Clinical and histopathologic features of biofilm-associated chronic rhinosinusitis with nasal polyps in Chinese patients.

BACKGROUND: Biofilms have given new insights to the understanding of pathogenesis of chronic rhinosinusitis (CRS). However, the link between biofilms formation and local inflammatory response remains poorly defined in CRS with nasal polys. The aim of this study was to determine the potential association of the presence of biofilms in the nasal mucosal tissues with clinical features in Chinese patients, which had CRS with nasal polyps (CRSwNP). METHODS: A total of 19 patients with CRSwNP and 12 patients with non-CRS were subjected to endoscopic surgery and their nasal mucosal tissue specimens were examined histologically and by scanning electron microscopy (SEM). Their demographic and clinical features were recorded. RESULTS: Thirteen (68.4%) out of the 19 specimens from patients with CRSwNP, but none from control patients, were positive for biofilms that displayed typical characteristics of bacterial and fugal structures. The presence of biofilms in the nasal mucosal tissues was associated with significantly greater values of purulent nasal discharge and preoperative Lund-Kennedy scores, higher levels of serum total IgE and percentages of subjects with endoscopic surgery (ESS) history in patients with CRSwNP, and more severe inflammation in the nasal mucosal tissues of patients with CRSwNP. CONCLUSION: Our study demonstrated the presence of biofilms in the nasal mucosal tissues of many patients, contributing to the understanding of the pathogenic process of CRSwNP in Chinese patients.

[Changes of the ultrastructures of temporomandibular joint after removal of emotional stress factors in rats].

OBJECTIVE: To examine the changes of the ultrastructures of temporomandibular joint after removal of the emotional stress factors in rats. METHODS: Thirty-two 12-week-old male Wistar rats were divided into two groups randomly, experimental group and control group. Each group was divided into two subgroups according to execution time, 9-week subgroup and 12-week subgroup with eight rats in each subgroup. Chronic unpredictable stress animal model were firstly established in experimental group in the first 6 weeks, then all the stimulation factors removed and breed normally. After 9 weeks, rats in 9-week subgroup were killed. After 12 weeks, rats in 12-week subgroup were killed. All condyles and articular discs were dissected and observed by scanning electron microscope. RESULTS: There was some recovery in condyles and articular discs in experimental group under scanning electron microscope. The gelatum on the surface of condyles increased, collagen fibrils became regular and deep layer collagen fibrils less exposed. There were no such obvious changes on the surface of condyles and articular discs in control group. CONCLUSIONS: The ultrastructure injures of temporomandibular joint in rats induced by emotional stress could be reversed if the stress factors were removed.

[Influence of chronic unpredictable emotional stress on the ultrastructures of temporomandibular joint in rats].

OBJECTIVE: To study the influence of emotional stress on the ultrastructures of temporomandibular joint in rats. METHODS: Sixteen male Wistar rats, aged 12 weeks, were divided into 2 groups randomly, experimental group and control group. Animal model of chronic unpredictable stress was established in the experimental group. After 6 weeks, all rats in both groups were killed and their condyles and articular discs were dissected, taken out and observed by scanning electron microscope. RESULTS: Under scanning electron microscope, the surface gelatum of the condyles in experimental group were cracked, and collagen fibrils in disorder and broke, and deep layer collagen fibrils were exposed. The articular discs' surface gelatum were still integrated, but the gyri dispose were irregular. In control group, there were no such changes in condyles and articular discs. CONCLUSIONS: The long-term emotional chronic unpredictable stress could induce the injuries of temporomandibular joint in rats in ultrastructure level.

[Evidence of bacterial biofilms in chronic rhinosinusitis].

