The correlation between FeNO and nNO in allergic rhinitis and bronchial asthma.

ABSTRACT: This study aimed to evaluate the correlation between fractional exhaled nitric oxide (FeNO) and nasal nitric oxide (nNO) in allergic rhinitis (AR) and patients with or without bronchial asthma (BA).A total of 90 patients who were diagnosed with persistent AR (AR group, n = 30), BA (BA group, n = 30), or allergic rhinitis with bronchial asthma (AR-BA) (AR-BA group, n = 30), were enrolled in this study, along with 30 healthy adult volunteers (control group, n = 30). The participants were further divided into 2 groups based on the results of a skin-prick test (SPT): a highly atopic group (SPT = 3+ and above) and a moderately atopic group (SPT = 2+ and below). All participants underwent FeNO and nNO measurement, an absolute blood eosinophil count, total serum immunoglobulin measurement, and horizontal baseline lung capacity determination.The results showed that the FeNO levels in the 3 observation groups were significantly higher than those in the control group (P < .01), and in the BA group they were significantly higher than in the AR-BA group (P < .01). The levels of nNO in both the AR group and the AR-BA group were higher than those in the control group and the BA group (P < .01), but there was no significant difference between the AR group and the AR-BA group (P > .05). The levels of nNO in the BA group were also significantly different from those in the control group (P < .01).FeNO and nNO are positively correlated with the degree of AR in patients with BA; therefore, nNO levels can be used as an inflammatory marker of AR in patients with BA. FeNO can also be used as an inflammatory marker of AR in patients complicated with BA as a warning indicator of asthma.

A novel Rap-Phr system in Bacillus velezensis NAU-B3 regulates surfactin production and sporulation via interaction with ComA.

Several quorum sensing systems occurring in Bacillus subtilis, e.g. Rap-Phr systems, were reported to interact with major regulatory proteins, such as ComA, DegU, and Spo0A, in order to regulate competence, sporulation, and synthesis of secondary metabolites. In this study, we characterized a novel Rap-Phr system, RapA4-PhrA4, in Bacillus velezensis NAU-B3. We found that the rapA4 and phrA4 genes were co-transcribed in NAU-B3. When rapA4 was expressed in the heterologous host Bacillus subtilis OKB105, surfactin production and sporulation were severely inhibited. However, when the phrA4 was co-expressed, the RapA4 activity was inhibited. The transcription of the surfactin synthetase srfA gene and sporulation-related genes were also regulated by the RapA4-PhrA4 system. In vitro results obtained from electrophoretic mobility shift assay (EMSA) proved that RapA4 inhibits ComA binding to the promoter of the srfA operon, and the PhrA4 pentapeptide acts as anti-activator of RapA4. We also found that the F24 residue plays a key role in RapA4 function. This study indicated that the novel RapA4-PhrA4 system regulates the surfactin synthesis and sporulation via interaction with ComA, thereby supporting the bacterium to compete and to survive in a hostile environment. KEY POINTS: •Bacillus velezensis NAU-B3 has a novel Rap-Phr quorum sensing system, which does not occur in model strains Bacillus subtilis 168 and B. velezensis FZB42. •RapA4-PhrA4 regulates surfactin production and sporulation. •RapA4-PhrA4 interacts with the ComA protein from ComP/ComA two-component system.

Effects of glucocorticoid on the expression and regulation of aquaporin 5 in the paranasal sinus of rats with chronic rhinosinusitis.

Aquaporins (AQPs) are water-specific membrane channel proteins that regulate water homeostasis for cells and organisms. AQP5 serves an important role in the maintenance of mucosal water homeostasis, and potentially contributes to mucosal edema and inflammation formation in chronic rhinosinusitis (CRS). The aim of the present study was to explore the expression pattern of AQP5 and the effect of glucocorticoids on AQP5 expression in rats with CRS. The rats were randomly divided into three equal groups, as follows: CRS, dexamethasone (dexa) treatment and control groups. A polyvinyl acetal material containing Staphylococcus aureus was inserted into the left nasal cavity of each rat from the CRS and dexa groups. On the 90th post-operative day, the dexa group received dexamethasone (2 mg/kg/day) via intraperitoneal injection for 7 days. The controls did not receive any treatment. The expression of AQP5 in the sinonasal mucosa was determined using immunohistochemistry and quantitative PCR. The immunoreactivities of AQP5 were primarily noted in the epithelial lining and glandular cells, the vascular endothelium and in the goblet cells in the sinonasal mucosa. The AQP5 mRNA expression level was significantly higher in the dexa group than in the control and CRS groups (P=0.006 and P=0.014, respectively). However, no significant difference was indicated between the CRS and control groups (P=0.760). In conclusion, the current study suggests that glucocorticoids induce AQP5 expression in the sinonasal mucosa of CRS rats, which highlights AQP5 as a potential target in the diagnosis and treatment of CRS.

