The ultraviolet A-activated photosensitizer 2-(4-aminophenyl)-7-methoxybenzothiazole suppresses proliferation and induces apoptosis of keloid fibroblasts: a potential adjunctive therapy for keloids.

BACKGROUND: Photodynamic therapy (PDT), a therapeutic approach employing a photosensitizer and a specific wavelength of light, is an emerging option for treating neoplastic and nonneoplastic diseases. Keloids are fibroproliferative dermal lesions characterized by the proliferation of fibroblasts. Recently, PDT has been demonstrated as a potential treatment for keloids. AIM: To investigate the effects of our newly synthesized photosensitizer 2-(4-aminophenyl)-7-methoxybenzothiazole (6d) plus ultraviolet (UV)A irradiation (6d-UVA) on proliferation and apoptosis in keloid fibroblasts (KFs). METHODS: Fibroblasts cultured from normal skin and keloids were treated with 6d-UVA. Relevant assays including 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, 5-bromo-2'-deoxyuridine incorporation assay, immunofluorescence assay and flow cytometry analysis were performed. RESULTS: The combination of 6d (2.0 or 5.0 µmol/L) and UVA 0.5 J/cm(2) significantly decreased the viability and proliferation of KFs but not normal fibroblasts (NFs). Cell cycle analyses showed significant G0/G1 arrest and increased sub-G1 distribution in NFs induced by UVA-activated 6d at 5.0 µmol/L (hereafter referred to as 6d-UVA). This treatment also significantly induced generation of intracellular reactive oxygen species (ROS), loss of mitochondrial membrane potential (ΔΨm), and increased expression of active caspase-3. Pretreatment with N-acetyl-L-cysteine (aROS scavenger) reversed the increased active caspase-3 expression induced by 6d-UVA, indicating the involvement of ROS in 6d-UVA-induced apoptosis. CONCLUSIONS: This study indicates that 6d-UVA treatment exerts antiproliferative and pro-apoptotic effects in KFs. We propose that 6d-UVA could be a potentially usefull ancillary method for keloid treatment.

Tacrolimus abrogates TGF-ß1-induced type I collagen production in normal human fibroblasts through suppressing p38MAPK signalling pathway: implications on treatment of chronic atopic dermatitis lesions.

BACKGROUND: Atopic dermatitis (AD) is a commonly encountered inflammatory skin disease. Although acute lesions of acute AD are characterized by intense inflammation, the hallmarks of chronic AD lesions include lichenified fibrosis and thickening of the upper dermis. The increased expression of transforming growth factor beta 1 (TGF-ß1), a well-known fibrogenic cytokine, is observed in chronic AD lesions. Tacrolimus (FK506) ointment has been reported to be effective for treating AD as well as some TGF-ß1-induced fibrotic diseases. OBJECTIVES: To evaluate the effect of tacrolimus on TGF-ß1-stimulated cultured normal human dermal fibroblasts and explore the potential signalling pathways involved. METHODS: Fibroblasts cultured from healthy adult human foreskins were treated with TGF-ß1 with or without tacrolimus. The impact on cell viability and proliferation were assessed by [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] (MTT) assay and BrdU incorporation assay respectively. Reverse transcription-polymerase chain reaction (RT-PCR), quantitative real-time PCR, enzyme-linked immunosorbent assay (ELISA) and western blotting were performed to evaluate the relevant expressions of mRNA or proteins in fibroblasts. RESULTS: Our results revealed that the increased expressions of transforming growth factor-ß receptor I (TGF-ßRI) and TGF-ßRII in TGF-ß1-treated fibroblasts were suppressed by tacrolimus treatment. In addition, tacrolimus significantly inhibited fibroblast proliferation enhanced by TGF-ß1. TGF-ß1 increased type I collagen production, and this enhancing effect was suppressed by tacrolimus. The down-regulation of MMP-1 and up-regulation of TIMP-1 induced by TGF-ß1 were reversed by tacrolimus. The increase in phosphorylated p38 mitogen-activated protein kinase (p38MAPK) expression stimulated by TGF-ß1 was down-regulated by tacrolimus. Moreover, the fibroblasts treated with p38MAPK inhibitor significantly reduced type I collagen expression induced by TGF-ß1. CONCLUSIONS: The present results demonstrated that tacrolimus significantly inhibited physiological functions of fibroblasts enhanced by TGF-ß1 in vitro. Clinically, we propose that topical tacrolimus may not only reduce AD recurrence but also ameliorate dermal fibrosis often seen in chronic AD lesions.