OBJECTIVE: To observe the presence of bacterial biofilms in mucosal specimens in patients operated for chronic rhinosinusitis. METHODS: A total of 12 subjects undergoing endoscopic sinus surgery were included. The control group was 6 patients with obstructive sleep apnea-hypopnea syndrome. Six patients with chronic rhinosinusitis were enrolled in the study group. Mucosa of uncinate process, ethmoid bulla or maxillary sinus was obtained during endoscopic sinus surgery. All the samples were prepared using standard methods for scanning electron microscopy. Patients' information such as age, gender, symptoms, sinus CT, endoscopic examination, skin prick test were recorded in detail. RESULTS: Standard-preparation scanning electron microscopy displayed denuded epithelium and disarrayed cilia in chronic rhinosinusitis patient's mucosa. In the study group, bacterial biofilms of different morphology were seen in five samples; One sample showed filamentous structure like fungi. Using strict scanning electron microscopy morphologic criteria, 83.3% samples in the study group were found to have micrographic evidence of biofilms. No bacterial biofilms were detected in the control group. CONCLUSIONS: In the patients undergoing surgery for chronic rhinosinusitis, different degree of mucosal injury could be found. Bacteria biofilms of different life stages were demonstrated to be present. No bacterial biofilms were detected in the control group.

Encapsidating artificial human papillomavirus-16 mE7 protein in human papillomavirus-6b L1/L2 virus like particles..

BACKGROUND: Human papillomaviruses (HPVs) can infect squamous or mucosal epithelia and cause cervical cancer or genital warts. Coinfection with multiple HPV types is a common finding of many epidemiological studies. Therefore, it is necessary to develop a vaccine, which can eradicate established HPV infections and prevent other HPV infections. In this study, we generated chimeric virus like particles (cVLPs) composed of HPV-6b L1, HPV-6b L2 and one artificial HPV-16 mE7 proteins. METHODS: The artificial HPV-16 mE7 gene was designed by codon modification, point mutation and gene shuffling then chemically synthesized and subcloned behind HPV-6b L2. HPV-6b L1 and L2-mE7 were expressed in insect cells by using Bac-to-Bac system. The generated cVLPs were purified by CsCl gradient ultracentrifuge and analyzed by immunoblot, electron microscope and haemagglutination assay. RESULTS: The HPV-6b L1 and L2-mE7 proteins were well expressed in insect cells and could selfassemble into cVLPs, whose diameter was about 55 nm and similar to that of HPV-6b L1/L2 VLPs. Intact cVLPs could be recognized by H6.M48 neutralizing monoclonal antibody and HPV-6b L2 polyclonal antibody, while the denatured cVLPs, but not the intact cVLPs, were reactive to HPV-16 E7 polyclonal antibody. HPV-6b L1/L2-mE7 cVLPs haemagglutinated mouse erythrocytes as efficiently as HPV-6b L1/L2 VLPs did. CONCLUSIONS: The insertion of the 158 amino acid HPV-16 mE7 protein behind L2 did not disrupt the correct assembling of cVLPs. The morphological characteristics and haemagglutinating activity of cVLPs were similar to those of HPV-6b L1/L2 VLPs. The cVLPs retained conformational B cell epitopes of HPV-6 VLPs and HPV-16 mE7 protein had an internal location in the cVLPs. Therefore, large modified E7 protein with higher immunogenicity could be incorporated into cVLPs by fusing to the C-terminus of L2, which would help to improve the therapeutic effects of L1/L2-E7 cVLPs.

[Isolation and identification of SARS-coronavirus in nasal and throat swabs collected from clinically diagnosed SARS patients].

OBJECTIVE: To isolate and identify SARS-coronavirus in nasal and throat swabs collected from clinically diagnosed severe acute respiratory syndrome (SARS) patients. METHODS: Nasal and throat swab specimens were inoculated onto well of 24-well plate containing confluent monolayers of Vero and MRC-5 cells. Isolates were identified with serology, electron microscopy and genome sequence. RESULTS: One hundred and fifty-eight nasal and throat swabs specimens from 79 SARS patients in Peking Union Medical College Hospital between April and May, 2003 were cultured for SARS-coronavirus. Cytopathic effect (CPE) was found in three nasal swab specimens inoculated in Vero cells. Acute and convalescent phase serum specimens collected from SARS patients were found with seroconversions and/or a fourfold or greater rises in indirect fluorescence antibodies (IgG and IgM) titers when the 3 isolates (infected Vero cells) were used as antigen. Coronavirus was observed in the culture supernatant by negative-stain electron microscopy. Genome sequence confirmed the isolates were SARS-coronavirus. CONCLUSIONS: The 3 isolates from nasal and throat swabs samples collected from 79 clinically diagnosed SARS patients were SARS coronavirus.

[Effect of negative pressure suction on rabbit optic nerve and retina].

