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Objective To establish a double-antibody sandwich enzyme linked immunosorbent assay (DAS-ELISA) for the rapid detection of germ tube-specific antigens of Candida albicans,and to evaluate its specificity and sensitivity.Methods A DAS-ELISA was established with the monoclonal antibody McAb03.2C1-C2 as the primary antibody,and horseradish peroxidase (HRP) labeled McAb03.2C1-C2 as the secondary antibody.The established assay was used to detect germ tube-specific antigens of Candida albicans in sera from 5 patients with systemic Candida albicans infection and from 6 rabbit models at 12,24,48,72hours,on week 1,2 after infection with Candida albicans.Results A good liner relationship was observed between the absorbance value at 495 nm and antigen concentrations when the titer of McAb03.2C1-C2 was 1 ∶ 4000 and the concentration of coated antigen varied from 1.25 to 40 μg/ml.The specificity and sensitivity of the DAS-ELISA were 95％ and 92％ respectively in the detection of germ tube-specific antigens in the rabbit models.The results of detection with DAS-ELISA in serum specimens from the patients were consistent with those with the routine method.Conclusions A DAS-ELISA is primarily established for the rapid detection of germ tube-specific antigens of Candida albicans,and has shown a satisfactory sensitivity and specificity in the animal model experiment.
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Objective To study the survival and melanogenic potential of human melanocytes reversibly immortalized via SV40T antigen gene and Cre/loxP system in Guinea pigs. Methods The supernatants of retrovirus vector Cre-ERT2 were used to infect melanocytes which had been successfully transfected by SV40TAg gene (MCT), then the expression of Cre recombinase was induced with tamoxifen in infected cells; subsequently, the surviving cells, which were named as MCTC, were subjected to expansion culture. Guinea pigs were utilized to establish animal models of vitiligo, then MCTC and primary melanocytes were transplanted respectively into the animal models. The repigmentation at the transplanted area was observed with naked eyes successively until 3 months after the transplantation when tissue samples were obtained from implanted area and nonimplanted area of guinea pigs and subjected to Masson-Fontana silver stain and Hematoxylin-eosin stain for the analysis of melanocyte distribution and melanin deposition in epidermis. Results Repigmentation started 4 weeks after the transplantation, and dark or brown patches, which ranged in size from 0.5 to 1 cm, were observed in the implanted area 3 months after the transplantation. The repigmentation rate was of no significant difference between pigs transplanted with MCTC and those with primary melanocytes (82.5% vs 76.7%, P > 0.05). Pathological examination revealed melanin deposition in the basal layer of epidermis and some hair follicles in transplanted area. Conclusions SV40T antigen gene combined with Cre/loxP site-specific recombinase system can induce the reversible immortalization of human melanocytes, and the immortalized melanocytes have a favorable profile of biological safety and similarity in survival rate and melanogenic potential to primary melanocytes.
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The patient,a 51-year-old veterinarian,was hospitalized for a two-month history of painful oral ulcers without fever.Physical examination revealed two superficial ulcers in the palate.Laboratory examination showed a decrease in peripheral blood leucocytes as well as a hyperplasia of bone marrow.No abnormality was found by X-ray radiography or B-mode ultrasonography.The paileat tested negative for anti-HIV antibody.Classification Of CD+ cells showed that CD3+ cells amounted to 0.559,CD4+ cells 0.289,CD8+ cells 0.207,CD4:CD8 ratio 1.4,and all the parameters were in a normal range.Histopathological analysis of the ulcer tissue revealed a chronic inflammatory granuloma with clustered yeast-like cells.Fungal culture of the sample from ulcer tissue at 25℃on the slant of PDA yielded white filamentous colony;typical rudder-like macroconidias were observed with electronic microscopy following microculture;and the isolate showed a biphasic growth pattern.PCR was performed to amplify the internal transcribed spacer 1 (ITS1)and D1/D2 region of the isolated fungus followed by sequence analysis,and a homology of 97%and 99%was observed in the sequence of ITS1 and D1/D2 region,respectively,between the isolate and standard strain of Histoplasma capsulatum.A diagnosis of primary oral histoplasmosis Was made.The lesions subsided after 2-month treatment with oral itraconazole 400 mg per day.
