interaction with other DNA molecules and behavior in an external electric field.

Baseline characteristics of cervical auscultation signals during various head maneuvers.

Cervical auscultation (CA) is an emerging method of assessing swallowing disorders that is both non-invasive and inexpensive. This technique utilizes microphones to detect acoustic sounds produced by swallowing activity and characterize its behavior. Though some properties of swallowing sounds are known, there is still a need for a complete understanding of the baseline characteristics of cervical auscultation signals as well as how they change due to the patient's head motion, age, and sex. In order to examine these parameters, data was collected from 56 healthy adult participants that performed six different head movement tasks without swallowing. After preprocessing the signal, features were extracted. Dependent variables were time domain, frequency domain and time-frequency domain features. Statistical tests showed that only the skewness and peak frequency were not statistically different for all tasks. The peak frequency results indicate that head movement does not significantly affect the microphone signal, and that it is unnecessary to filter out the lowest frequency components. No sex differences were observed in the extracted features, but several features exhibited age dependence.

Multiscale modeling of two-dimensional contacts.

A hybrid simulation method is introduced and used to study two-dimensional single-asperity and multi-asperity contacts both quasistatically and dynamically. The method combines an atomistic treatment of the interfacial region with a finite-element method description of subsurface deformations. The dynamics in the two regions are coupled through displacement boundary conditions applied at the outer edges of an overlap region. The two solutions are followed concurrently but with different time resolution. The method is benchmarked against full atomistic simulations. Accurate results are obtained for contact areas, pressures, and static and dynamic friction forces. The time saving depends on the fraction of the system treated atomistically and is already more than a factor of 20 for the relatively small systems considered here.

Characterization of torovirus from human fecal specimens.

The toroviruses, Berne virus (BEV) and Breda virus (BRV), are recognized pathogens of horses and cattle, respectively. Torovirus-like particles (TVLPs) that are immunologically related to BRV have been reported as etiological agents of gastroenteritis in humans. Of the toroviruses, only BEV has been shown to replicate in cell culture. Hence, these agents can be routinely detected only by electron microscopy (EM), although serological testing has been used as well. Our studies have provided supporting evidence that the TVLPs detected in the stool specimens of pediatric patients with gastroenteritis are human toroviruses. By EM, these particles are morphologically similar to BEV and BRV. Thin-section electron microscopy revealed that TVLPs contain toroidal-shaped nucleocapsids. Viruses purified from human fecal specimens agglutinate rabbit erythrocytes. BRV antiserum as well as convalescent sera from patients with gastroenteritis whose stools contain TVLPs were shown to contain antibodies that react with purified TVLPs as demonstrated by hemagglutination inhibition, immunoelectron microscopy, and immunoblotting. RNA extracted from partially purified TVLP preparations is amplifiable by RT-PCR using primers bracketing a 219-base region at the 3' end of the Berne virus genome. Sequence analysis of amplicons from five isolates showed a high degree of identity with the corresponding BEV sequence.

Longitudinal study of molecular epidemiology of small round-structured viruses in a pediatric population.

Small round-structured viruses (SRSV), recently designated members of the family Caliciviridae, can now be readily subtyped by amplification of a defined portion of their genome by reverse transcription-PCR and then by identification of the amplicons with specific probes by Southern blotting. A longitudinal survey (from 1991 to 1995) was conducted to determine the genotypes of the SRSV present in pediatric stool specimens from patients with sporadic cases of gastroenteritis. It was found that almost all viruses were of the G-2 genotype, and on probing, the subtype P2-B was predominant but the frequencies of the different subtypes varied from year to year. A survey of the genotypes of SRSV from community outbreaks from 1995 showed that the G-2 genotype was also predominant and that the distribution of its subtypes was similar to that seen in sporadic cases of diarrhea in pediatric patients over the same time period. It was concluded that there is a succession of subtypes of SRSV in our pediatric population over time. This distribution of genotypes in sporadic cases of pediatric gastroenteritis may reflect the distribution in community outbreaks occurring at the same time.

Characterization of proteoglycan accumulation during formation of cartilagenous tissue in vitro.

In order to study proteoglycan retention and accumulation, we optimized a chondrocyte cell culture system in which isolated bovine articular chondrocytes accumulate extracellular matrix to form a continuous layer of cartilagenous tissue. The tissue can attain a thickness of up to 110 microns by 35 days. The cells synthesize large keratan sulfate containing proteoglycans and type II collagen indicating that the chondrocytes maintain their phenotype in these culture conditions. Matrix accumulation is enhanced by increased cell density and the presence of serum and ascorbic acid. The amount of proteoglycans synthesized by the chondrocytes increases up to day 21 and then decreases to the same levels as are synthesized during the first week of culture. The percentage of newly synthesized proteoglycans retained in the matrix increases from 20% on day 6 to a maximum of 85% by day 35. The proteoglycan and collagen content in the tissue increases with time in culture. The changes in the percentage of proteoglycans retained parallels the increase in proteoglycan content. After day 35, there is no further increase in the amount of proteoglycans and collagen nor in the percentage of newly synthesized proteoglycans retained in the extracellular matrix. These studies demonstrate that the cultures are going through two phases: one of matrix accumulation and then one of maintaining the existing matrix. The period of matrix accumulation occurs between days 10-21 whereas matrix maintenance is observed after day 35. Using this culture system to study proteoglycan accumulation and maintenance during these culture periods may prove useful in identifying the mechanisms regulating these processes.

[A retrospective investigation of patients with gastric cancer in ward patients].

A retrospective investigation of 1,079 patients with gastric cancer among 181,055 patients in a general hospital showed that 0.59% of all the ward patients suffered from gastric cancer (6:1, male preponderant) , with the age ranging from 41-70, and the peak age was between 50-60. Most (98.24%) were progressive, others (1.76%) were early gastric cancers. Many of the patients with clinical courses over 1 year before hospitalization had histories of gastropathy. Pathological examination of the early gastric cancers showed that the cancers in 15 (78.94%) out of 19 patients were due to malignant change of ulcers. However, our results were not quite similar to previous reports in the distribution, limit, number and microscopic classification of the lesions. There was no obvious discrepancy in lesion display and diagnostic accuracy among the three methods, e.g., regular examination, double contrast examination, and the endoscopy examination (P greater than 0.05). The accuracy of diagnosis might be improved by using multiple examinations of stomach and combination of gastrointestinal examination with endoscopy.