10.3969/j.issn.2095-4344.2013.24.024

Using immunoenzymatic technique,we have observed the localization ofimmunoglobulins in human palatine tonsils,of patients suffering from chronictonsilitis and hyperplasia.The result as follows:1.Epithelium:The outer surface of tonsil is lined by stratified squamous epithelium,the epithelium rarely shows keratinization and lacks a well-defind and continuousbasement membrane.In the basal layer of epithelium,it is reticular structure andspaces between the cords of epithelium cells are filled with Ig~+ lymphocytes andplasma cells,the reticular structure is particularly pronounced near the crypts.Thisspecial structure is regarded as an expression of active immune function.In thesuperficial layer of epithelium,the predominate cells are Ig~- small lymphocytes,maybe T lymphocytes.2.Follicle:All follicles have germinal centres,especially hyperplastic tonsils.The follicle has polarity,each follicle may be divided into three Zones(a b c),twozones(a b)form the centre of the follicle and the thired(c)is the mass of smalllymphocytes“capping”and enclosing the centre.The upper limits of zone(c)areoften indistinct,from the presence of many lymphocytes in the epithelium.Thedistribution of Ig~+ cells in follicle are different from their three zones.In the centre,IgG cells are most numerous,next most numerous are IgA~+ cells,and considerablyfewer IgM positive cells.In the c zone,only a few Ig~+ cells are seen(IgG IgM).Itis found that the germinal centre may produce more than one type of Ig,it differsfrom the report of Sordat,but quite agrees with Curran's result.However the numbersof cells containing the various types of Ig show a great difference.3.Other lymphoid tissue:The tissue between the follicles are rich in small Tlymphocytes and a few Ig~+ containing cells,this area was called thymic dependentarea.At an older age,this region is always enlarged,but the density of the cellswill decrease,meanwhile much connective tissues are seen in the stroma.

Radioactive seed implantation in pancreatic cancer is a very effective treatment. Its indications, contraindications and complications have been improved gradually. Radioactive seeds can be implanted precisely under CT or ultrasound-guided. Treatment of seeds can be combined with chemotherapy, radiofrequency ablation and other means to improve the clinical efficacy.

Objective To analyze the morbidity of the old people with hypertension and metabolic syndrome (MS),the characteristics of MS components distribution,and correlation hypertension and MS.Methods 438 old patients over 60 with hypertension were selected randomly from cheek-up crowd in the physical examination center of Affiliated Zhongshan Hospital of Dalian University in 2013.Abdominal girth,height,body mass were measured,and then calculated BMI.The indicators such as FPG,TC,TG,HDL-C,LDL-C were detected and analyzed.Results The total morbidity of MS in the old people with hypertension was 37.7％,39.0％ from male people,36.7％ from female people,and there was no difference between genders(x2 =0.46,P ＞ 0.05).The level of BMI,abdominal girth,FPG,and TG in the old people with MS were higher than the people without MS(t =4.83,8.53,5.08,7.29,all P ＜0.05),and HDL-C was lower than the people without MS(t =-9.67,P ＜ 0.05).The level of FPG in women was higher than that in men apparently(x2 =5.82,P ＜ 0.05),and the level of HDL-C was lower than that in men apparently (x2 =8.73,P ＜ 0.01).The risk factors to MS include BMI,abdominal girth,FPG,TG (OR =2.139,1.106,2.156,2.315,all P ＜0.05),and HDL-C is protective factor to MS(OR =0.039,P ＜0.05).Conclusion Hypertension might increase MS morbidity of old patients.Hypertension related with MS closely.The risk factors include BMI,abdominal girth,FPG,TG,on the other hand HDL-C is protective factor to MS.

Objective To investigate the molecular mechanisms of cross-resistance to azoles in a clinical isolate of Aspergillus fumigatus. Methods A. fumigatus was isolated from a patient with invasive aspergillosis.Clinical Laboratory Standard Institute M38-A2 broth microdilution method and E-test method were used to determine the minimum inhibitory concentrations (MICs) or minimum effective concentration (MEC) of itraconazole, voriconazole, amphotericin B, posaconazole and caspofungin for the A. fumigatus isolate. DNA was extracted from the isolate and subjected to the amplification of cyp51A gene encoding the target enzyme of azole antifungal agents followed by sequence analysis. Results The broth microdilution test showed that the MEC of caspofungin was 0.5 mg/L, and MICs of itraconazole, voriconazole and amphotericin B were ≥ 16 mg/L,8 mg/L and 1 mg/L, respectively, for this isolate; while E-test assay revealed that the MICs of caspofungin,itraconazole, voriconazole, amphotericin B and posaconazole were 0.047 mg/L, ≥32 mg/L,≥32 mg/L, 12 mg/L and ≥32 mg/L, respectively. Sequence analysis showed an insertion of a 34-bp tandem sequence in the promoter region of the cyp51A gene as well as a T364A point mutation causing the substitution of leucine 98 (L98H). In addition, there were some other mutations in the cyp51A gene of this isolate, such as A137T,G585A, C814A, G836C, T991C and A1350G, which could result in corresponding amino acid substitutions.Conclusions An A. fumigatus strain with cross-resistance to azole antifungal agents is isolated. There is an insertion of a 34-bp tandem sequence into the promoter region as well as a T364A point mutation in the cyp51A gene, which contribute to the cross resistance to azole antifungal agents including itraconazole, voriconazole,and posaconazole. In addition, other mutations causing amino acid substitutions have also been detected in the cyp51 A gene of this isolate.