Increased expression of toll-like receptors 2 and 4 and related cytokines in persistent allergic rhinitis.

OBJECTIVES: Toll-like receptors (TLRs) are the crucial components of host defenses and supposed to play a role in nasal inflammation such as chronic rhinosinusitis and seasonal allergic rhinitis. This study was performed to investigate the expression patterns of TLRs and related cytokines in persistent allergic rhinitis (PER). STUDY DESIGN: Experimental study of human nasal tissue. SETTING: Academic medical center. SUBJECTS AND METHODS: Nasal biopsy specimens were obtained from 21 patients with PER and 21 controls from December 2012 to September 2013. Messenger RNA (mRNA) expression of TLR1-9, interleukin (IL)-1, IL-6, IL-8, IL-12, interferon (IFN)-α, and tumor necrosis factor (TNF)-α was determined by quantitative reverse transcription polymerase chain reaction. The cellular localizations as well as protein expression of TLR2 and TLR4 were further detected by immunohistochemistry and Western blotting, respectively. RESULTS: TLR1-9 mRNA could be determined in nasal mucosa. Compared with the controls, mRNA expression of only TLR2 and TLR4 was significant higher in patients with PER (P < .05). In addition, mRNA expression of IL-6 and IL-8, but not IL-1, IL-12, IFN-α, and TNF-α, was upregulated in patients with PER vs control subjects (P < .05). However, these increased cytokines were not correlated with either TLR2 or TLR4 in patients with PER. Protein expression of TLR2 and TLR4 was consistent with mRNA levels (P < .05). The cellular distributions of TLR2 and TLR4 were localized in nasal epithelium, subepithelial glands and capillary endothelial cells, and immune cells. CONCLUSION: TLR2 and TLR4 are increased in patients with PER and may be one of the major contributors to the persistence and aggravation of allergic inflammation in PER.

Inhibition of Myo6 gene expression by co­expression of a mutant of transcription factor POU4F3 (BRN­3C) in hair cells.

An eight­base pair (bp) deletion in the Pou4f3 gene in hair cells is associated with DFNA15, a hereditary form of hearing loss. To explore the pathological mechanisms underlying the development of DFNA15, the effect of the mutation in Pou4f3 on the activity of the myosin VI (Myo6) promoter, was investigated. The upstream regulatory sequence of Myo6 (2625 bp), consisting of an 1899 bp upstream sequence and a 727 bp intron 1 sequence, was amplified using polymerase chain reaction and subcloned into the pGL3­Basic vector expressing firefly luciferase. For verification of inserted fragments, plasmids were subjected to restriction analysis and then sequenced. HEK293T human embryonic kidney cells were transiently transfected with renilla luciferase­thymidine kinase vectors expressing Renilla luciferase and the Myo6 promoter­driven firefly luciferase expressing vectors along with pIRES2­enhanced green fluorescent protein (EGFP)­Pou4f3 (expressing wild­type Pou4f3) or pIRES2­EGFP­Pou4f3 (expressing the truncation mutant of Pou4f3). The relative luciferase activities were measured to determine the activity of the Myo6 promoter. The Myo6 promoter activity was not affected by co­expression of wild­type Pou4f3, as indicated by the comparable relative luciferase activities in the presence of the pIRES2­EGFP­Pou4f3 and the empty control vectors. However, co­expression of mutated Pou4f3 significantly inhibited the activity of the Myo6 promoter to almost half of that of the control (P<0.001). The data suggests that mutated Pou4f3 has a negative role in the promoter activity of Myo6, and by extension, the expression of myosin VI, and this may be an underlying mechanism of DFNA15 hearing loss.

Myosin light chain kinase regulates hearing in mice by influencing the F-actin cytoskeleton of outer hair cells and cochleae.