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Objective To detect the expression and function of cysteinyl leukotriene receptors (CysLTRs)in keratinocytes.Methods Human keratinocytes were isolated from the tissue of foreskin by digestion with dispase Ⅱ and trypsin,and subjected to primary culture.By using confocal laser scanning microscopy and reverse transcriptase PCR,the localization and expression of CysLTRs were studied in kemtinocytes.respectively.Some primarily cultured keratinocytes were pretreated with leukotriene D4 (30 nmoi/L),MK571(300 nmol/L),and BAYu9773 for 5 minutes followed by the detection of intmcellular calcium level using the Ca2+ indicator dye Fura-2/AM as well as cell proliferation bv MTT assay.Results The expressions of CysLTR1 and CysLTR2 were observed in cultured keratinocytes,and they were mainly located on cell membrane,partly in cytoplasm and nuclei.Compared with non.stimulated cells,a significant increase Was noted in the expression of CysLTRs,especially in the nuclei of keratinocytes stimulated by LTD4(P＜0.05),together with an elevation in intracellular calcium level(42.27±3.00 mmol/L,P＜0.01)and acceleration in cell proliferation (P＜0.01).However,both MK571 and BAYu9773 could completely block the effect of LTD4 on intmcellular calcium level and cell proliferation.and there was no significant difference in the blocking effect between MK571 and BAYu9773.Conclusions Functional CysLTRs are expressed in human keratinocytes.and they carl increase the intracellular calcium level in,and cell proliferation of,keratinocytes.
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OBJECTIVE To supervise the environmental fungal load and species distribution in transplantation department and intensive care unit of the Southwest Hospital,and to analyze the relationship with season,temperature,humidity,ventilation and personnel activities.METHODS Data from Dec 2005 to Jan 2006 were collected from liver transplantation department(LTD),cerebral surgery intensive care unit(CSICU) and central intensive care unit(CICU).Air,surfaces and tap water were sampled twice a month at each department.RESULTS The air fungal load was 123.63 CFU/m3,139.90 CFU/m3,7 CFU/m3 and 217.71 CFU/m3 at LTD,CSICU,CICU and outdoor,respectively.The five most prevalent fungi collected from air and surface were Penicillium spp,Cladosporium spp,Alternaria spp,Aspergillus spp and Saccharomyces spp in turn.The five most prevalent fungi collected from water were Saccharomyces spp,Candida spp,Aspergillus spp,Penicillium spp and Rhodotorula spp in turn.The fungal load in LTD was positively correlated with the average temperature and the average humidity;the fungal load in CSICU was correlated with the average temperature and the average humidity,but the correlation between air fungal load and personnel activities wasn′t observed.CONCLUSIONS It demonstrated the fungi are found in the environment of the hospital including air,surface and water.The air fungal load varies throughout the year.The crest-time is May to June and September to October.Air fungal load is lower in winter and higher in summer and autumn.The correlation between air fungal load and temperature and humidity is observed.
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This paper discusses the problems, clinical application and limitations of argon-helium cryosurgical scalpel and liquid nitrogen cryosurgical scalpel. The feasibility of self-absorption liquid-CO2 cryosurgical scalpel is analyzed. The result shows that self-absorption liquid-CO2 cryosurgical scalpel can be applied to cryosurgery.
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Objective To establish a simple and practical method for isolating and purifying nucleosomes. Methods Nuclei were isolated from chicken erythrocytes, and then digested with staphylococcal nuclease. After centrifugation, the supernatant of digestion was separated and centrifugated on sucrose gradients. Results Nucleosomes with good stability were isolated properly by gradient centrifugation. Conclusion This method for the isolation and purification of nucleosomes is simple and effective, which might contribute to the further researches of the roles of nucleosomes in the pathogenesis of systemic lupus erythematosus.
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Objective To investigate whether the sort of drugs causing drug eruptions complicated with drug-induced liver dysfunction are the same as those causing drug eruptions. Methods Sixty-one cases of drug eruption complicated with drug-induced liver dysfunction in 261 cases confirmed as drug eruptions from January 1998 to March 2003 were analyzed retrospectively. Results The major hepatotoxic drugs in these cases were antivirus drugs (60%, 9/15), antituberculosis drugs (66.67%, 8/12), zyloric (55.56%, 5/9), and some traditional Chinese medicines (31.58%, 6/19). Conclusion The sort of drugs causing drug eruptions complicated with drug-induced liver dysfunction are the same as those causing drug eruptions, which should be taken into consideration in clinical medication.
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Objective To investigate the therapeutic efficacy of HPV16 E7 peptide pulsed dendritic cells (DCs) in recurrent condyloma acuminate (CA). Methods A total of 32 cases of recurrent CA (more than 3 times) were divided into the treatment group and the control group. Patients (11 cases, HPV16 +and HLA A2 +) in the treatment group received treatment with DCs, while the other 21 cases in the control group were treated with interferon. A follow up of 6 months was conducted in all patients. The pathological lesions, the peripheral T cell subpopulations of the patients, and the therapeutic efficacy before and after treatment were observed. Results The size of the lesions became smaller or disappeared in the treatment group. The infiltrated lymphocytes increased, but the koilocytotic cells decreased in the lesions. No significant change in the peripheral T cell subpopulations was found before and after therapy. The recurrence rates in the treatment group and the control group were 18.2% and 61.9%, respectively. Conclusion The therapy by E7 peptide pulsed dendritic cells can improve the local immune status in the skin and reduce the recurrence rate significantly in patients with recurrent CA.
