Evaluation of a Novel Formaldehyde-Free Fixation Solution for the Fixation of Mouse Organs.

Objective: A new aldehyde-free fixative has been developed and its effect has been compared to traditional formaldehyde fixative in terms of the fixation effect and HE staining of the heart, liver, lung, and kidney. The air in the experimental area was examined to evaluate its impact on the environment and human health. Methods: The organs from mice of groups 1-6 were taken respectively (thickness of liver and lung was 3 mm). After the heart and kidney capsule were removed, the organs were longitudinally cut along their maximum surface, and half was taken. Thereafter, the tissue fixation effect was observed by Hematein and Eosin (H＆E) staining and the total protein content of tissue was examined by the ultramicro spectrophotometer. Additionally, the volatility ratio of the new fixative and the traditional formaldehyde is compared. Result: The results showed that there was no significant difference between the fixation effect of the new aldehyde-free fixation and the traditional formaldehyde fixative on mouse organs and the air quality in the experimental area was found to be significantly better when the new aldehyde-free fixative is used than when the traditional formaldehyde fixative is used. Conclusion: Traditional formaldehyde fixative in HE staining can be replaced by the new environment-friendly formaldehyde-free fixative, however further special staining of fixed tissue and immunohistochemical studies are needed.

Downregulation of Linc00173 increases BCL2 mRNA stability via the miR-1275/PROCA1/ZFP36L2 axis and induces acquired cisplatin resistance of lung adenocarcinoma.

BACKGROUND: LINC00173 had been reported as a cisplatin (cis-diamminedichloroplatinum, DDP) chemotherapy-resistant inducer in small-cell lung cancer (SCLC) and lung squamous cell carcinoma (LUSC). This study aimed to display reverse data for LINC00173 as a DDP chemosensitivity-inducing factor in lung adenocarcinoma (LUAD). METHODS: LINC00173 was screened from the Gene Expression Omnibus database (GSE43493). The expression level of LINC00173 in LUAD tissues and cell lines was detected using in situ hybridization and quantitative reverse transcription-polymerase chain reaction. Colony formation, cell viability, half-maximal inhibitory concentration, flow cytometry, and xenograft mouse model were used to evaluate the role of LINC00173 in the chemosensitivity of LUAD to DDP. The mechanism of LINC00173 in DDP resistance by mediating miR-1275/PROCA1/ZFP36L2 axis to impair BCL2 mRNA stability was applied, and co-immunoprecipitation, chromatin immunoprecipitation, RNA antisense purification, RNA immunoprecipitation, and luciferase reporter assays were performed. RESULTS: LINC00173 downregulation in patients with DDP-resistant LUAD was correlated with poor prognosis. Further, LINC00173 expression was significantly reduced in DDP-resistant LUAD cells and DDP-treated human LUAD tissues. Suppressed LINC00173 expression in LUAD cells enhanced DDP chemoresistance in vivo and in vitro, while restored LINC00173 expression in DDP-resistant LUAD cells markedly regained chemosensitivity to DDP. Mechanistically, DDP-resistant LUAD cells activated PI3K/AKT signal and further elevated the c-Myc expression. The c-Myc, as an oncogenic transcriptional factor, bound to the promoter of LINC00173 and suppressed its expression. The reduced LINC00173 expression attenuated the adsorption of oncogenic miR-1275, downregulating the expression of miR-1275 target gene PROCA1. PROCA1 played a potential tumor-suppressive role inducing cell apoptosis and DDP chemosensitivity via recruiting ZFP36L2 to bind to the 3' untranslated region of BCL2, reducing the stability of BCL2 mRNA and thus activating the apoptotic signal. CONCLUSIONS: This study demonstrated a novel and critical role of LINC00173. It was transcriptionally repressed by DDP-activated PI3K/AKT/c-Myc signal in LUAD, promoting DDP-acquired chemotherapeutic resistance by regulating miR-1275 to suppress PROCA1/ZFP36L2-induced BCL2 degradation, which led to apoptotic signal reduction. These data were not consistent with the previously described role of LINC00173 in SCLC or LUSC, which suggested that LINC00173 could play fine-tuned DDP resistance roles in different pathological subtypes of lung cancer. This study demonstrated that the diminished expression of LINC00173 might serve as an indicator of DDP-acquired resistance in LUAD.