FK506 inhibits the enhancing effects of transforming growth factor (TGF)-ß1 on collagen expression and TGF-ß/Smad signalling in keloid fibroblasts: implication for new therapeutic approach.

BACKGROUND: Keloid is a unique proliferative disorder of fibroblasts resulting from derailment of the typical wound healing process. Due to lack of animal models for therapeutic testing, treatment of keloids remains a clinical challenge. Transforming growth factor (TGF)-ß1-related signalling plays a key role in keloid formation. As tacrolimus (FK506) has been reported to inhibit the effects of TGF-ß1 on cultured fibroblasts, we hypothesized that FK506 may be useful in treating keloids. OBJECTIVES: To explore the effects of FK506 on TGF-ß1-stimulated keloid fibroblasts (KFs) in terms of proliferation, migration and collagen production and to investigate the regulatory pathways involved. METHODS: Fibroblasts derived from keloids were treated with TGF-ß1 with or without FK506. Relevant assays including 5-bromo-2'-deoxyuridine incorporation assay, in vitro scratch assay, reverse transcription-polymerase chain reaction (PCR), quantitative PCR and Western blotting were performed. RESULTS: The proliferation and migration of KFs were significantly higher than those of normal fibroblasts. FK506 markedly inhibited KF proliferation, migration and collagen production enhanced by TGF-ß1. The increase in TGF-ß receptor I and II expression in TGF-ß1-treated KFs was suppressed by FK506 treatment. TGF-ß1 increased the phosphorylation of Smad2/3 and Smad4 in KFs, and this enhancing effect was abrogated by FK506. In addition, FK506 significantly increased the expression of Smad7 which was suppressed by TGF-ß1 treatment. CONCLUSIONS: Our results demonstrate that FK506 effectively blocks the TGF-ß/Smad signalling pathway in KFs by downregulation of TGF-ß receptors and suggest that FK506 may be included in the armamentarium for treating keloids.

Misoprostol dose and route after mifepristone for early medical abortion: a randomised controlled noninferiority trial.

OBJECTIVE: To compare 400 and 800 microg sublingual or vaginal misoprostol 24 hours after 200 mg mifepristone for noninferiority regarding efficacy in achieving complete abortion for pregnancy termination up to 63 days of gestation. DESIGN: Placebo-controlled, randomised, noninferiority factorial trial, stratified by centre and length of gestation. Misoprostol 400 or 800 microg, administered either sublingually or vaginally, with follow up after 2 and 6 weeks. SETTING: Fifteen obstetrics/gynaecology departments in ten countries. POPULATION: Pregnant women (n = 3005) up to 63 days of gestation requesting medical abortion. METHODS: Two-sided 95% CI for differences in failure of complete abortion and continuing pregnancy, with a 3% noninferiority margin, were calculated. Proportions of women with adverse effects were recorded. OUTCOME MEASURES: Complete abortion without surgical intervention (main); continuing live pregnancies, induction-to-abortion interval, adverse effects, women's perceptions (secondary). RESULTS: Efficacy outcomes analysed for 2962 women (98.6%): 90.5% had complete abortion after 400 microg misoprostol, 94.2% after 800 microg. Noninferiority of 400 microg misoprostol was not demonstrated for failure of complete abortion (difference: 3.7%; 95% CI 1.8-5.6%). The 400-microg dose showed higher risk of incomplete abortion (P < 0.01) and continuing pregnancy (P < 0.01) than 800 microg. Vaginal and sublingual routes had similar risks of failure to achieve complete abortion (P = 0.47, difference in sublingual minus vaginal -0.7%, 95% CI -2.6-1.2%). A similar pattern was observed for continuing pregnancies (P = 0.21). Fewer women reported adverse effects with vaginal than sublingual administration and with the 400-microg dose than the 800-microg dose. Of the women, 94% were satisfied or highly satisfied with the regimens, 53% preferred the sublingual route and 47% preferred the vaginal route. CONCLUSIONS: A 400-microg dose of misoprostol should not replace the 800-microg dose when administered 24 hours after 200 mg mifepristone for inducing abortion in pregnancies up to 63 days. Sublingual and vaginal misoprostol have similar efficacy, but vaginal administration is associated with a lower frequency of adverse effects.

