[Analysis of clinical features and surgical outcomes of petrous bone cholesteatomas].

Objective: To analyze the clinical features and surgical outcomes of petrous bone cholesteatomas (PBCs). Methods: Data from 39 PBCs patients treated in the Department of Otorhinolaryngology, Xijing Hospital from September 2011 to December 2017 were reviewed retrospectively, including 23 males, 16 femals, aged 12-71 years old, with the median age of 37. Clinical classifications, surgical methods, facial and hearing function, and intraoperative and postoperative complications were made summary analysis. Results: In this study, five patients were congenital PBCs and 34 patients were acquired PBCs. The common clinical symptoms were hearing loss (100%, 39/39), ear discharge/pus (89.7%, 35/39) and facial paralysis (46.2%, 18/39). According to Sanna's classification, 14 cases were supralabyrinthine, including three cases underwent transcochlear (TC) approach, six cases underwent transotic (TO) approach and five underwent translabyrinthine (TL) approach. 10 cases were infralabyrinthine, including eight cases underwent subtotal petrosectomy, one case underwent TO approach and one underwent TL approach.10 cases were massive, including seven cases underwent TC approach, three cases underwent TO approach. Five cases were infralabyrinthine-apical, including two cases underwent TC approach, two cases underwent TO approach, and one case underwent endoscope assisted infratemporal fossa type B. The degree of facial nerve (FN) dysfunction from high to low was massive (6/10), supralabyrinthine (8/14), infralabyrinthine-apical (2/5) and infralabyrinthine (2/10). 19 cases involved in facial nerve operation, three cases underwent FN decompression, four cases underwent FN rerouting, four cases underwent nerve grafting, and one case underwent facial-hypoglossal anastomosis. Preoperative FN involvement in 18 cases, and the FN function was improved in 14 cases after surgery. The improved rate of postoperative FN function was 77.8%. The bone conducted hearing retained 50.0% (14/28) postoperatively. Five cases with cerebrospinal fluid leak were managed by inserting free muscle plugs and cavity obliteration. Two cases with the cholesteatomas matrix involved the sigmoid sinus and the jugular bulb, and occlusion of the sigmoid sinus was performed. Postoperatively, two patients presented with synkinesis. The patients were followed up for 40 to 115 months, and there was no recurrence. Conclusions: There are no specific clinical manifestations for PBCs, thus, it is difficult in early diagnosis and treatment. According to Sanna's classification, preoperative FN and hearing function, the best surgical approach should be selected with minimal recurrences and perioperative morbidity.

[Crouzon syndrome with secretory otitis media: a case report and literature review].

Summary Clinical data from a case of Crouzon syndrome with secretory otitis media in our department was collected and the related literatures were reviewed. Whole exome sequecing and Sanger sequencing were performed to analyze genetic cause. The 6-year old patient with Crouzon syndrome had snoring and mouth breathing during sleep for 2 years, and was found hearing loss for 2 weeks. The results of endoscopy showed adenoid hypertrophy and secretory otitis media of both ears. And CT scan proved chronic rhinosinusitis. Myringotomy and adenoidectomy were done under general anesthesia. The follow-up at 6 months showed normal sleep and hearing level. A heterozygous fibroblast growth factor receptor 2 missense mutation（c.1061C>G, p.S354C） in exon 8 was identified in this patient.

[Analysis of predictors for Frey syndrome after parotidectomy].

Objective:To analysis the data of the patients with parotid tumors, clarify the contributing factors of Frey syndrome, and to evaluate the role of soft tissues membrane SIS in prevention of Frey syndrome after parotidectomy. Method:The data of 95 patients who suffered from parotid tumors and underwent parotidectomy were included in this study. The relationship between the patients' age, sex, tumor location ,tumor size, disease pathology, type of resection, SIS application and the incidence of Frey syndrome were statistically analyzed . Result:The incidence of Frey syndrome after parotidectomy for 95 patients was 25.3%. Age, sex, tumor location and size, disease pathology, type of resection did not appear to be associated with development of Frey syndrome（P>0.05）. SIS application was the only statistically significant factor（P<0.01）, and SIS could prevent Frey syndrome after parotidectomy. Conclusion:Frey syndrome is one of the common complicationsafter parotidectomy. Implantation of SIS is an effective method for prevention of Frey syndrome after parotidectomy.