Herpes Simplex Virus 1 MicroRNA miR-H28 Exported to Uninfected Cells in Exosomes Restricts Cell-to-Cell Virus Spread by Inducing Gamma Interferon mRNA.

An earlier report showed that herpes simplex virus 1 (HSV-1) expresses two microRNAs (miRNAs), miR-H28 and miR-H29, late in the infectious cycle. The miRNAs are packed in exosomes and, in recipient cells, restrict the transmission of virus from infected cells to uninfected cells. We now report that (i) miR-H28 induced the synthesis of gamma interferon (IFN-γ) in both infected cells and cells transfected with miR-H28, (ii) IFN-γ accumulated concurrently with viral proteins in infected cells, (iii) IFN-γ was produced in HEp-2 cells derived from cancer tissue and in HEK293T cells derived from normal tissue, and (iv) HSV-1 replication was affected by exposure to IFN-γ before infection but not during or after infection. The results presented in this report support the growing body of evidence indicating that HSV-1 encodes functions designed to reduce the spread of infection from infected cells to uninfected cells, possibly in order to maximize the transmission of virus from infected individuals to uninfected individuals.IMPORTANCE In this report, we show that IFN-γ is produced by HSV-1 viral miR-H28 and viral replication is blocked in cells exposed to IFN-γ before infection but not during or after infection. The inevitable conclusion is that HSV-1 induces IFN-γ to curtail its spread from infected cells to uninfected cells. In essence, this report supports the hypothesis that HSV-1 encodes functions that restrict the transmission of virus from cell to cell.

Effect of Loss-of-function of the Herpes Simplex Virus-1 microRNA H6-5p on Virus Replication.

To date, 29 distinct microRNAs (miRNAs) have been reported to be expressed during herpes simplex virus infections. Sequence analysis of mature herpes simplex virus-1 (HSV-1) miRNAs revealed five sets of miRNAs that are complementary to each other: miR-H6-5p/H1-3p, miR-H6-3p/H1-5p, H2-5p/H14-3p, miR-H2-3p/H14-5p, and miR-H7/H27. However, the roles of individual miRNAs and consequences of this complementarity remain unclear. Here, we focus on two of these complementary miRNAs, miR-H6-5p and miR-H1-3p, using loss-of-function experiments in vitro and in a mouse model of infection using an miRNA sponge approach, including tandem multiplex artificial miRNA-binding sequences that do not match perfectly to the target miRNA inserted downstream of a green fluorescent protein reporter gene. Infection with recombinant virus expressing the miR-H6-5p sponge reduced viral protein levels and virus yield. Decreased accumulation of viral proteins was also observed at early stages of infection in the presence of both an miR-H6-5p inhibitor and plasmid-expressed miR-H1-3p. Moreover, establishment of latency and reactivation did not differ between the recombinant virus expressing the miR-H6-5p sponge and wild-type HSV-1. Taken together, these data suggest that miR-H6-5p has an as-yet-unidentified role in the early stages of viral infection, and its complement miR-H1-3p suppresses this role in later stages of infection. This report extends understanding of the roles of miRNAs in infection by herpes simplex viruses, supporting a model of infection in which the production of virus and its virulent effects are tightly controlled to maximize persistence in the host and population.

Traffic-related air pollution induces non-allergic eosinophilic airway inflammation and cough hypersensitivity in guinea-pigs.