Two mifepristone doses and two intervals of misoprostol administration for termination of early pregnancy: a randomised factorial controlled equivalence trial.

OBJECTIVE: To compare the efficacy of 100 mg and 200 mg of mifepristone and 24- and 48-hour intervals to administration of 800 microg vaginal misoprostol for termination of early pregnancy. DESIGN: Placebo-controlled, randomized, equivalence trial, stratified by centre. SETTING: 13 departments of obstetrics and gynecology in nine countries. POPULATION: 2,181 women with 63 days or less gestation requesting medical abortion. METHODS: Two-sided 95% CI for the risk differences of failure to complete abortion were calculated and compared with 5% equivalence margin between two doses of mifepristone and two intervals to misoprostol administration. Proportions of women with adverse effects were compared between the regimens using standard testes for proportions. OUTCOME MEASURES: Rates of complete abortion without surgical intervention and adverse effects associated with the regimens. RESULTS: Efficacy outcome was analysed for 2,126 women (97.5%) excluding 55 lost to follow up. Both mifepristone doses were found to be similar in efficacy. The rate of complete abortion was 92.0% for women assigned 100 mg of mifepristone and 93.2% for women assigned 200 mg of mifepristone (difference 1.2%, 95% CI: -1.0 to 3.5). Equivalence was also evident for the two intervals of administration: the rate of complete abortion was 93.5% for 24-hour interval and 91.7% for the 48-hour interval (difference -1.8%, 95% CI: -4.0 to 0.5). Interaction between doses and interval to misoprostol administration was not significant (P = 0.92). Adverse effects related to treatments did not differ between the groups. CONCLUSIONS: Both the 100 and 200 mg doses of mifepristone and the 24- and 48-hour intervals have a similar efficacy to achieve complete abortion in early pregnancy when mifepristone is followed by 800 micrograms of vaginally administered misoprostol.

Hyperglycaemic conditions decrease cultured keratinocyte mobility: implications for impaired wound healing in patients with diabetes.

BACKGROUND: Elevated blood glucose in patients with diabetes mellitus (DM) leads to complications including poor wound healing. Proper keratinocyte migration and proliferation are the crucial steps during re-epithelialization. We hypothesize that the impaired wound healing in patients with DM is due to the disruption of proper re-epithelialization. OBJECTIVES: We aimed to explore the effects of high glucose on keratinocytes in terms of cell migration and proliferation. METHODS: Keratinocytes were cultivated in normal and high glucose conditions. Their viability was evaluated by MTS assay. Transwell migration and in vitro scratch assays were used to evaluate their mobility. The mRNA expressions and activities of matrix metalloproteinase (MMP)-2 and MMP-9 were determined. The mRNA of their respective physiological inhibitors, tissue inhibitor of MMP (TIMP)-1 and TIMP-2, was also evaluated. Immunofluorescent staining and Western blotting were used to examine the expression of phosphorylated focal adhesion kinase (pp125(FAK)). The impacts of high glucose on keratinocyte proliferation were assessed by 5-bromo-2'-deoxyuridine incorporation assay. RESULTS: High glucose treatment did not affect keratinocyte viability up to 3 days. In contrast, the mobility of keratinocytes, the activities and gene expressions of MMP-2 and MMP-9, the expression of pp125(FAK), and the cell proliferation after 5 days were significantly downregulated after hyperglycaemic treatments while the mRNA expression of TIMP-1 increased. CONCLUSIONS: Under hyperglycaemic conditions, keratinocytes demonstrate reduced migration and decreased proliferation capacities. These impairments of keratinocyte functions are likely to result in inadequate re-epithelialization. These defective physiological events provide a reasonable explanation for the poor wound healing commonly observed in patients with DM.

Comparison of four different dosages of injectable bromocriptine retard for puerperal ablactation.

