1999年 · 第112卷第06期

OBJECTIVE To examine the in vitro activities of five antifungal agents against common pathogenic Exophiala species; to detect the minimum inhibitory concentration (MIC) discrepancies among different drugs and different species; to evaluate the role of the influence factors in MIC determination; and to establish a standard method for the antifungal susceptibility testing of Exophiala spp. METHODS MICs of itraconazole (ICZ), fluconazole, ketoconazole, amphotericin B (AmB) and 5-flucytosine against 5 species (57 strains) of Exophiala spp were determined by modified NCCLS M27-A broth microdilution method. Two inoculum sizes (0.5×103-2.5×103 CFU/ml and 0.5×104-2.5×104 CFU/ml) and three incubation times (4, 7 and 10 days) were evaluated. The minimum fungicidal concentrations (MFC) of itraconazole against 20 Exophiala strains were also detected. RESULTS All the tested Exophiala species were sensitive to AmB and ICZ. MICs varied among different species and drugs. Resistant strains to different drugs existed in this genus. The proper inoculum density was about 0.5×103 CFU/ml to 2.5×103 CFU/ml, and the 4-day incubation time was suitable for more than 90% of the strains. CONCLUSIONS AmB or ICZ may serve as the first choice in treatment of phaeohyphomycosis. A standard microdilution procedure for MIC detection of Exophiala species is established in this experiment. The inoculum size and incubation time are the essential factors in the standardization of antifungal susceptibility testing of filamentous fungi. We hope this experiment may correlate well with the clinical treatment of phaeohyphomycosis caused by Exophiala species.

OBJECTIVE To investigate Mycoplasma genitalium (Mg) infection in high risk populations of sexually transmitted diseases (STDs) in China by means of culture, Mg-DNA detection, observation under an electron microscope (EM) and an atomic force microscope (AFM). METHODS 227 urogenital specimens taken from both patients in clinics and high risk populations of STDs in Beijing, Nanjing and Shanghai areas were cultured in SP-4 medium for isolation of Mg. Suspicious positive strains were identified first by PCR technique which could amplify the special DNA fragments of Mg adhesion protein (Mg-Pa) and 16S-rRNA sequences. The morphologic features of Mg were observed under EM and AFM. RESULTS Eight strains were isolated from the 227 specimens, the total positive rate was 3.52%. The isolated strains met the criteria of growth and metabolic conditions required by Mg. Specific fragments of Mg-DNA (374 bp based on Mg-Pa and 402 bp on 16S-rRNA primers, respectively) could be amplified from both the isolates and the genital secretions. Under EM and AFM, Mg showed different shapes, including flask, pear, spindle and globe shape. The typical shape of Mg was flask or pear shape with projecting neck portion and broadened terminal structure under EM and with constrictive ring between the neck and body under AFM. CONCLUSIONS Mg infection exists in high risk populations of STDs in China. We highly suggest the use of AFM in dermatological study.

OBJECTIVE To address whether circulating T-cells from the patients with primary and metastatic malignant melanoma show altered chemotaxis to monocyte-chemotactic protein 1 (MCP-1) and its relation to tumor infiltrating lymphocyte (TIL) and metastasis. METHODS Chemotactic response of T-cells towards MCP-1 and immuno-histochemistry study of TIL and tumor-associated-macrophages (TAM) were investigated in patients with primary and metastatic melanoma compared to patients with basal cell carcinoma and healthy persons. RESULTS T-cells from patients with primary and metastatic melanoma showed a significantly decreased chemotactic migration towards MCP-1 and T-cells from patients with basal cell carcinoma showed normal chemotactic response. Immuno-histochemistry study showed that there was no correlation between the number of TIL and the decreased chemotaxis of circulating T-cells to MCP-1 in patients with primary melanoma. CONCLUSIONS Circulating T-cells from patients with primary and metastatic melanoma showed a MCP-1-specific decrease in chemotactic migration. This may be due to abnormal expression or modulation of MCP-1-receptor expression on these cells.

OBJECTIVE To investigate the role of allele loss on chromosome 9 in the pathogenesis of basal cell carcinoma (BCC) by examining the loss of heterozygosity in sporadic basal cell carcinoma and other cutaneous tumors. METHODS DNA samples were isolated from the tumors, the matched adjacent normal skin and the blood of patients with sporadic basal cell carcinoma (14), squamous cell carcinoma (25) and Bowen's disease (5) by phenol-chloroform extraction. In our study, we used two microsatellite markers on chromosome 9, one was locus D9S319 (9p21), and the other was D9S299 (9q22.2-22.3). After PCR was performed, the samples were electrophoresed through 5% denatured polyacrylamide gels which were dried and exposed to Fuji XR films. RESULTS Loss of heterozygosity with D9S319 (9p21) marker was not observed in the 10 informative cases of sporadic basal cell carcinoma, 19 squamous cell carcinoma and 4 Bowen's disease. Allelic deletion of D9S299 was not seen in the 21 informative cases of squamous cell carcinoma and 4 Bowen's disease. D9S299 (9q22.2-22.3) allele loss occurred in 2 of the 10 informative cases of sporadic basal cell carcinoma, indicating that there might be a susceptible tumor suppressor gene on chromosome 9, and loss of this allele might play an important role in the development of basal cell carcinoma. Inactivation of Drosophila patch (PTCH) (MSSE gene ESS1) in BCC might be necessary, if not sufficient, for BCC carcinogenesis. CONCLUSIONS Chromosome 9q22.2-22.3 may contain a putative tumor suppressor gene, and the loss of this gene may play an important role in the development of sporadic basal cell carcinoma.