[Surgical management of petrous bone cholesteatoma and facial nerve protection].

Objective: To discuss the therapeutic scheme of petrous bone cholesteatoma(PBC) and the technique of facial nerve reconstruction. Method: The data of 28 patients who underwent surgery for PBC in our center were analyzed retrospectively. All patients were diagnosed radiologically with PBCs and reconfirmed pathologically after surgery. The surgical approach was discussed basing Sanna's classification of PBCs, and the facial nerve outcomes were analyzed moreover. Result: PBC cases 11 were supralabyrinthine, 4 infralabyrinthine, 3 infralabyrinthine-apical, 10 massive and none apical. The facial nerve was involved in 50% of the cases. The translabyrinthine approach were used in 3 cases. The transotic approach was used in 7 cases in this series.The transcochlear approach type was applied in 12 cases. The Infratemporal fossa type B approach and subtotal petrosectomy were employed in 2 cases and 4 cases respectively. Active management of the nerve(rerouting, anastomosis, or grafting) was required in 14 cases, postoperative facial nerve function were inproved in 10 cases(71.43%). Conclusion: The appropriate surgery approach was vitally important to radical disease clearance in PBCs. The facial nerve preservation was preceded hearing preservation. Active facial nerve management were beneficial to facial nerve recovery..

[The study for the characteristics of secondary acquired cholesteatoma].

Objective:Retrospctive analysed the Characteristics and outcomes of surgical treatment of 18 patients who were diagnosed as secondary acquired cholesteatoma (SAC).Method:Patients with SAC accepted operations were enrolled in this study. Then the factors such as sex, age, cource of history, otorrhea before operation, the size of perforation of tympanic membrane, entry site of epithelium, extension direction, ossicular destruction, tympanosclerosis, tympanum tympani tendon involvement, stage of cholesteatoma（JOS, 2015, Japan），degree of gasification of mastoid（JOS，2015，Japan），air conductive threshold, bone conductive threshold, air-bone gap, the healing of tympanic membrane and the auditory improvement were analyzed.Result:Eighteen patients were enrolled in this study, with course of history range from 2 months to 50 years (average: 20.20±16.31) years. There were 14 cases with wet ear before operation. All patients were conformed with perforation of membranal tensa by otoendoscopic photography before operation, with nearly total in 5 ears,large size in 10 ears,medium size in 2 ears and small size in 1 ear. Ossicular erosion were found in 13 patients (malleus involved in 6, incus involved in 12 and stapes involved in 4). Thirteen patients with tendon of tensor tympani involved and 9 patients with tympanosclerosis were conformed during operation. The epithelium entered through the malleus manubrium to the promontory in 13 cases, through the edge of the perforation in 3 cases and through the incus long process to around stapes in 1 case. Cholesteatoma invasion extend to anterosuperior area in 3 cases, posterosuperior area in 3 cases,both in anterosuperior and posterosuperior area in 12 cases. The cholesteatomas classified: stage Ⅰ in 11 cases, stage Ⅱ in 7 cases. Mastoid gasification classified MC0 in 6 cases, MC1 in 10 cases and MC2 in 2 cases. The average air conductive threshold was (56.32±10.15) dB, bone conductive threshold was (20.76±6.22) dB and air-bone gap (35.56±9.84) dB.Tympanic membrane healed in all patients during following up, without recurrent of cholesteatoma, and the post-operative air conductive threshold (43.02±14.96) dB and air-bone gap (21.04±12.90)dB were improved significantly(P<0.05).Conclusion:Most of SAC were secondary to nearly total or large perforation of membranal tensa (83.33%), with relative long history of chronic otitis media (average 20.20 ± 16.31) years and otorrhea before operation. The epithelium entered mainly through the malleus manubrium to the promontory，then through edge of the perforation，by extending anterosuperior and posterosuperior area and usually accompanied with tendon of tensor tympani involved, ossicular destruction, and poor mastoid gasification and tympanosclerosis. The characteristics of SAC were different from other type of cholesteatoma which need further researches.

Quantitation of hepatitis B virus DNA in serum by ammonium sulfate precipitation and molecular hybridization.