The clinical efficacy, tolerance and effect on plasma prolactin levels of four different dosages of intramuscular bromocriptine retard were compared. 108 Chinese puerperas with a mean body weight of 58 kg, who chose not to breast-feed following vaginal delivery, were randomized into four equal groups. The patients in each group were given intramuscular bromocriptine retard: 20, 30, 40 or 50 mg. The injection was well tolerated by all, except for two patients who developed small haematomas at the site of the injection and 2 patients who complained of dizziness. Ablactation occurred in 100% of the patients in the 40 mg group, but was successful in only 92 and 91% of the patients in the 20 and 30 mg groups, respectively. There were two cases of suboptimal response in the 50 mg group, despite marked reduction in plasma prolactin levels. Both these patients had developed moderate breast engorgement before they received the injection. The difference in response among the four groups was not statistically significant. We recommend that the injection be given prior to the development of breast engorgement.

Detection of IgM antibodies from cerebrospinal fluid and sera of dengue fever patients.

During the dengue epidemic from late 1987 to 1989, 6 specimens of cerebrospinal fluid (CSF) and sera for IgM detection were collected from 4 cases virologically confirmed dengue patients who had neural symptoms. Another 20 serum specimens, which had been diagnosed as dengue infection either virologically or serologically, were sent to the laboratory from Kaohsiung Medical College Hospital. All these specimens were also taken to detect the existence of IgM. The results showed that IgM could be detected from 14 out of 20 serum specimens. One of the positive specimens showed IgM can last up to 252 days after onset of illness. In addition, IgM was detected from both CSF and sera of all four dengue patients with neural symptoms. The IgM titer in CSF (less than or equal to 1:20) was always lower than that in serum (greater than or equal to 1:80). Two cases with sequentially collected specimens showed the fading of IgM titer in CSF. As a matter of fact, it became undetectable about a month after onset of illness, which is apparently different from the situation in serum.

[Value of CA125 (carbon hydrate tumor-associated antigen) and endometrial antibodies for the detections of endometriosis].

Levels of CA125 and endometrial antibodies (EMAb) in serum and peritoneal fluid were determined in 42 cases, including 28 cases of endometriosis as trial group and 14 infertile patients without endometriosis as controls. Results showed that the levels of CA125 and EMAb in peritoneal fluid were higher significantly than in serum, but no difference were found between two groups. Serum EMAb levels were significantly higher in the trial group than in the controls. There was no significant difference in the levels of serum CA125 between two groups. The positive rate of serum CA125 and EMAb were 71.43% and 82.14% respectively. The sensitivity of CA125 and EMAb measurements in the diagnosis of endometriosis were 71.43% and 82.14%, and the specificity were 57.21% and 57.14% respectively.

Phenotypic characteristics of dengue 2 virus persistently infected in a C6/36 clone of Aedes albopictus cells.

A C6/36 cell culture persistently infected by dengue 2 virus was established and retained for over 20 weeks. No CPE was observed at any stage of infection. However, some giant cells up to 2- or 3-fold the diameter of normal cells appeared in the established culture. Extracellular virus titers fluctuated with a periodic tendency while the antigen level remained constant, suggesting some viral particles evolved into noninfectious defective interfering particles. Plaque size tended to change during the passage of cultures. The passage 8 virus mainly produced plaques relatively smaller than those produced by the passage 1 virus. Nevertheless, the plaque sizes produced by the passages 18 virus were somewhat mosaic. A temperature-sensitive mutant was detected in the virus released from passages 8 and 18. In addition, passages 8 and 18 produced the lowest virus titers. When persistently infected viruses were grown in Vero cells at 37 degrees, at passage 1 there was a high titer which decreased 5 days postinoculation, whereas at passages 8 and 18 titers were undetectable until 4 days postinoculation. It seems that dengue 2 virus persistently infected in C6/36 cells could have been genetically altered. This will be tested by nucleic acid analysis of the viruses.

Detection of dengue virus antigens in cultured cells by using protein A-gold-silver staining (pAgs) method.