OBJECTIVE To establish a test model for phototoxic agents using the method of chemiluminescence. METHODS The phototoxicity of pipemidic acid, doxycycline, griseofuvin and chlorpromazine was detected. These agents and distilled water were irradiated with ultraviolet A (UVA) in the presence of nicotinamide adenine dinucleotide reduced (NADH), and the duplicated samples were incubated in the dark as dark controls. Then luminol was added to the test samples, and the chemiluminescent value was counted and calculated. RESULTS Chemiluminescent values of pipemidic acid, doxycycline and griseofuvin were significantly higher than those in controls. The result of linear regression analysis showed that phototoxic intensity was linear correlated with UVA dosage. The regression coefficient for distilled water was 0.56, indicating that the luminescent value (LV) rose slightly after UVA irradiation. For pipemidic acid, griseofuvin and doxycycline, the regression coefficients reached 76.96, 254.33 and 92.61 respectively, significantly increased in comparison with those of negative controls (P < 0.01). CONCLUSIONS Phototoxicity of pipemidic acid, doxycycline and griseofuvin can be detected with the method of chemiluminescence.

OBJECTIVE To elucidate the therapeutic mechanism of topical camptothecin (CPT) in treating psoriasis and to detect the effects of CPT on keratinocyte proliferation, differentiation and apoptosis. METHODS Mitotic numbers in mouse vaginal epithelium at estrus and numbers of scale with granular layer per 100 scales in mouse tail epidermis were determined in vivo. Experiments on cultured normal human keratinocytes were performed using the methods of crystal violet staining, absorbance-cell number converting, cell counting and quantitation of morphologic changes during differentiation, transglutaminase assay and nucleosomal enrichment assay. RESULTS Inhibition of cell proliferation and promotion of cell differentiation by camptothecin were showed in animal models and were reconfirmed in cultured keratinocytes. Apoptosis was induced by camptothecin and was showed by activation of "tissue" transglutaminase and increase in nucleosomes. The endonuclease activity was reduced by an endonuclease inhibitor aurintricarboxylic acid. CONCLUSIONS The therapeutic effects of camptothecin on psoriasis can at least partly be explained by its multiple effects on DNA as a topoisomerase inhibitor.

OBJECTIVE To further investigate the effects of ultraviolet (UV)-irradiated urocanic acid (UCA) on T lymphocytes, the effects of cis-UCA on T lymphocyte IL-10 secretion was studied. METHODS Four groups of normal lymphocytes were cultured in RPMI-1640 containing 15% fetal calf serum. Besides negative and phytohemagglutinin (PHA) positive control, the cis-UCA and trans-UCA groups were set. The number of lymphocytes was 1.5 x 10(6). PHA was 0.05 mg/ml in each group. Both cis-UCA and trans-UCA were detected by 1.00, 0.75, 0.50 and 0.20 mmol/L respective concentrations. IL-10 was detected by ELISA method after 48 hours' culture. IL-10 mRNA expression level was assayed by RT-PCR. RESULTS The results indicated cis-UCA could reduce IL-10 secreted by T lymphocytes much more than trans-UCA (t = 2.91, P<0.05) could. It was also found that the concentrations of cis-UCA were negatively correlated with IL-10 levels (r = -0.99, tr = 9.23, P<0.01). The IL-10 mRNA expression of T lymphoctytes was inhibited by cis-UCA. CONCLUSIONS Our investigations revealed that cis-UCA, a major UV receptor in the stratum comeum of epidermis, could inhibit T lymphoctes secreting IL-10 by suppressing IL-10 mRNA expression, finally affects the humoral immune response.

OBJECTIVE To better understand the potential effect of ultraviolet light on the photosensitivity of patients with lupus erythematosus (LE), to elucidate the mechanisms of SSA/Ro antibody formation after UV exposure, and to investigate the role of this autoantibody in the pathogenesis of skin lesions. METHODS Primary human keratinocytes were cultured in Medium-154. After ultraviolet-B light (UVB) irradiation, the keratinocytes were treated with affinity-purified anti-SSA/Ro sera and stained with FITC-labeled goat-anti-human IgG and propidium iodide (PI), followed by enzyme digestion with RNase, RNase-free DNase or RNase plus DNase. As target cells, the irradiated keratinocytes were incubated with affinity-purified anti-SSA/Ro sera, with or without fresh human sera as complement. The supernatants of irradiated keratinocytes were analyzed with ELISA method for SSA/Ro antigens. RESULTS UVB irradiation induced apoptotic blebs on the cell surface. The blebs were composed of ribonucleoproteins and contained SSA/Ro antigens. SSA/Ro antigens expressed on UVB irradiated keratinocytes bound to affinity-purified anti-SSA/Ro sera, leading to complement-dependent cytotoxicity. However, no SSA/Ro antigens were detected in the supernatants. CONCLUSIONS SSA/Ro, a ribonucleoprotein antigen expressed on UVB irradiated keratinocytes, may be recognized and presented to immune cells by a direct cell-cell contact other than be eliminated into the circulation.

