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Objective To compare the performance of 2％ (w/v) agarose gel,2％ (w/v) Metaphor agarose gel and 10％(w/v) nondenaturating polyacrylamide gel in the PCR-restriction fragment length polymorphism (RFLP) analysis of mycobacterial heat shock protein 65 (hsp65) gene.Methods This study included 8 Mycobacteria strains,including clinical isolates and standard strains of Mycobacteria tuberculosis and Mycobacterium intracellulare.Bacterialsuspension of these strains was prepared with the concentration of bacterial cells varying from 10 to 106per milliliter.PCR was performed to amplify the hsp65 gene with a pair of universal primers followed by the digestion of amplicons with two restriction endonucleases,BstE Ⅱ and Hae Ⅲ.Then,the restriction enzyme-digested fragments were subjected to electrophoresis in 2％ agarose gel,2％ Metaphor agarose gel and 10％ nondenaturating polyacrylamide gel respectively.Results As analysis of variance showed,the three gel electrophoresis methods were statistically different in sensitivity for the RFLP analysis of mycobacterial hsp65 gene (F =36.379,P ＜ 0.01).Least significance difference (LSD) procedure revealed that the 2％ agarose gel-based electrophoresis was less sensitive than the 2％ Metaphor agarose gel-and 10％ non-denaturing polyacrylamide gel-based electrophoresis (both P ＜ 0.01),and no significant differences were observed between the 2％ Metaphor agarose gel-and 10％ non-denaturing polyacrylamide gel-based electrophoresis (P ＞ 0.05).Conclusion The 2％ Metaphor agarose gel-and 10％nondenaturating polyacrylamide gel-based electrophoresis methods appear to be more sensitive than the 2％ agarose gel-based electrophoresis method for the PCR-RFLP analysis of mycobacterial hsp65 gene.
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Objective To investigate the imbalance between T helper 17 (Th17) cells and CD4+CD25+ regulatory T (Treg) cells in the peripheral blood of patients with psoriasis vulgaris and its significance. Methods Peripheral blood were collected from 48 patients with psoriasis vulgaris and 32 normal human controls. Pasoriasis area and severity index (PASI) was used to assess the disease severity in these patients. Flow cytometry was performed to determine the percentage of Th17 and Treg cells in peripheral blood, enzyme linked immunosorbent assay (ELISA) to measure the levels of serum interleukin (IL) -17 and IL-10. Results There was a significant increase in the percentage of Th17 cells [(2.70 ± 1.43)% vs. (0.86 ± 0.25)%, P< 0.01] and serum IL-17 level (90.65 ± 29.61 ng/L vs. 48.82 ± 5.49 ng/L, P < 0.01), but a decrease in the percentage of Treg cells [(3.63 ± 1.14)% vs. (7.87 ± 1.26)%, P< 0.01] and serum IL-10 level (17.78 ± 4.09 ng/L vs. 23.76 ± 3.82 ng/L, P <0.01) in patients with psoriasis vulgaris compared with the normal controls. The ratios of Thl7 to Treg cells and serum IL-17 to IL-10 level were significantly higher (0.95 ± 0.76 vs. 0.12 ± 0.06, 5.78 ± 3.19 vs. 2.16 ±0.68, both P < 0.01) in the patients than in the normal controls. The PASI score in patients was positively correlated with the percentage of Th17, serum level of IL-17, Th17/Treg ratio and IL-17/IL-10 ratio (r = 0.97,0.93, 0.99 and 0.97, all P < 0.01), but negatively correlated with the percentage of Treg cells and serum IL-10 level (r = -0.87, -0.90, both P < 0.01). Conclusion The imbalance between Th17 and Treg cells may play an important role in the pathogenesis of psoriasis vulgaris.
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Objective To test the drug susceptibility of 16 Mycobactena tuberculosis isolates from patients with cutaneous tuberculosis,and to provide a basis for the treatment of this entity.Methods Sixteen strains of mycobacterium were isolated from patients with cutaneous tuberculosis.and they were consistently identified as M.tuberculosis by biochemistry and molecular biology.Absolute concentration method was used to test the susceptibility of these isolates to isoniazid.streptomycin.rifampicin and ethambutol.For two strains resistant to streptomycin,PCR and sequencing were performed to analyse the mutation of rpsL gene.Results Out ofthe 16 strains of M tuberculosis.2 were resistant to streptomycin but sensitive to isoniazid,rifampicin and ethambutol,and 14 were sensitive to isoniazid,streptomycin,rifampicin and ethambutol.Sequencing of the rpsL gene revealed a mutation of AAG to AGG at codon 43 in one streptomycinresistant strain and a substitution of CGC bv CAC at codon 54 in another resistant strain.Conclusions In past five years,the resistance ratio of M tuberculosis was low in patients with cutaneous tuberculosis,and streptomycin resistance predominated in these strains.