Myosin light chain kinase (MLCK) phosphorylates myosin regulatory light chains to facilitate its interaction with actin filaments and produce contractile activity. The outer hair cells (OHCs) in the ear contain large amounts of actin and a variety myosins. The stereociliary and somatic motility of OHCs are closely related to hearing. It appears likely that MLCK may play an important role in acoustic trans-duction. In this study, we analyzed, both in vivo and in vitro, the OHCs of mice bearing a specific deletion of the MLCK gene and the OHCs of control mice. The phenotype was assessed by auditory function [acoustic brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs)], inner ear morphology and histology. MLCK-deficient mice aged 6-7 months showed impaired hearing, a 5- to 10-dB sound pressure level (SPL) increase in the ABR thresholds, when responding to clicks and tones of different frequencies (8 and 16 kHz) (P<0.05). The DPOAE amplitudes of 3-month-old MLCK-deficient mice decreased significantly (>10 dB SPL) at low frequencies (4, 5 and 6 kHz). The OHCs in the MLCK-deficient mice increased with abnormal stereocilia. The staining of F-actin and the phosphorylation of the regulatory light chain in MLCK-deficient OHCs was weak. Our results indicate that MLCK may regulate the structure and the motility of stereocilia through F-actin polymerization.

[A guinea pigs model of otitis media with effusion caused by reversible Eustachian tube obstruction].

OBJECTIVE: To develop a animal model for acute otitis media with effusion (OME). METHODS: In 22 guinea pigs, the left nasal orifice of Eustachian tube was approached via a transpalatal incision and obstructed with polyvinyl acetal material. Right ears were set as the control. Then all the ears were evaluated by otomicroscopy every day. Seven, 14 and 21 days after the intervention, six guinea pigs were killed for histologic study. RESULTS: Of the 22 guinea pigs included in this study, 20 ears (90.9%) were found to have effusion 3 - 7 days after the operation, two cases were excluded for purulent otorrhea 10 days postoperatively. The epithelium initially developed hyperplasia, and the submucosa showed vascular and lymphatic dilatations with inflammatory cells infiltration. None of the contralateral control ears had evidence of disease by otomicroscopic examination and histologic study. CONCLUSION: This experimental methods provoked reproducible pathologic characteristics similar to those for otitis media with effusion.

[Clinicopathologic characteristics and chromosomal abnormalities in salivary mucosa associated lymphoid tissue lymphomas].

OBJECTIVE: To study the morphological and genetic characteristics in salivary gland marginal zone B cell lymphoma of mucosa associated lymphoid tissue (MALT) lymphomas. METHODS: Twenty-eight cases of MALT lymphomas of salivary gland were collected from Department of Pathology, Cancer Hospital of Fudan University. Morphological review based on HE sections, and specific chromosomal abnormalities were detected by two-color interphase fluorescent in situ hybridization (FISH). Four different probes were available to detect for API2-MALT1 fusion gene, bcl-10, IgH and MALT1 gene, respectively. RESULTS: There were 16 females and 12 males, median age was 52. In those cases, 18 originated from parotid gland, 6 from submandibular and 4 from sublingual gland. Ten were localized mass and 18 were masses diffusely involved the glands. According to the clinical information, only 8 cases showed symptoms of dry mouth, dry nose or dry eye. Pathological findings showed that all cases had a dense lymphoid infiltration and obliteration and atrophy of acini and ducts. Twenty-two (78.6%) showed prominent monocytoid B cells and more often formed broad halos around epithelial islands. Eighteen (64.3%) showed clusters of lymphoblastic cells or plasma cells, Russel' and Dutcher' body were easily seen. Ten (35.7%) showed nerve or blood vessel infiltration. Interphase FISH showed that 3 cases harbored t(11;18) and 2 cases harbored trisomy 18, but none of all found IgH and bcl-10 translocations. After operation, 22 patients' follow-up information was available. One case died on 15 months later after operation, the rest of 21 cases were alive. Except surgical resection, patients did not get systematic radio-or chemotherapy. Eight to fifteen months after operation, 8 cases found recurred nodules on the original resected sites or cervical lymph nodes, but did not get repeated biopsy. All follow-up time was from 23 to 54 months. CONCLUSIONS: Most salivary MALT lymphomas are arising from parotid glands. Most patients do not have the symptoms of the Sjogren's syndrome. The final diagnosis depends on the pathological findings, the number and distribution of monocytoid B cells and clusters of plasmacytoid cells are hints for diagnosis of salivary MALT lymphomas, invasion of blood vessels or nerve also help for malignant diagnosis. t(11;18) and trisomy 18 may be the main chromosomal abnormalities in salivary gland MALT lymphomas, but with low morbidity. This genetic characteristic may connect with the low malignancy and slow progression in biological behavior.

Anti-tumor immunity against nasopharyngeal carcinoma by means of LMP2A-specific cytotoxic T lymphocytes induced by dendritic cells.