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Objective To study the effects of recombinant chimeric toxin Dsg3EC_(1-2)PE40 on T and B cells from PV patients. Methods The recombinant protein Dsg3EC_(1-2)PE40 was expressed on BL21TrxB (DE3) cells, then identified and purified. ELISPOT assay was used to detect and quantitate autoantibody-producing B cells in different concentrations of recombinant chimeric toxin, and MTT assay and ~3H-TdR assay to observe the metabolism and proliferation of T cells from PV patients in vitro. Results The purity of expressed protein Dsg3EC_(1-2)PE40 was up to 80%. The number of anti-Dsg3 antibody-producing B cells in PBMC from PV patients decreased by 40% with treatment of Dsg3EC_(1-2)PE40, which was significantly lower (P
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Objectives To construct a cDNA subtractive library of dermal papilla cells (DPCs) in anagen with suppression subtractive hybridization (SSH) and clone differentially expressed genes related to DPCs in anagen. Methods Total RNA was isolated from DPC of anagen and telogen follicles. Then ds cDNAs were synthesized in turn using SMART cDNA synthesis technique. After cDNAs from anagen and telogen follicle DPCs were hybridized with each other twice and underwent two rounds of nested PCR, PCR products were ligated with arms of T/A plasmid vectors to set up the subtractive library. Selected clones were verified by reverse Nothern blot and DNA sequencing, and the acquired sequences were analyzed for homology based on Genbank nucleotide database. Results cDNA subtractive library of DPCs in anagen follicle was set up successfully with high subtractive efficiency. Thirty-five genes were identified with 22 known functional genes and 13 unknown functional genes. Conclusions These results demonstrate the effectiveness and sensitivity of SSH in detecting differentially expressed genes from a small amount of clinical samples. Information about such alterations in gene expression might be useful for elucidating the genetic events in hair follicle growth regulation.
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Objective To investigate the biological activities of a conditioned medium for human dermal papilla. Methods Culture medium of the lower passage human dermal papilla cells was collected as the conditioned medium. The growth pattern and the growth curve of the higher passage human dermal papilla cells cultured with conditioned medium were observed in vitro. And the morphology of the co-culture of the higher passage human dermal papilla cells and the lower passage human dermal papilla cells was observed. Results The higher passage human dermal papilla cells, which was cultured with conditioned medium from the lower passage human dermal papilla cells, showed aggregative growth pattern. And the growth curve of the higher passage human dermal papilla cells was much better than that in the control groups (P
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Objective To study the bacteriological characteristics and the pathogenesis of Staphylococcus aureus (S. aureus) on eczema and atopic dermatitis (AD). Methods A multi-center randomized, double blind bacteriological study on the lesions and non-lesional skin of patients with eczema (207) and AD (119) were carried out. The antibiotic sensitivity and the bacteriophage typing were performed on all the S. aureus isolated from the patients. Results There were statistical differences in the positive rate of the culture, the ratio and the colonization of S. aureus between the lesion and the non-lesional skin in eczema (P
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Objective To investigate the role of IL-2,IFN-? and IL-10 in pemphigus acantholysis. Methods Acantholysis was observed histopathologically in the skin organ culture model of pemphigus after interacting with different concentrations of IL-2, IFN-? and IL-10 for 24 h?48 h and 72 h. Results The acantholysis was promoted by IL-2 and IFN-?, and the severity of acantholysis was related to the concentrations of IL-2 and IFN-?. The effect of IFN-? was weaker than that of IL-2. IL-10 could inhibit the acantholytic effect of IFN-? significantly, and inhibit the acantholytic effect of IL-2 when its concentration was higher than 100 pg/mL. Conclusions Th1 cytokines can promote acantholysis induced by antibody of pemphigus (Pab) while Th2 cytokines can inhibit the acantholysis induced by Pab, and the effect of Th1 cytokines. Th2 lymphocytes may play an important role in the pathogenesis of pemphigus.
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Objective To screen and analyze genes differentially expressed within dermal papillae cells(DPC)with aggregative behavior.Methods Total RNA was extracted from DPC with and without ag-gregative behavior,and double-stranded cDNA were synthesized by using SMART cDNA synthesis.The cD-NA fragments of differentially expressed genes in dermal papillae cells with aggregative behavior were isolat-ed by suppression subtractive hybridization,sequencing,and then subtracted library was set up.Positive clones were screened by PCR method and verified by cDNA dot blot and then analyzed through homologous retrieving.Results A subtractive cDNA library of DPC with aggregative behavior was successfully construct-ed.The results of screening and cloning of the library showed that DPC with aggregative behavior could ex-press genes related to homologous aggregation,regnlation of growth,differentiation and development,and sig-nal transduction proliferation and cycle control,which included known genes(capping protein,paladin,vas-cular endothelial growth factor),hematopoietic stem/progenitor cells(HSPC)related genes(HSPC011and HSPC016)and a new gene.Conclusions The construction of subtractive library of DPC lays solid founda-tion for screening and cloning new and specific genes related to aggregative behavior of DPC.Several genes may cooperatively involve in homologous aggregation,and regnlation of growth of DPC.Among these genes,capping protein and palladin may be closely related to aggregative behavior of DPC,and VEGF and HSPC re-lated clones may be responsible for the status of higher proliferation of DPC.