OBJECTIVE: To study the effect of acute intraocular pressure (IOP) elevation on the optic nerve and retina in laser in situ keratomileusis (LASIK). METHODS: Acute IOP was increased to 65 mm Hg for different period of time (30 s, 1 min and 3 min) by using scleral suction on rabbit eyes. The tissues of retina and optic nerve of immediately extirpated group (instant group), of 2 weeks later extirpated group (recovery group) both after the negative pressure suction and of normal rabbit eyes were examined with electron and light microscope. RESULTS: After the negative pressure suction for 30 s, 1 min, and 3 min respectively, the optic nerve and retinal cells changed lightly; some part of optic nerve and retinal cells changed; optic nerve fibers and retinal cells changed sharply. CONCLUSION: Ultrastructural changes of retina and optic nerve may be induced by acute IOP elevation in LASIK. The longer the time, the more apparent the changes.

Expression of human papillomavirus type 6 L1 and L2 isolated in China and self assembly of virus-like particles by the products.

To study variations of genome late region of human papillomavirus type 6 (HPV-6) isolated in China and assembling capabilities of the encoded capsid proteins, HPV-6 L1 and L2 sequences were cloned and used for expression in Bac-to-Bac baculovirus expression systems (Gibco BRL). Based upon L1 and L2 overlapping sequence two sequences (GenBank accession number AY015006, AY015008) of HPV-6 late region (2869 bp long) were assembled and classified into HPV-6b by phylogenetic analysis. Compared with prototype sequence, nine point mutations were found, including four missense mutations. L1, instead of L2, could self-assemble into virus-like particles (VLPs) in Sf9 nucleus. VLPs self-assembled by L1 alone (L1-VLPs) and by L1 plus L2 (L1+L2-VLPs) were purified and further characterized. Both types of VLPs were spherical particles with a diameter of approximately 50 nm. L1+L2 VLPs comprising L1 and L2 in the molar ratio of about 4:1 possessed the HPV-6 L1 VLP surface and conformational epitopes. In co-expression assay with a series of MOI combination of L1 and L2 recombinant baculoviruses (total MOI=10), existence of L2 of certain level enhanced L1 production by 0.8 fold and VLP production by 3 - 4 folds under experimental conditions. In conclusion, variation rate of HPV-6 genome late region is less than 0.28% and the substitutions A to G at position 7081 and G to A at 7099 may represent region characteristics. The cloned HPV-6 L1 and L2 sequences can be expressed efficiently in Sf9 cells, and the expressed products (L1 or L1+L2) can self-assemble into VLPs that resemble naturally occurring virions.

[Construction of herpes simplex virus type I glycoprotein D DNA vaccine and its preliminary study].

OBJECTIVE: The goal of this study was to construct a eukaryotic expression plasmid containing the gene encoding herpes simplex virus type I glycoprotein D (HSV-1, gD) and evaluate its utility for DNA immunization in mice. METHODS: The gD gene was amplified from viral DNA using PCR with EcoR I and BamH I restriction sites encoded on 5' and 3' ends, respectively. The PCR fragment was inserted into the transfer vector pGEM-T Easy. gD was then cut from this vector and inserted into the EcoR I and BamH I sites in the pcDNA3.1 at the multiple cloning sites (MCS). The recombinant plasmid, pcDNA3.1-gD1, was transfected into COS-7 cells using Lipofectamine according to the manufacture's instructions. The expression of the glycoprotein D was analyzed by immunoblotting of the cell lysates. 4-6 weeks old BALB/C mice were given two injections at tibia anterialis muscle, each containing 100 micrograms of plasmid DNA, on days 0 and 15. pcDNA3.1 was used as negative control. Blood samples were taken from all mice at weeks 0, 2, 4, and 6 after the first inoculation. Standard indirect ELISA was employed to evaluate the levels of specific total Ig in serum. RESULTS: The recombinant plasmid was confirmed with restriction digestion and sequencing to contain target gene segment and expressed in COS-7 cells in vitro shown by Western blotting. The pcDNA3.1-gD1 immunized group induced specific antibody response as compared to the negative control, and the titer was about 1:2000. CONCLUSIONS: The recombinant plasmid pcDNA3.1-gD1 is potential to be used as a candidate vaccine, for the treatment of HSV-1 infection.