OBJECTIVE: Adoptive immunotherapy with specific cytotoxicity T lymphocytes (CTLs) induced by dendritic cells (DCs) pulsed with tumor antigens plays a crucial role in the immunity against tumor. The purpose of this study was to assess the feasibility and efficacy of latent membrane protein 2A (LMP2A)-specific CTLs immunity against nasopharyngeal carcinoma (NPC) in vitro and in vivo. METHODS: DCs were generated by culturing the monocytes purified from human peripheral blood mononuclear cells (PBMCs) in cytokine cocktail containing granulocyte/macrophage colony-stimulating factor (GM-CSF), interleukin-4 (IL-4), and tumor necrosis factor-alpha (TNF-alpha). The phenotype of mature DCs was analyzed by fluorescence activated cell sorter (FACS). Mature DCs transfected with EBV-LMP2A recombinant adenovirus were co-cultured with autologous PBMCs to induce LMP2A-specific CTLs. The expression of the surface antigens such as CD3, CD4, CD8 and CD56 on CTLs were detected by FACS. The specific cytotoxicity of CTLs on target CNE cells expressing EBV-LMP2A was confirmed using cytotoxicity assay. The anti-tumor effect of LMP2A-specific CTLs in vivo was assessed in the mice tumor models implanted with CNE cells expressing EBV-LMP2A. RESULTS: Mature DCs expressed typically morphologic characteristics and high level of surface markers (CD1a, CD83, CD86, CD80 and HLA-DR). LMP2A-transfected DCs could induce LMP2A-specific CTLs consisting of a majority of CD4+ and CD8+ T cells. Cytotoxicity assay confirmed that the LMP2A-specific CTLs displayed significant cytotoxicity on target CNE cells compared with the controls. The study in vivo demonstrated that the treatment using these specific CTLs retarded the growth of established tumor in the treated mice. CONCLUSION: These findings suggest that DCs transfected with EBV-LMP2A recombinant adenovirus can elicit LMP2A-specific CTLs that have a specific killing effect on NPC in vitro and in vivo. The results provide experimental basis for the further immunotherapy of NPC in clinical trails.

[Anti-nasopharyngeal carcinoma effect in vivo and in vitro of cytotoxicity T lymphocyte induced by Ad5-latent membrane protein 2A].

OBJECTIVE: To study the biological characteristics of cytotoxicity T lymphocyte (CTL) induced by dendritic cell (DC) transfected with the Epstein-Barr virus latent membrane protein 2A (EBV-LMP2A) recombinant adenovirus. To establish nasopharyngeal carcinoma (NPC) animal models expressing LMP2A and investigate the anti-tumor effect of LMP2A specific CTL in vivo. METHODS: The mononuclear cells were isolated from human peripheral blood mononuclear cells (PBMC) and cultured with the cytokines [granulocyto-monocyte colony stimulating factor (GM-CSF), interleukin-4 (IL-4) and tumor necrosis factor-alpha TNF-alpha]. The expression of surface markers on mature DC was detected by fluorescence activated cell sorter FACS. Mature DC were transfected with LMP2A recombinant adenovirus. Under the help of interleukin-2 (IL-2), LMP2A specific CTL were induced by coculturing LMP2A-transfected DC with autologous PBMC. The population of CTL was detected by FACS. NPC animal models were constructed by implanting CNE cells expressing LMP2A subcutaneously into BALB/c nude mice. After intra-tumoral injection of LMP2A specific CTL, the size of tumor was measured. The tumors were removed after 30 d and subjected to histological examination. RESULTS: Mature DC displaying typical characteristics of morphology and phenotype were obtained from monocytes cultured in the medium containing GM-CSF, IL-4 and TNF-alpha. The LMP2A specific CTL induced by transfected DC were composed of mainly CD4+ and CD8+ T cells. The NPC animal models were constructed three weeks after implanting CNE cells. The study in vivo indicated that the tumors treated with LMP2A specific CTL grew slowly compared with control. Tumor volume of treated groups was significantly smaller than that of controls. The histological sections showed local necrosis and infiltration of lymphocyte in tumor tissue. CONCLUSIONS: Typically mature DC could be generated in vitro by culturing monocytes with the cytokines. LMP2A-specific CTL could be induced by LMP2A transfected DC in vitro. NPC mice models could be constructed by implanting CNE cells. LMP2A specific CTL could inhibit the growth of implanted tumor and produce an anti-tumor effect in vivo.