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Objective To assess the levels of nucleosomes released from peripheral blood mononu-clear cells(PBMC)of patients with systemic lupus erythematosus(SLE),and their relationship with auto-antibodies as well as disease activity.Methods Levels of both nucleosomes released from PBMC and vari-ous auto-antibodies were detected by ELISA in sera from SLE patients.The disease severity was evaluated using SLEDAI(systemic lupus erythematosus disease activity index)system.Results Levels of nucleosomes released from PBMC were significantly higher in patients with active SLE than those of patients with inactive disease and normal controls(39.39?25.70,13.44?8.82,and11.73?7.87IU/mL,respectively).There was a significant positive correlation between nucleosome levels and SLEDAI scores,serum ds-DNA auto-an-tibody levels,and low C3levels.Conclusion Nucleosomes released from apoptotic PBMC of patients with SLE is closely correlated to disease activity,which implies that nucleosomes may play an important role in the pathogenesis of SLE.
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Objective To investigate the role of cytotoxic T lymphocyte associated molecule-4Ig(CTLA-4Ig) in the prevention of C57BL/6 mice autoimmune hepatitis. Methods The C57BL/6 mice were intraperitoneally immunized with C57BL/6 mice liver-specific protein in complete Freund's adjuvant. At the same time CTLA-4Ig were given to observe the pathologic alteration of C57BL/6 mice liver. Results With the increase of time of immunization, the results in the treatment group were similar to those of the control group; but inflammatory cell infiltration, hepatic cell swelling, focal necrosis and severe hepatocyte damage were found in the pathologic model group. There was a significant difference between the pathologic model group and control one. Conclusion Autoimmune hepatitis of C57BL/6 mice can be effectively prevented by CTLA-4Ig.
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Objective To analyze the clinicopathological features of angiolymphoid hyperplasia with eosinophilia (ALHE). Methods The pathological specimens of 7 cases of ALHE collected in our department from 1950 to 1999 were sectioned, stained and observed. Results There were 3 pathological characteristics in ALHE: ①massive hyperplasia of capillaries in the dermis; ②the endothelial cells proliferated and swelled, projecting into vascular cavity like tombstones; ③mixed infiltration of lymphocytes and eosinocytes in the vessels. Conclusion ALHE is a disease with local benign proliferated vessels, whose etiology and pathogenesis is still unknown. It is necessary to grasp the pathological changes of ALHE to distinguish it from other diseases.
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Objective To investigate the actions of extra cellular medium in growth and differentiation of hair follicle and to look for growth adjusting factors for dermal papilla cells (DPC). Methods Dermal papilla cells were isolated and cultivated with two steps method and the cells were identified by immunohistochemical staining for actin. Influence was examined on the adhesion and growth of dermal papilla cells by chondroitin sulfate A, chondroitin sulfate C and heparin sulfate. Results Two steps method of enzyme digestion for isolating and cultivating dermal papilla cells was an efficient method and large amount of dermal papilla of high purity were harvested with this method. The method is very simple and easy to manege with. Increased adhesion and growth of dermal papilla cells were observed in specimen treated with chondroitin A and heparin sulfate. No significant effects was observed in the cells treated with chondroit in sulfate C. Conclusion Some extra cellular medium can regulate the adhesion and growth of dermal papilla cells and therefore influence the growth and development of hair follicle.
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Objective To investigate the cause of excessive collagen accumulation in keloid tissue. Methods The ultrastructure of keloid was observed by transmission electron microscope. New formed collagen in keloid was localized with ABC immunohistochemical staining. Type I procollagen mRNA level in keloid tissue was determined by dot blot hybridization using human pro-al (I)collagenspecific cDNA probe. Results Numerous fibroblasts with abundant, well developed rough endoplasmic reticulum were exhibited in the ultrastructure of keloid. The type I procollagen mRNA levels were significantly increased in kreloid tissue. Immunohistochemical staining showed increased expression of new formed, type I procollagen in keloid tissue. Conclusion the fibroblasts are activated in collagen synthesis in active keloid. The enhanced collagen synthesis by fibroblasts is a critical factor leading to the overabundant collagen accumulation in keloid.