OBJECTIVE To evaluate the effects of nitric oxide inhibitor on prostacyclin (PGI2) biosynthesis and the role of PGI2 in hyperhemodynamics of portal hypertension. METHODS Sprague Dawley rats were divided into four groups: intrahepatic portal hypertension (IHPH) by injection of CCl4, prehepatic portal hypertension (PHPH) by stenosis of the portal vein, end-to-side portacaval shunt (PCS), and sham-operated controls (SO). Animals of each group were subdivided into 2 groups: systemic administration of nitric oxide inhibitor L-NMMA and vehicle. The radioactive microsphere method was used for hemodynamic study. The level of plasma PGI2 (6-keto-PGF1α) was measured by radioimmunoassay. RESULTS The characteristics of hyperdynamic circulatory state including increased cardiac output and splanchnic blood flow, decreased mean arterial blood pressure, total peripheral vascular resistance, and splanchnic vascular resistance were observed in IHPH, PHPH and PCS rats. The magnitude of hyperhemodynamics was in the order of PCS>PHPH>IHPH rats. The hyperdynamic circulatory state in IHPH, PHPH and PCS rats could be effectively reversed by L-NMMA to the baseline values of hemodynamics in SO rats. The baseline concentrations of plasma 6-keto-PGF1α (ng/ml) in PHPH, IHPH, PCS, and SO rats were 6.93±2.43, 5.09±2.27, 2.36±1.01 and 1.56±0.61, respectively. The concentrations of plasma 6-Keto-PGF1α in PHPH, IHPH and PCS rats were significantly higher than those in SO rats. Moreover, the concentrations were significantly higher in PHPH and IHPH rats than in PCS rats (P<0.05). After administration of L-NMMA, the concentrations of plasma 6-Keto-PGF1α (ng/ml) in PHPH, IHPH, PCS and SO rats were 7.69±2.98, 5.68±2.66, 5.50±0.79, 5.02±2.86, respectively. As compared to the baseline value, the concentrations of 6-keto-PGF1α rats were slightly increased in IHPH, PHPH rats (P>0.05), but significantly increased in PCS and SO rats (P<0.05). CONCLUSIONS In this study, the hyperdynamic circulatory state in portal hypertensive rats and portacaval shunt rats was completely reversed by L-NNMA to normal, but the level of 6-keto-PGF1α was still elevated. The results indicate that PGI2 is not involved in hyperhemodynamics of portal hypertension.

OBJECTIVE To evaluate the effect of early intervention on the intellectual development of the premature infants. METHODS Premature infants at gestational age of 28-36.9 weeks were randomly divided into two groups: intervention and conventional care groups. Normal newborn infants during the same period were included in the control group (routine care). Up to March 1996, 156 cases were over the age 1.5-2 years (corrected age), 52 in the intervention group, 51 in the conventional care group and 53 in the normal control group. Parents were taught to carry out the 0-2 year intervention program, which included motor, cognitive, speech development and social behavior. Every three months, height, weight and head circumference were measured. At the age of one and a half and two years, all infants in the three groups received infant development tests of Child Development Center of China (CDCC) scale. The examiner did not know which infant had received intervention. RESULTS There was no significant difference in biological factors among the two premature groups and in cultural and social factors among the three groups. Intelligence tests at the age of one and a half and two years showed that the average mental development index (MDI) in the intervention group was 13.8 and 14.6 higher than those in the conventional care group and the differences were significant. The psychomotor development index (PDI) was 5.2 and 4.7 higher but the differences were not significant. The MDI and PDI in the intervention group and normal control were quite close, but at two years, the MDI and PDI in the intervention group were 5.7 and 7.3 higher than those in the normal control and the differences were significant (P &lt; 0.05). Compared with the normal control, the MDI in conventional care group at one and a half and two years of age were 11.5 and 8.9 lower. The difference was very significant. There were four cases of mental retardation, whose mental development index (MDI) was less then 70 in the conventional care group, but none in the intervention group. CONCLUSIONS Early intervention can promote intellectual development of the premature infants and may be beneficial to the prevention of mental retardation. Early and intensive intervention can produce better results. Bringing parent's initiative into full play through deepening their understanding of the importance of early intervention is the key to success.

With the improvement in the living standards of the Chinese people, more attention has been drawn to the importance of dermatology and the need for dermatological service. This fact has resulted in the advancement of dermatology in China in the recent years.