Construction of exosome-loaded LL-37 and its protection against zika virus infection.

Zika virus (ZIKV) is an enveloped, single-stranded and positive-stranded RNA virus of the genus Flavivirus in the family Flaviviridae. ZIKV can cross the placental barrier and infect the fetus, causing microcephaly, congenital ZIKV syndrome, and even fetal death. ZIKV infection can also lead to testicular damage and male sterility. But no effective drugs and vaccines are available up to now. Previous studies have shown that the cathelicidin antimicrobial peptide LL-37 can protect against ZIKV infection. However, LL-37 is a secreted peptide, which can be easily degraded in vivo. We herein constructed exosome-loaded LL-37 (named LL-37-TM-exo and TM-LL-37-exo) using the transmembrane protein TM to load LL-37 onto the membrane of exosome. We found that exosome-loaded LL-37 could significantly inhibit ZIKV infection in vitro and in vivo, and LL-37-TM-exo had stronger antiviral activity than that of TM-LL-37-exo, which could significantly reduce ZIKV-induced testicular injury and sperm injury, and had broad-spectrum antiviral effect. Compared to free LL-37, exosome-loaded LL-37 showed a better serum stability, higher efficiency to cross the placental barrier, and stronger antiviral activity. The mechanism of exosome-loaded LL-37 against ZIKV infection was consistent with that of free LL-37, which could directly inactivate viral particles, reduce the susceptibility of host cells, and act on viral replication stage. Our study provides a novel strategy for the development of LL-37 against viral infection.

Activation of STAT6 by intranasal allergens correlated with the development of eosinophilic chronic rhinosinusitis in a mouse model.

Eosinophilic chronic rhinosinusitis (ECRS) is a chronic inflammatory disease characterized by prominent eosinophilic infiltration along with a T-helper-2 (Th2) response. It has been well documented that signal transducer and activator of transcription 6 (STAT6) is a nuclear transcription factor that mediates Th2-type immunity and is implicatory of STAT1 and STAT3 in the pathogenesis of allergic airway diseases. However, little is known about the association between STATs and ECRS. Here, we explored the relationship between STAT1, STAT3, and/or STAT6 and eosinophilic inflammation accompanied by Th2-type immunity in a mouse model of ECRS. An ovalbumin (OVA)-staphylococcal enterotoxin B (SEB)-induced ECRS murine model was first established. The mucosal histological alterations were determined using hematoxylin and eosin staining. The number of eosinophils in peripheral blood was measured using a blood cell analyzer. The cytokine (IL-4, IL-5, IL17 A and IFN-γ) expression levels in the sinonasal mucosa and total and OVA-specific IgE from serum were measured using ELISA. Then, the protein levels of STAT1, STAT3, STAT6, phosphorylated STAT1 (p-STAT1), p-STAT3, p-STAT6, T-box expressed in T-cells (T-bet), GATA binding protein 3 (GATA-3), and retinoic acid receptor-related orphan receptor γ (RORγt) in the sinonasal mucosa were examined by immunohistochemical staining or Western blotting. Local administration of OVA combined with SEB (OVA + SEB) induced multiple polyp-like lesions, accompanied by prominent eosinophilic infiltration in the sinonasal mucosa. The OVA- and OVA+SEB-treated groups showed significantly higher eosinophil counts from peripheral blood and total and OVA-specific IgE levels from serum than those in the PBS- and SEB-treated groups. The levels of p-STAT6 were markedly increased by OVA + SEB exposure, as well as GATA-3, IL-4, and IL-5, but did not affect STAT6, p-STAT1, p-STAT3, T-bet, RORγt, IFN-γ, or IL-17A. Furthermore, an eosinophil count in the sinonasal mucosa showed a positive correlation with the level of p-STAT6 in the ECRS mouse model. Signal transducer and activator of transcription 6 signaling could be activated in the OVA+SEB-induced ECRS model and might be a crucial signal transducer in the development of Th2-skewed ECRS.

Investigation of mercury adsorption and cyclic mercury retention over MnOx/γ-Al2O3 sorbent.