BACKGROUND: The pathogenesis and pathophysiology of eosinophilia-related chronic cough such as non-asthmatic eosinophilic bronchitis and cough variant asthma are still not clear. OBJECTIVE: This study is to examine the potential role of traffic-related air pollution (TRAP) in eosinophilic inflammation and cough responses. METHODS: Non-sensitized guinea-pigs were exposed to TRAP in an urban traffic tunnel or kept in a filtered air environment for 7 or 14 days. Reflexive cough was measured using citric acid and allyl isothiocyanate (AITC) challenges, respectively. Spontaneous cough counting was determined using audio recording and a waveform analysis. Airway inflammation was evaluated using differential cells in bronchoalveolar lavage fluid (BALF) and lung histopathology. To further elucidate the relationship between airway inflammation and cough hypersensitivity, a subgroup of those exposed for 14 days received a dexamethasone treatment. RESULTS: Compared to reflexive cough count (mean (95% confidence interval) in 10 minutes) provoked by the AITC challenge for the unexposed animals (3.1 (1.7-4.5)), those were increased significantly following both the 7-day (12.0 (6.8-17.2), P < 0.01) and the 14-day (12.0 (6.4-17.6), P < 0.01) TRAP exposure. The effect provoked by the citric acid challenge was more profound following the 14-day exposure (26.0 (19.5-32.5) vs 3.8 (1.5-6.0) for the control, P < 0.001). TRAP exposures enhanced spontaneous cough events, caused a significant increase of eosinophils and neutrophils in BALF and resulted in a dramatic eosinophilic infiltration in submucosal layer of trachea and bronchus, which can be inhibited significantly by dexamethasone treatment. CONCLUSIONS & CLINICAL RELEVANCE: TRAP exposures induced cough hypersensitivity and non-allergic eosinophilic inflammation of airways in guinea-pigs. This study highlights the potential mechanisms of eosinophilia-related chronic cough that can be induced by traffic-related air pollution.

SOCS4 expressed by recombinant HSV protects against cytokine storm in a mouse model.

Oncolytic viruses are genetically engineered viruses designed for the treatment of solid tumors, and are often coupled with the antitumor immunity of the host. The challenge of using oncolytic herpes simplex virus (oHSV) as an efficacious oncolytic agent is the potential host tissue damage caused by the production of a range of cytokines following intratumoral oHSV injection. An HSV­suppressor of cytokine signaling 4 (SOCS4) recombinant virus was created to investigate whether it inhibits cytokine storm. Recombinant HSV­SOCS4 and HSV­1(F) were used to infect mice, and levels of several representative cytokines, including monocyte chemoattractant protein­1, interleukin (IL)­1ß, tumor necrosis factor­α, IL­6 and interferon γ, in serum and bronchoalveolar lavage fluid (BALF) of infected mice were determined, and immune cells in BALF and spleen were enumerated. Lung damage, virus titers in the lung, body weight and survival rates of infected mice were also determined and compared between the two groups. The cytokine concentration of HSV­SOCS4­infected mice was significantly decreased compared with that of HSV­1(F)­infected mice in BALF and serum, and a smaller number of cluster of differentiation (CD)11b+ cells of BALF, and CD8+CD62L+ T cells and CD4+CD62L+ T cells of the spleen were also identified in HSV­SOCS4­infected mice. HSV­SOCS4­infected mice exhibited slight lung damage, a decrease in body weight loss and a 100% survival rate. The results of the present study indicated that SOCS4 protein may be a useful regulator to inhibit cytokine overproduction, and that HSV­SOCS4 may provide a possible solution to control cytokine storm and its consequences following induction by oncolytic virus treatment.

Effects of leukotriene D4 and histamine nasal challenge on airway responsiveness and inflammation in persistent allergic rhinitis patients.

BACKGROUND: Both histamine and leukotrienes are implicated in the pathogenesis of allergic rhinitis (AR), although the pattern and severity of the nasal response to these two potent inflammatory mediators may differ, which has not been adequately studied in patients with persistent AR. OBJECTIVE: We sought to compare the differential effects of nasal challenge with leukotriene D4 (LTD4 ) and histamine on the airway response and inflammation in patients with AR. METHODS: An open-label, crossover study was performed in 25 persistent AR patients (AR group) and 16 healthy subjects (control group). Participants randomly underwent histamine and LTD4 nasal provocation within a two-week interval. Nasal symptoms according to a visual analogue scale (VAS), fractional exhaled nitric oxide (FENO), nasal lavage, induced sputum, and spirometry were evaluated before and after nasal challenge. RESULTS: Nasal airway resistance (NAR) increased significantly after both LTD4 and histamine nasal challenge in AR patients (P < .05). The potency of LTD4 was 142-fold higher than that of histamine in increasing NAR (P < .001). The nasal symptom score induced by histamine challenge was significantly higher than that triggered by LTD4 (3.42 ± 0.83 vs. 1.16 ± 0.94, P < .05) in the AR group. LTD4 and histamine nasal challenge led to a significant increase in neutrophils in the nasal lavage and induced sputum (P < .05) in AR patients. There were no significant differences in the changes of eosinophils before and after LTD4 and histamine nasal challenges in nasal lavage and induced sputum. No significant changes in NAR, the induced symptom score, or inflammatory cells in the nasal lavage and sputum were found in the control group. CONCLUSIONS: LTD4 and histamine nasal challenge caused different patterns and severities of nasal symptoms, which correlated with symptoms (TSS) that affect patient's daily life. LTD4 was far more potent than histamine at increasing the NAR, while histamine nasal challenge induced more sneezing and nasal discharge. These results may guide the prescription of anti-histamine or anti-leukotriene agents for treating different AR phenotypes.