We developed a method to quantitate hepatitis B virus (HBV) DNA in serum by ammonium sulfate fractionation and DNA hybridization. Serum samples were precipitated with 45% saturated ammonium sulfate, resuspended in buffer, and spotted on a nylon membrane. Following denaturation in alkali, HBV DNA sequences on the membrane were detected by hybridization with a 32P-labeled DNA probe of the entire HBV genome. Bound radioactivity was measured with liquid scintillation counting. Ammonium sulfate fractionation of positive samples increased assay sensitivity by 10-30-fold compared to no treatment. Sensitivity for detection of cloned HBV DNA added to negative serum was 0.2 pg. Recovery of cloned HBV DNA added to negative serum before fractionation was equivalent to direct spotting of DNA onto the membrane in the absence of serum. This method enhanced HBV DNA recovery from serum into small volumes, thereby expanding the potential analytic range of spot hybridization assays.

Chemiluminographic detection of von Willebrand factor multimeric composition.

Diagnosis of von Willebrand's disease (vWD) requires quantitation of von Willebrand factor (vWF) in plasma plus qualitative assessment of the vWF multimers according to molecular size ranges. Characterization of vWF multimeric size distributions is typically done using sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE) followed by immunoblotting in the gel with radiolabeled antibody against vWF and autoradiographic exposure. We applied a western blot technique to vWF multimeric analysis. It included SDS-AGE, electroblotting onto a membrane, and chemiluminescent detection using rabbit anti-human vWF as primary antibody and goat anti-rabbit IgG as secondary antibody conjugated with horseradish peroxidase. Using this method, 18 to 20 vWF multimers were regularly resolved in normal plasma with exposure times of 2 to 4 sec compared to 4 hr or longer by autoradiography. Sensitivity of detection was at least 4-fold enhanced by chemiluminescence compared to radiolabel. Specificity of the assay was confirmed by analysis of plasma samples known to be deficient to different degrees in the larger vWF multimers. The chemiluminographic assay for vWF multimers is superior to the autoradiographic one because it is more sensitive, avoids use of radioactivity, and has shorter total assay time (under 2 days versus five radiolabel).

Interaction of Epstein-Barr viral (EBV) origin of replication (oriP) with EBNA-1 and cellular anti-EBNA-1 proteins.

We have previously shown that 12-O-tetradecanoylphorbol-13-acetate (TPA) which activates expression of the latent genome of the Epstein-Barr virus (EBV) in Burkitt lymphoma cells induces the synthesis of two cellular anti-EBNA-1 competitor proteins, anti-EBNA-1.1 and anti-EBNA-1.2. Both anti-EBNA-1 proteins can uncouple the specific binding of the EBNA-1 to the region required for EBV plasmid maintenance (oriP). Here, we show by DNase I footprinting that the binding sites on oriP for the EBNA-1 and the anti-EBNA-1 proteins were indistinguishable. The proteins bound to the 30-bp tandem repeats of the oriP. Glycerol-gradient centrifugation and gel retardation assay revealed that a 60-kDa protein formed the anti-EBNA-1.1-DNA complex and a 40-kDa protein formed the anti-EBNA-1.2-DNA complex.

Induction of anti-EBNA-1 protein by 12-O-tetradecanoylphorbol-13-acetate treatment of human lymphoblastoid cells.

Binding of the Epstein-Barr virus (EBV) nuclear antigen (EBNA-1) to BamHI-C DNA was studied by affinity column chromatography followed by immunoblotting with human serum specific for EBNA-1. Two species of EBNA-1 (68 and 70 kilodaltons) were identified in nuclear extracts of the EBV-positive Burkitt's lymphoma cell line Raji and not in nuclear extracts of the EBV-negative Burkitt's lymphoma cell line BJAB. Both EBNA-1s bound specifically to the region required for EBV plasmid DNA maintenance (oriP) located in the BamHI-C fragment. Upon treatment with 12-O-tetradecanoylphorbol-13-acetate, which activates latent EBV genome in Raji cells, the 68-kilodalton EBNA-1 was uncoupled from binding to EBV oriP. Nuclear extracts from 12-O-tetradecanoylphorbol-13-acetate-treated BJAB cells also uncoupled the binding of both EBNA-1s to oriP. DNA-cellulose column chromatography identified two protein species which competed for and uncoupled the binding of EBNA-1 to oriP. The two cellular competitors we called anti-EBNA-1 proteins had molecular masses of 60 and 40 kilodaltons, respectively. They were not found in nuclear extracts of BJAB cells not activated by 12-O-tetradecanoylphorbol-13-acetate.