In this study mercury sorbent based on manganese oxides impregnated γ-alumina was synthesized. Mercury retention characteristics were investigated by mercury speciation and thermal desorption experiments. No gaseous mercuric oxide was observed in mercury speciation experiments with a mercury mass balance ratio of 89.11%. Maximum mercury desorption peak at 480 °C indicated that mercury was adsorbed in the form of mercuric oxide. Three cycles of mercury retention were tested with different thermal treatment in-between to evaluate the cyclic performance. Changes in surface phase and manganese chemistry before and after thermal treatment were characterized by XRD and XPS. Deterioration in mercury retention capacity was observed after thermal desorption at 500 °C, which was interpreted with reduced initial adsorption rate calculated by Pseudo-second order kinetic model. XPS studies suggested that atomic ratios of Mn4+/(Mn4++Mn3+) decreased from 73.2% to 32.3% and 33.9% after thermal desorption in N2 and air, respectively. The reduction of MnO2 to Mn2O3 was irreversible thus the mercury retention capacity could not be restored by thermal desorption at high temperatures. Spent sorbents that were reactivated at 200 °C in air without thermal desorption at 500 °C possessed considerable cycling performance for mercury retention due to the preserved Mn4+.

Staphylococcal enterotoxin burden determines the type of T helper cell response and pathology of the maxillary sinus mucosa in rabbits.

Staphylococcal enterotoxin (SE) induces T lymphocyte activation along with nasal allergic inflammation during rhinosinusitis, but it is under debate on which types of T helper (Th) cells respond exclusively or whether they respond synergically. We hypothesize that their responses may vary based on dose of SE. To test this hypothesis, we initiated to determine the nature of the T cell response and pathological feature upon repeated exposure to staphylococcal enterotoxin A (SEA) at different doses in the maxillary sinus of rabbits. SEA (0.6 or 60 ng) was instilled daily into the left maxillary sinus of rabbits for 28 days. The right maxillary sinus receiving normal saline was used as control. Mucosal histological changes were examined by hematoxylin-eosin and toluidine blue staining. Tissue expression of myeloperoxidase (MPO), eosinophil cationic protein (ECP), T-box expressed in T cells (T-bet), and GATA binding protein 3 (GATA-3) were examined using immunohistochemistry. Mucosal levels of representative pro-inflammatory cytokines were measured using ELISA. SEA at 60 ng/day induced acute rhinosinusitis, as confirmed by CT scan. Histopathologic examination revealed epithelial disruption, subepithelial edema, and inflammatory cell infiltration. MPO and T-bet expression, as well as interleukin (IL)-2 and interferon (IFN)-γ levels, were up-regulated. However, 0.6 ng/day SEA did not cause discharge. Histological examination revealed prominent eosinophilic infiltration. ECP and GATA-3 expression, as well as IL-4 and IL-5 levels, were increased at this lower dose. In conclusion, SEA induces acute rhinosinusitis associated with a Th1-type immune response at high dose, and a predominantly Th2-biased allergic inflammation at low dose.

[Effects of staphylococcal enterotoxin of different concentrations on the expression of GATA-3 and Th1/Th2 cytokines in the maxillary sinus mucosa of rabbits].