Objective To investigate the relationship between multidrug resistance of X-ray irradiated human nasopharyngeal squanous carcinoma cell line CNE1 and platelet endothelial cell adhesion molecule-1 (PECMA-1)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT)/Bcl-2 signaling pathway.Methods ①The CNE1 cells were cultured in incubator.Exponentially growing CNE1 cells were exposed to 50 Gy ionizing radiation administered in 25 fractions,2 Gy per fraction,once every two days,and the CNE1/R cells were collected.②CNE1/R cells were cultured for 24 h,and divided into three groups:the control group,experimental group one (CNE1/R cells were processed through 1 ∶ 600 PECAM-1 antibody for 24 h) and experimental group two (CNE1/R cells were processed through 10 μmol/L PI3K inhibitor LY294002 for 24 h).③The half maximal inhibitory concentration (IC50) values of the control group and two experimental groups of CNE1/R cells were detected by methyl thiazolyl tetrazolium (MTT).④The mRNA expressions of PI3K and Bcl-2 in the control group and experimental group one were measured by semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR).⑤The mRNA expressions of AKT and Bcl-2 in the control group and experimental group two were measured by semi-quantitative RT-PCR.Results ①The IC50 of the control group,experimental group one and two were (2.99 ± 0.02) μg/ml,(1.50 ± 0.03) μg/ml and (1.60 ±0.05)μg/ml,and the difference among the three groups was statiatically signficant (F =4 381.263,P =0.000).The IC50 of the two experimental groups were obviously lower than that of the control group (t =116.885,P =0.000;t =73.006,P =0.000).②The semi-quantitative values of PI3K in the control group and experimental group one were 1.66 ±0.01 and 0.90 ±0.05 (t =50.394,P =0.000),and the semi-quantitative values of Bcl-2 were 1.66 ±0.02 and 0.71 0.05 (t =183.165,P =0.000),and the differences were statistically significant.③The semi-quantitative values of AKT of control group and experimental group two were 1.53 ± 0.01 and 0.72 ± 0.01 (t =135.281,P =0.000),and the semi-quantitative values of Bel-2 were 1.66 ± 0.02 and 0.63 ± 0.02 (t =128.814,P =0.000),and the differences were statistically significant.Conclusion PECAM-1 induced multidmg resistance of human nasopharyngeal squamous cell carcinoma CNE1 cells may be related to the activity of Bcl-2 through the signal pathway of PI3K/AKT.

Objective:To investigate the efficiency of Agrobacterium tumefaciensmediated transfor-mation of Aspergillus fumigatus by using pyrG as a recessive selectable marker.Methods: FAP1 and SHO1 genes target sequences,composed of a selectable marker pyrG and the flanking sequences of the FAP1 and the SHO1 genes,were cloned into a binary plasmid pDHt/sk,respectively.The produced plasmids were transformed into A.tumefaciens.The A.tumefaciens and uracil auxotroph A.fumigatus were cocultured in induction medium without uricil and uridine at 24 ℃ for 48 h.To inhibit growth of A.tumefaciens and to select transformants,the cultures were transferred to 37 ℃ and incubated for another 48 h.Results: In this study,A.tumefaciens-medidated transformation of A.fumigatus produced high homologous recombination rates,which was 44%(7 of 16) for FAP1 and 35%(7 of 20) for SHO1.Conclusion: Our study showed that A.tumefaciens-medidated transformation by using pyrG as a recessive selectable marker is an efficient tool for target gene deletion of A.fumigatus.