Identification of Marek's disease virus nuclear antigen in latently infected lymphoblastoid cells.

A new Marek's disease virus (MDV) nuclear antigen (MDNA) was identified in two MDV-transformed T-lymphoblastoid cell lines, MKT-1 and MSB-1, derived from chickens bearing tumors induced by MDV. This MDNA was not detected in MSB-1 cells maintained in iododeoxyuridine, which activates the latent MDV genome. Moreover, it was not found in chicken embryo fibroblasts undergoing productive and cytolytic infection with MDV. Expression of MDNA is not related to strain pathogenicity in chickens, because chicken embryo fibroblasts productively infected with the pathogenic RBIB strain or the nonpathogenic CV-1 strain of MDV did not express this antigen. DNA-protein immunoprecipitation studies revealed that MDNA bound to two sites in the 190,00-base-pair (bp) MDV genome. One of these loci identified by MDNA obtained from MKT-1 and MSB-1 cells corresponded to a 476-bp segment within the short unique region of BamHI-A MDV DNA. A second locus located in a 280-bp segment within the short inverted repeat region of BamHI-A was also identified by MDNA from MSB-1 cells but not by MDNA obtained from MKT-1 cells. Analyses of the nucleotide sequence by DNase digestion showed that MDNA protected a 60-bp segment spanning a 22-bp palindromic sequence of the short unique region and a 103-bp sequence encompassing a 32-bp palindrome in the short inverted repeat region of BamHI-A MDV DNA.

A novel EBNA-1 titration method and putative anti-EBNA-1 protein.

A novel and rapid EBNA-1 titration method has been developed which uses immunoprecipitation of specific DNA-protein complexes with EBNA-1-positive serum. The method is more sensitive than the conventional immunofluorescence method and has potential value as a diagnostic reagent for clinical laboratories. TPA induction of putative anti-EBNA-1 protein of cellular origin is discussed, which may play a key role for the shift from latent to lytic replication of EBV.

Alteration in the ribonucleic acids in rat liver induced by a methionine-free total parenteral nutrition solution.

The effect of infusion of a methionine-free total parenteral nutrition solution for 7 d on ribonucleic acids in liver of rats were investigated. The control solution contained leucine, valine, isoleucine, lysine, phenylalanine, threonine, tryptophan, arginine, histidine, glycine, methionine, glucose and vitamins and minerals. Deprivation of a methionine is known to increase the activity of RNA polymerase I. Infusing the methionine-free solution resulted in the accumulation of RNA molecules larger than 28S in the liver nuclei and resulted in a higher rate of rRNA synthesis than in rats infused with the control solution. A methionine deficiency did not impede either the processing of 45S pre-rRNA or transport of 28S and 18S rRNA into cytoplasm. When rats were infused with the methionine-free solution for 7 d followed by the control solution for 2 d, the level of RNA in the nucleus as well as the rate of RNA polymerase I were similar to the levels in rats receiving the control solution for 9 d. There were no significant changes in the rate of DNA synthesis due to nutritional manipulations.

Effect of ethionine on synthesis and methylation of ribosomal ribonucleic acid in regenerating rat liver.

Ethionine, a hepatocarcinogen, was administered into rats 24 h before partial hepatectomy and immediately thereafter. Hepatic precursor ribosomal RNA (pre-rRNA) obtained 20 h after the operation of rats injected with ethionine and adenine resulted in methyl deficiency as judged by the incorporation of [3H]methyl group of S-adenosylmethionine into nuclear rRNA by partially purified rRNA methylase. The ethionine and adenine treatment causes methyl deficiency of nuclear rRNA at 2'-hydroxyribose sites of cytidine and uridine, but not at base sites. Although the ethionine and adenine treatment produced no significant change in total hepatic RNA synthesis in vivo assayed by the incorporation of labeled orotate, a one-third increase in nuclear rRNA synthesis as well as a one-third decrease in microsomal rRNA synthesis was found under the treatment. These results suggest that the undermethylation at 2'-hydroxyribose of pre-rRNA in liver nucleus, which is caused by ethionine and adenine administration into rats, causes an inhibition of the processing of nuclear pre-rRNA to cytoplasmic rRNA.