OBJECTIVE: The aim of this study was to determine the expression of GATA-3 and the level of Th1 and Th2 cytokines upon repeated exposure to staphylococcal enterotoxin B(SEB) of different concentrations in the maxillary sinus mucosa of rabbits. METHOD: The rabbits were randomly divided into 2 groups (24 rabbits per group): low-dose SEB group and high-dose SEB group. The low-dose SEB group and high-dose SEB group received daily injections of 0.6 ng of SEB (2 ml) and 60 ng of SEB (2 ml) into the left maxillary sinus of rabbits for 28 days, respectively. Concurrent treatment of the right maxillary sinus with normal saline was used as a control. Six rabbits chosen randomly in two groups were killed on days 3, 7, 14, and 28, and to obtain the sinus mucosa from the two-side maxillary sinuses for measurement. Mucosal levels of IL-2, IL-4, IL-5, and IFN-γ were measured using ABC-ELISA. Tissue expression of GATA-3 were examined using Real-time PCR and immunohistochemistry. RESULT: IFN-γ and IL-2 levels were significantly elevated in the high-dose SEB group compared with the low-dose SEB and control groups on days 7, 14, and 28 (P < 0.05). However, IL-4 and IL-5 levels were markedly enhanced in the low-dose SEB group compared with the high-dose SEB and control groups on days 14 and 28 (P < 0.05). Real-time PCR showed that the expression of GATA-3 mRNA in the low-dose SEB group was markedly enhanced, and immunohistochemical staining illustrated that the number of GATA-3 positive cells was markedly increased in the low-dose SEB group as compared with the high-dose SEB group (P < 0.05). No significant differences were observed in GATA-3 expression between the high-dose SEB and the control groups (P > 0.05). CONCLUSION: SEB promoted Th1 cytokines production at high concentrations, and enhanced Th2 cytokines expression and Th2 immune response at low concentrations.

[The staphylococcal enterotoxin burden determines the ultrastructure of ciliated epithelia and inflammatory changes in maxillary sinus mucosa of rabbits].

OBJECTIVE: To investigate the ultrastructure of ciliated epithelia and inflammatory changes upon repeated exposure to staphylococcal enterotoxin A (SEA) of different concentrations in the maxillary sinus mucosa of rabbits. METHODS: The rabbits were randomly divided into 2 groups (24 rabbits per group): low-dose SEA group and high-dose SEA group. The low-dose SEA group and high-dose SEA group received daily injections of 0.6 ng of SEA (2 ml) and 60 ng of SEA (2 ml) into the left maxillary sinus of rabbits for 28 days, respectively. Concurrent treatment of the right maxillary sinus with normal saline was used as control. Six rabbits chosen randomly in two groups were examined by computed tomography (CT) scans and then sacrificed to obtain the sinus mucosa from the two-side of maxillary sinuses for histological assessment on days 3, 7, 14 and 28. To characterize the inflammatory changes of the sinus mucosa examined using light microscope, hematoxylin and eosin (HE) and toluidine blue staining was performed. Scanning and transmission electron microscopy were performed to observe ultrastructure of ciliated epithelia in the maxillary sinus mucosa. SPSS 13.0 software was used to analyze the data. RESULTS: On days 14 and 28, CT images showed opacification of the left maxillary sinus in the high-dose SEA group. The percentage of epithelial disruption was (22.73 ± 5.72) % and (30.79 ± 4.30)% in the high-dose SEA group respectively, and were significantly greater than those in the low-dose SEA group (5.12% ± 1.98% and 5.38% ± 1.64%, q value was 10.079 and 19.132) and control group (4.08% ± 1.29% and 4.81% ± 1.62%, q value was 11.016 and 19.592, respectively, all P < 0.01). The subepithelial thickness in the high-dose SEA group was (113.34 ± 14.81)µm and (120.86 ± 12.35) µm respectively, and were significantly different from those of the low-dose SEA group [(71.08 ± 10.39)µm and (81.63 ± 9.32)µm, q value was 8.090 and 8.782] and control group [(37.45 ± 7.67)µm and (38.79 ± 7.68)µm, q value was 15.759 and 19.541, all P < 0.01]. Viewed under the electron microscope, loss of cilia was observed, a few compound cilia and cytoplasmic protrusion were found, an obvious stretching of the endoplasmic reticulum and an obvious turgescence of the mitochondria was also observed. However, in the low-dose SEA group on days 14 and 28, CT scan of the left maxillary sinus showed transparency; light microscopy observations of the maxillary sinus mucosa showed the number of eosinophils was markedly increased as compared with the high-dose SEA and control groups, the differences were significant (q value was 5.871 and 6.766 on day 14, and q value was 7.572 and 8.970 on day 28, respectively, all P < 0.05). But no significant differences were observed in epithelial disruption between the low-dose SEA and the control groups on days 14 and 28 (q value was 1.512 and 0.859 respectively, all P > 0.05); inordinate array and adhesion of cilia was observed, but cilia loss, compound cilia, cytoplasmic protrusions, mitochondrial swelling and endoplasmic reticulum stretching were not found. CONCLUSIONS: SEA may induce allergic inflammation of the sinus mucosa without damaging the structure of ciliated epithelia at low concentration. Whereas SEA impairs the structure of mitochondria and endoplasmic reticulum in ciliated epithelial cells at high concentration, and results in cilia loss and epithelial disruption, which may be one of the main reasons to induce acute sinusitis.