Effects of leukotriene D4 nasal challenge on bronchial responsiveness and inflammation in asthmatic patients with allergic rhinitis.

BACKGROUND: In asthmatic patients with allergic rhinitis (AR), increased cysteinyl leukotrienes (CysLTs) production in the secretion of nasal mucosa has been associated with greater bronchial hyperresponsiveness (BHR) after nasal allergen challenge. However, the role of CysLTs in eliciting BHR after nasal allergen challenge has not been evaluated. The aim of this study is to evaluate the effect of LTD4 nasal challenge on BHR and inflammation in asthmatic patients with AR. METHODS: In this self-controlled study, fifteen eligible consecutively recruited subjects underwent methacholine (Mch) bronchial provocation test before and 30 minutes after LTD4 nasal provocation test. The cumulative concentration of LTD4 inducing a 60% increase in nasal airway resistance (PC60NAR) was calculated. The mean values of cumulative doses inducing a 20% decrease in forced expiratory flow in one second (PD20FEV1) for Mch before and after nasal challenge were compared. Fractional exhaled nitric oxide (FeNO), differential inflammatory cell counts in nasal lavage and induced sputum before and after nasal challenge were compared. RESULTS: House dust mites were the major allergens accounting for 10/15 (66.7%) of asthmatic patients with AR. The PC60NAR for LT was (8.39±3.48)×10-3 mg·mL-1. The PD20FEV1 before and after nasal challenge was 3.05±3.81 and 2.70±3.81 µmol, respectively (P=0.45). The percentages of eosinophils were (38.36±23.14)% and (45.70±24.86)% in nasal lavage, and (17.51±11.05)% and (24.29±16.52)% in induced sputum before and 24 hours after nasal challenge. The neutrophil counts were (60.64±23.14)% and (53.30±24.46)% in nasal lavage, and (53.83±23.27)% and (56.19±22.28)% in induced sputum before and 24 hours after nasal challenge. The values of FeNO were 40 [35] and 43 [30] ppb before and 24 hours after nasal challenge. No severe adverse effects were reported during the tests. CONCLUSIONS: Although most asthmatic patients with AR were sensitive to LTD4 nasal challenge, LTD4 nasal provocation tests do not confer any major effect on BHR. LTD4 might not play a vital role in eliciting bronchial responsiveness induced by nasal allergen challenge.

Carbocisteine attenuates TNF-α-induced inflammation in human alveolar epithelial cells in vitro through suppressing NF-κB and ERK1/2 MAPK signaling pathways.

AIM: We previously proven that carbocisteine, a conventional mucolytic drug, remarkably reduced the rate of acute exacerbations and improved the quality of life in the patients with chronic obstructive pulmonary disease. In this study we investigated the mechanisms underlying the anti-inflammatory effects of carbocisteine in human alveolar epithelial cells in vitro. METHODS: Human lung adenocarcinoma cell line A549 was treated with TNF-α (10 ng/mL). Carbocisteine was administered either 24 h prior to or after TNF-α exposure. The cytokine release and expression were measured using ELISA and qRT-PCR. Activation of NF-κB was analyzed with Western blotting, immunofluorescence assay and luciferase reporter gene assay. The expression of ERK1/2 MAPK signaling proteins was assessed with Western blotting. RESULTS: Carbocisteine (10, 100, 1000 µmol/L), administered either before or after TNF-α exposure, dose-dependently suppressed TNF-α-induced inflammation in A549 cells, as evidenced by diminished release of IL-6 and IL-8, and diminished mRNA expression of IL-6, IL-8, TNF-α, MCP-1 and MIP-1ß. Furthermore, pretreatment with carbocisteine significantly decreased TNF-α-induced phosphorylation of NF-κB p65 and ERK1/2 MAPK, and inhibited the nuclear translocation of p65 subunit in A549 cells. In an NF-κB luciferase reporter system, pretreatment with carbocisteine dose-dependently inhibited TNF-α-induced transcriptional activity of NF-κB. CONCLUSION: Carbocisteine effectively suppresses TNF-α-induced inflammation in A549 cells via suppressing NF-κB and ERK1/2 MAPK signaling pathways.