Objective:To construct the sho1 gene mutant of Aspergillus fumigatus,and to investigate the function of sho1 in adaptation to environmental changes.Methods:Saccharomyces cerivisiae sho1 gene homolog was identified by tblastn searches in A.fumigatus genome database.A PCR fragment,composed of the 5' flanking sequence(approximately 1 kb) of sho1,sho1 ORF,and its 3' flanking sequence(approximately 1 kb),was subcloned into a vector pDHt/SK2 with ApaⅠ/Xba Ⅰ enzyme to produce a recombinant plasmid PA.The selected marker pyrG cut from pLAX223 with Nco Ⅰ/Pst Ⅰ was cloned into PA,and PB was produced.The plasmid PB was transformed into competent Agrobacterium tumefaciens EHA105 by using the freeze/thaw method.The strain of A.tumefaciens was designated as At sho1.Then sho1 was produced via ATMT.The radial growth was observed and compared between sho1 and wild type(wt) on MM,CM and BHI medium,and the sensitivity of sho1 and wt to amphotericin B,itraconazole,voriconazole,and caspofungin investigated.Results:Radial growth had visible difference between sho1 and wt on MM,CM and BHI medium.sho1 mutant was sensitive to high osmotic pressure but not to low osmotic pressure.The susceptibility to antifungal drugs was similar between the sho1 and wt.Conclusion:Slow growth of the sho1 mutant has nothing to do with the different medium.The sho1 gene plays a role in response to osmotic pressure in A.fumigatus,but sho1 has a minor role in the susceptibility to the antifungal drugs despite the slow growth.

Objective To investigate the in vitro susceptibility of 16 strains of Exophiala dermatitidis to 6 commonly used antifungal agents. Methods The Glinical and Laboratory Standards Institute (CLSI)M27-A2 protocol was carried out to determine the MIGs of terbinafine (TRB), itraconazole (ITC), amphotericin B (AMB), fluconazole (FLC), voriconazole (VRC), and caspofungin (GAS) to 16 strains of E. dermatitidis, and E-test was performed to determine those of VRG, ITC and AMB. Besides, the minimal fungicidal concentrations (MFGs) of the above antifungal agents to the 16 strains of E. dermatitidis were further assessed.The activity of TRB in combination with ITC and VRG against E. dermatitidis was also estimated. Results The MIC ranges of TRB, VRC, ITC, AMB, FLC, and CAS were 0.125 - 0.25 mg/L, 0.25 - 0.5 mg/L, 2.0 mg/L,2.0 mg/L, 16 - 32 mg/L and 16 - 64 mg/L respectively as shown by M27-A2 microdilution assay, while the MIC ranges of VRG, ITG and AMB, as determined by E-test, were 0.032 - 0.094 mg/L, 0.047 - 0.5 mg/L and 0.125 - 3.0 mg/L, respectively. The MFC ranges of TRB, VRC, ITG, AMB and FLG were 0.125 - 0.5 mg/L,0.25 - 0.5 mg/L, 2.0 - 4.0 mg/L, 2.0 - 4.0 mg/L and 16 - 64 mg/L, respectively. No synergism in the acitivity against E. dermatitidis was observed for the combination of TBR with ITC or VRC. Conclusion E. dermatitidis is susceptible to TRB, ITC, AMB, and VRC, but less sensitive to both FLC and GAS.

Objective To construct a mutant strain of Aspergillus fumigatus with the mutant pbs2 gene, and to investigate the effect of the mutant gene on the morphology of A. fumigatus, the variation of os-motic pressure and the sensitivity of oxidative stress. Methods S. cerevisiae pbs2 gene homologs was identi-fied in A. fumigatus genome database. A PCR fragment, composed of the 5' flanking sequence(approximate-ly 1 kb) ofpbs20RF and its 3' flanking sequence (approximately 1 kb), was subcloned into a vector pDHt/SK with Xho Ⅰ/Xba Ⅰ enzyme to produce a recombinant plasmid PA. The selected marker pryG amplified from pLAX223 was cloned into PA with BamH Ⅰ/Pst Ⅰ enzyme to produce plasmid PB. PB was transformed into Agrobacterium tumefac/ens and named plasmid At pbs2. Then At pbs2 was transformed into A. fumigatus strain AF293.1 (pyrG) via ATMT to produce the transformants △pbs2. To observe the growth rate and the phenotype on the MM containing different oxidative stress and osmotic pressure between the △pbs2 and the wild type. Results There were no visible difference between △pbs2 and AF293 on the radial growth rate and morphology. △pbs2 mutant was not only more sensitive to osmotic pressure produced by sodium chloride and glycerol but also sensitive to oxidative stress produced by hydrogen peroxide than the wild type. Conclu-sion The pbs2 gene plays a role in osmotic pressure and oxidative stress signal tmnsductions in A. fumiga-tus but only involved in oxidative stress induced by hydrogen peroxide specifically. However, pbs2 gene has a minor effect on growth and morphology in A. fumigatus.