[Observation on ultrastructure of maxillary sinus mucosa in experimental acute sinusitis].

OBJECTIVE: To observe ultrastructure of maxillary sinus mucosa of experimental acute sinusitis in rabbits. METHOD: Twenty-five rabbits were randomly divided into experimental group (20 rabbits) and blank control group (5 rabbits). We established a rhinogenic model of experimental acute sinusitis in experimental group. Five rabbits chosen randomly in experimental group were sacrificed and dissected after 1, 2, 3, and 4 weeks, and the tissue (0.3 cm x 0.3 cm) of sinus mucosa were prepared for visualization by transmission electron microscope (TEM). Animals in blank control group were sacrificed after 1 week. RESULT: Under the transmission electron microscope, in the blank control group, cilia of maxillary sinus mucosa lined up in order without ciliary loss, no stretched endoplasmic reticulum or turgescent mitochondria was observed. However, in the experimental group, inordinate array and loss of cilia was observed, a few compound cilia and cytoplasmic protrusion were also found. Both endoplasmic reticulum and mitochondria were swelling, and the lymphocytes were infiltrating with fibroblast proliferation in the submucosa. There was statistically significant difference between the experimental group and the blank control group (P < 0.05). In the experimental group, the number of compound cilia increased from 1 to 4 weeks, and the amount of compound cilia of the mucosa at 3 and 4 weeks was significantly higher than that at 1 week (P < 0.05). Swelling of mitochondria and endoplasmic reticulum was severe at 2 weeks and abated gradually with time, the results at 2 weeks were different from those of experimental group at 4 weeks (P < 0.05). CONCLUSION: The obstruction of nasal sinuses and the bacterial infection might lead to ultrastructural changes of maxillary sinus mucosa, and these ultrastructural changes were believed to the important processes of pathological changes in acute sinusitis.

Effects of intranasal corticosteroids in the treatment of experimental acute bacterial maxillary sinusitis in rabbits.

OBJECTIVE: To investigate the possible effects of intranasal corticosteroids on the focal bacterial colonization and mucosal histomorphological changes in experimental acute bacterial maxillary sinusitis in rabbits. METHODS: Acute bacterial maxillary sinusitis was induced in 48 rabbits. They were divided into 4 groups: antibiotic group (group A), antibiotic and corticosteroid combination group (group B), corticosteroid group (group C), and control group (group D). Six rabbits in each group were sacrificed to obtain secretions from the maxillary sinuses for bacterial culture after they had been treated for 2 or 4 weeks. Maxillary sinuses were removed for whole-mount histological analysis. RESULTS: The differences between bacterial culture ratios in groups A and B compared with groups C and D were significant after treatment for 4 weeks (p < 0.05), but there was no significant difference between groups A and B (p > 0.05). Semi-quantitative analyses showed that epithelial ulceration and ciliary loss in groups A and B were less pronounced than that in groups C and D (p < 0.05). Infiltration by inflammatory cells diminished more significantly in group B than in groups C and D (p < 0.05). A significant difference in inflammatory cell infiltration between groups A and B was found at the fourth week (p < 0.05). Ultrastructural changes showed a similar trend in both group A and group B. CONCLUSIONS: Intranasal corticosteroids may lessen infiltration by inflammatory cells, and their combined application does not decrease the effects of antibiotic therapy in our study. In the treatment of acute bacterial maxillary sinusitis, intranasal corticosteroids cannot be a substitute for antibiotic treatment as a single therapy, but intranasal corticosteroids administered with antibiotics provide better efficacy.