[The synthetic peptide RGDSY-CTTHWGFTLC inhibits metastasis and proliferation of breast cancer cells in vitro].

OBJECTIVE: To study the effect of the synthetic peptide RGDSY-CTTHWGFTLC on the biological behavior of breast cancer MCF-7 cells in vitro. METHODS: MCF-7 cells were incubated with different concentrations of the synthesized peptide RGDSY-CTTHWGFTLC (RGDSY-CTT), the positive control peptide CTTHWGFTLC (CTT), or the negative control peptide STTHWGFTLS (STT) in fibronectin-coated 96-well plates for different time lengths, and the changes in cell adhesion, invasiveness, proliferation, apoptosis and cell cycle were detected using Transwell chamber assay, MTT assay, and flow cytometry. RESULTS: Incubation of the cells with 50, 100 and 200 µg/ml of RGDSY-CTT caused a significant concentration- dependent inhibition of the cell adhesion (cell adhesion rates of 85.1%, 74.1% and 63.8%, respectively) with stronger effects than CTT (P<0.05). At 100 and 200 µg/ml, RGDSY-CTT significantly inhibited the invasion (with inhibition rate of 41.8% and 63.9%, respectively) of MCF-7 cells with an effect similar to that by CTT (P>0.05). At 50, 100 and 200 µg/ml, RGDSY-CTT concentration-dependently suppressed MCF-7 cell proliferation (with cell proliferation rates of 98.8%, 82.4% and 63.0%, respectively), and this inhibitory effect was stronger than that of CTT at 100 and 200 µg/ml (P<0.05). The results of flow cytometry also demonstrated a stronger apoptosis-inducing effect of RGDSY-CTT (76.7%) than that in CTT, STT and the blank control groups (P<0.05). CONCLUSIONS: RGDSY-CTT can inhibit cell invasion, suppress adhesion and proliferation, and induce apoptosis in MCF-7 cells.

[Role of matrix metalloproteinase (MMP)-2 and -9 and vascular endothelial growth factor C in lymph node metastasis of breast cancer].

OBJECTIVE: To study the expression of matrix metalloproteinase (MMP)-2, MMP-9 and vascular endothelial growth factor (VEGF)-C in breast cancer and their role in lymph node metastasis. METHODS: Immunohistochemical staining was used to detect the expression of VEGF-C, MMP-2, MMP-9 and LYVE-1 in 84 cases of breast cancer, including 52 cases with and 32 cases without lymph node metastases. The recombinant vector (pSIREN-VEGF-C) was transfected into human breast cancer cell MCF-7 by liposome, and the RNA expression of MMP-2, MMP-9 and VEGF-C in MCF-7 cells after transfection was detected by PCR. RESULTS: The expressions of MMP-2, MMP-9 and VEGF-C were 98.1% (51/52), 88.5% (46/52), and 94.2% (49/52) respectively for the metastatic group, and 75.0% (24/32), 53.1% (17/32), and 65.6% (21/32) respectively for the non metastatic group, and there was significant difference between these groups (P < 0.05). The lymphatic vessel density between these two groups was also significantly different (P < 0.05). Increased expression of MMP-2, MMP-9 and VEGF-C was also associated with increased number of lymphatic vessels had also increased (P < 0.05). The expression of MMP-2, MMP-9 and VEGF-C in MCF-7 cells after gene transfection decreased significantly (P < 0.05). CONCLUSION: MMP-2 and MMP-9 in conjunction with VEGF-C, promote lymphangiogenesis and lymph node metastasis of breast cancer.