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Objective To develop a rapid method with high sensitivity and specificity to detect Mycobacterium avium.Methods A polymerase chain reaction(PCR)was developed.Its sensitivity and specificity were verified by sequential dilution of DNA of M.avium and other17Mycobacterium spp.,respectively.Simulation of clinical infection was established by mixing normal skin tissue with M.avium.The treatment of the tissue specimens was optimized in order to improve the sensitivity of the PCR assay.Results A fragment of427bp was amplified by the PCR assay with the strains of M.avium at the sensitivity of1?10 2 cells/mL.The other17Mycobacterium spp.were all negative.The sensitivity of the PCR assay decreased to1?10 4 cells/mL when M.avium was mixed with the homogenized skin tissue.The sensitivity recovered to1?10 2 cell/mL when the skin tissue was diluted to≥1∶4.Conclusion It is suggested that PCR be a rapid and reliable method for detection of M.avium.
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Objective To study level of the expression of RAR? mRNA in the skin lesions of patients with psoriasis vulgaris. Methods The biopsies were taken from skin lesions in 20 patients with psoriasis vulgaris and the skin of 10 normal controls, and levels of RAR? mRNA were investigated with RT-PCR. Results The level of RAR?mRNA was significanlly lower in the epidermis of patients with psoriasis vulgaris than that in the control(P
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1), attributable risk (AR)=13.33%; ⑤Even though the serum specimens were taken from PB patients, the Ab of majouity of patients converted to positive when relapse occurred. In the majority of patients relapsed the levels of IgM-AbL tended to be increasing or pesistently positive. Usually relapse occurred 1-2 years after IgM-AbL was converted to positive. Relapse occurred 11-30 years after the patients were cured. In very rare case downgrading(from tuberculous to borderline leprosy) occurred. ⑥The levels of IgM-AbL gradually decreased in all relapsed patients after effective treatment except one case whose IgM-AbL was persistently positive. Conclusions The above results indicate that the ND-ELISA might be useful in screening early M.leprae infection and in predicting and monitoring the relapse of leprosy, especially in multibacillary patients.
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Objective To develop a PCR-RFLP method for the identification of eight mycobacterial species. Methods PCR was performed targeting the gene encoding 65-kDa heat shock protein which was common to all mycobacteria. Two restriction enzymes, BstE Ⅱ and Hae Ⅲ, were used to digest the PCR products, and specific restriction patterns of different mycobacteria were obtained. Results The specific restriction patterns of different mycobacteria were identical to the data previously reported. Conclusion We could differentiate M. avium, M. intracellulare, M. kansasii, M. tuberculosis, M. scrofulaceum, M. marinum, M. fortuitum and M. chelonae in one experiment by PCR-RFLP.
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Objective To investigate the levels of cytokines in sera of patients with various types of psoriasis.Methods Six cytokines, sIL-2R、 IL-2、 -4、 -10、 -12 and IFN-γ , were detected by sandwich ELISA in the sera from 15 patients with guttate psoriasis, 23 plaque psoriasis, 9 pustular psoriasis, 9 arthropathic psoriasis and 9 erythrodermic psoriasis.Results Significantly higher levels of cytokines were observed in guttate psoriasis for sIL-2R (P<0.01), in plaque psoriasis for IL-4,-12 and sIL-2R (P< 0.05 or < 0.01), in pustular psoriasis for IL-4 and -10 (P< 0.05), in arthropathic psoriasis for IL-10 (P< 0.01), in comparison with the controls.The levels of the six cytokines were increased in erythrodermic psoriasis with no significant difference from the controls.The ratio of the average level of IL-4 to IFN-γ was 1.57 in pustular psoriasis, 0.61 in plaque psoriasis, 0.30 in gutatte psoriasis, 0.24 in erythrodermic psoriasis, and 0.02 in arthropathic psoriasis.Conclusion There is different expression of cytokines in sera of patients with various types of psoriasis.
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Objective To establish a polymerase chain reaction(PCR) method to amplify DNA of M.leprae from fixed and paraffin－ embedded tissue(FPET). Methods The DNA of M.leprae was released from FPET by using Texpat Kit and purified with 100％ alcohol. The primers RPOT(1) and RPUT(2) were used to conduct the PCR. Results A total of 32 samples were examined. Out of 32 samples with BI of more than 1＋ , 28 were positive for PCR. The PCR was negative in a sample with BI=0. The sensitivity of PCR reached a level of 0.04 pg DNA. Conclusion This PCR method is very useful for amplifying the DNA of M.leprae from FPET.
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Objective To explore whether there were differences in genotypes between strains of M.leprae in China or not. Methods Polymerase chain reaction (PCR) was used to detect the genotypes of M.leprae in samples from patients with leprosy in different parts of China. Results Out of 16 samples from 9 provinces, 91 bp DNA fragments were present in 7 samples and 97 bp DNA fragments in 9 samples. Conclusion Above- mentioned results suggest the genotyping differences in strains of M.leprae with different distribution in areas of China.