A protein A-gold-silver (pAgs) staining was developed to detect dengue virus antigens in cultured cells. The method can be carried out in either newly-subcultured or monolayered cells. Dengue virus-inoculated C6/36 clone of Aedes albopictus cells and human endothelial cells appeared brown-yellowish color on the peripheral membrane of the infected cells. In many cases, the infected C6/36 cells appeared darker than that of the infected endothelial cells. The positive results from the inoculated C6/36 cells usually appeared as early as 2 days post-inoculation for types 1, 2, and 4 of dengue viruses and 3 days for the dengue 3 virus. The same batch of specimens detected by direct immunofluorescence antibody test (DFA) showed positive 4 days post-inoculation for the types 2, 3, and 4 of dengue viruses and 6 days for the dengue 1 virus. The result also showed that all pAgs-positive specimens were also DFA-positive, but not vice versa. It suggested that pAgs is not only sensitive but also specific for dengue virus detection from inoculated cultured cells.

Identification and characterization of a novel benzo[a]pyrene-derived DNA adduct.

The aim of this study was to generate and identify a novel benzo[a]pyrene (BP)-derived DNA adduct found both in vitro and in vivo. To date, the majority of studies have focused on N(2)-[10 beta(7 beta,8a,9a-trihydroxy-7,8,9,10-tetrahydrobenzo[a]pyrene)yl]-deoxyguanosine (anti-BPDE-dG), the major adduct generated following bioactivation of BP. However, a second adduct is also formed following bioactivation of BP which has been speculated to result from further metabolism of 9-OH-BP. In order to identify this second reaction pathway, the ultimate DNA binding species, and the DNA base involved, we have synthesized and characterized a dG-derived DNA adduct arising from further bioactivation of 9-OH-BP in the presence of rat liver microsomes. Analysis of the adducted nucleotides was conducted using both the (32)P-postlabeling assay and capillary electrophoresis-mass spectrometry (CE-MS).

Changing prevalence of antibody to Dengue virus in paired sera in the two years following an epidemic in Taiwan.

To elucidate the epidemic pattern of a dengue outbreak in southern Taiwan during 1987-8, antibody prevalence rates were investigated in paired sera collected in both epidemic (Kaohsiung) and non-epidemic (Tainan) areas. In Kaohsiung, the IgG prevalence rate in 1989 was significantly higher (9.23%) than that in 1988 (5.29%) suggesting that new infections continuously appeared after the first bleeding in 1988. Although IgG antibody persisted in most infected blood samples, waning of antibody occurred in 6/355 (1.69%) of Kaohsiung sera. IgM antibody was only detected in Kaohsiung sera, suggesting that Tainan was not involved in the outbreak. Because IgG antibody was present in some samples collected in 1989, but not in 1988, from the non-epidemic area, sporadic infections perhaps occurred. Additionally, 4/355 (1.13%) of Kaohsiung sera showed IgM antibody positive in both 1988 and 1989. In turn, secondary infections may have occurred because of circulation of multiple-types of the virus. The possible relationship between low levels of dengue haemorrhagic fever (DHF) and the loss of IgG antibodies over time is also discussed.

[A study on transovarial transmission of dengue type 1 virus in Aedes aegypti].

The main purpose of this study is to determine the possibility of transovarial transmission of dengue type 1 virus, which was isolated from the serum of a patient with dengue fever during the 1987 dengue epidemic in southern Taiwan, in Aedes aegypti of Kaohsiung strain (KH). Parent female mosquitoes were inoculated with dengue 1 virus by intrathoracic inoculation technique. The F1 offspring adults collected from three sequential ovarian cycles were pooled to become 51, 13 and 14 pools, respectively. All pools were individually inoculated into C6/36 cells and then were detected by direct immunofluorescence antibody technique. Ten, five and three pools, respectively, among these three ovarian cycles turned out to be positive. It revealed that the minimum infection rates (MIR), were 1:254.6 (2,546 mosquitoes), 1:133.6 (668 mosquitoes) and 1:238 (714 mosquitoes), respectively. Meanwhile, the estimated filial infection rates were calculated to be 0.44%, 0.97% and 0.48%, respectively. Since viral antigen has been detected in the tissue of maturing eggs within ovarioles, the results exhibited the possibility that dengue type 1 virus can be transovarially transmitted to the next generation.