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Objective To genotype the clinically isolated strains of M.leprae by genome sequencing analysis. Methods PCR amplification was used to produce the 200 bp partial rpoT gene fragments from 2 standard strains and the isolated strains of M. leprae isolated from clinical specimens in 7 areas of China. The fragments were sequenced by BigDye Terminator Cycle Sequencing Reaction kit. Results① 12 rpoT－ 91/97bp DNA fragments and 2 rpoT－ 194/200 bp DNA fragments were obtained from 13 clinically isolated strains of M. leprae by PCR amplification;② based upon genome sequencing analysis, a component of 3－ copy or 4－ copy“ GACATC” repeat sequence was found in the nucleotide sequence of rpoT－ 194/200 bp DNA fragment. Conclusion The genome sequencing analysis can be used to objectively and accurately genotype M.leprae, and it is a useful tool for epidemiological study on transmission and infection of leprosy. However, the longer DNA fragments are necessary when sequencing analysis is conducted. Therefore this method needs further improvement because only the shorter DNA fragments amplified from paraffin－ embedded tissue.
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We report the results of detecting M. lepraespecific antigen--phenolic glycolipid I (PGL--I) with modified M-Dot-ELISA in sera from 75 cases of household contacts (HC)of leprosy patients, which were all seropositive by Gelatin Particle Agglutination Test (MLPA)and ELISA. The results indicate that: (1 ) 16/75 (21. 3 % ) are antigen positive. The rates of positivity in HC of MB patients are much higher than those in HC of PB patients,the difference (P
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Objective To investigate the levels of cytokines in sera of patien ts with various types of psoriasis. Methods Six cytokines, sIL－2R、 IL－2、－ 4、－10、－12 and IFN－? , were detected by sandwich ELISA in the sera from 15 patients with guttate psoriasis, 23 plaque psoriasis, 9 pustular psoriasis, 9 arthropathic psoriasis and 9 erythrodermic psoriasis. Results Significantly hig her levels of cytokines were observed in guttate psoriasis for sIL－2R (P
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Objective To study the level of expression of RAR?/RXR?mRNA in skin lesions of patients with psoriasis vulgaris.Methods The skin biopsies were collected fro mskin lesions in 20cases of psoriasis vulgaris and 10normal controls.Expression o f RAR?/RXR?mRNA was detected by RT -PCR.Results Expression levels of RXR?mRNA were 0.19760?0.0933in epiderm ides from psoriatic lesions,which were markedly lower than those in normal controls(0.5867?0.0132)(P0.05).Conclusion The results indicate that decreased expression of RXR?mRNA might be related to the pathogen esis of psoriasis.
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Objective To evaluate the inhibitory action of ethanolic extracts from196kinds of Chi-nese herbs on tyrosinase.Methods Tyrosinase inhibitory activity was determined by the dopachrome method using L-DOPA as the substrate and the amount of dopachrome in the reaction mixture was measured by spec-trophotometer.Results Nine Chinese crude extracts[Glycyrrhiza glabra L.,Melaphis chinensis(Bell.)Baker,Cryptotympana atrata Fabricius,Paeonia suffruticosa Andr.,Sophora flavescens Ait.,Spirea thunbegii Sieb.et Bl.,Xanthium sibiricum Patr.,Morus alba L.,Rheum palmatum L.]showed potent inhibitory action on tyrosi-nase compared with positive control arbutin(1mmol/L,P0.05).Conclusion The results suggest that20kinds of crude extracts from Chinese herbs[Glycyrrhiza glabra L.,Melaphis chinensis(Bell.)Baker,Cryptotympana a-trata Fabricius,Paeonia suffruticosa Andr.,Sophora flavescens Ait.,Spirea thunbegii Sieb.et Bl.,Xanthium sibiricum Patr.,Morus alba L.,Rheum palmatum L.,Artemisia anomala S.Moore,Hemistepta lyrata Bunge,Lycium chinensis Mill.,Dipsacus asper wall.,Gastrodia elata Bl.,Forsythia suspensa(Thunb.)Vahl.,Acer gin-nala Maxim.,Glycyrrhiza uralensis Fisch,Patrinia sabiosaefolia Fisch.,Buxus sinica(Rehd.et Wils.)Ching,Lonicera japonica Thunb.,]inhibit tyrosinase and may be used as depigmentaty agents for pigmentary skin dis-eases caused by abnormal tyrosinase activity.
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Objective To develop a rapid method with high sensitivity and specificity to detect 4 mycobacterial species(M. tuberculosis, M. avium, M. intracellulare and M. kansasii) which are the most common opportunistic Mycobacteria in AIDS patients. Methods The sensitivities and specificities of PCR were determined with different primer pairs targeting various mycobacterial genes. Multiplex PCR with combination of 4 primer pairs was used to detect the template mixtures of either 1, 2 or 3 mycobacterial DNA. Sensitivities of multiplex PCR were measured. Results Specific DNA fragments of 4 mycobacterial species mentioned above could be detected by PCR and sensitivities ranged from 1 ? 101 ~ 1 ? 102 cells/mL, while the other 17 mycobacterial strains were all PCR-negative. Multiplex PCR could amplify the corresponding 1, 2 or 3 DNA fragments, depending on the number of template DNA added, and sensitivities of multiplex PCR ranged from 1 ? 102 ~ 1 ? 103 cells/mL. Conclusions Multiplex PCR is a rapid, sensitive and specific method for differentiation and detection of Mycobacteria.