Intercellular gap formation induced by thrombin in confluent cultured bovine retinal pigment epithelial cells.

PURPOSE: Thrombin is formed at the site of intraocular hemorrhage and may be important in the development of progressive retinal damage. The authors observed that thrombin-treated bovine retinal pigment epithelial (RPE) cell cultures develop intercellular gaps and initiated this study to examine in detail the effects of thrombin on RPE cell morphology, adhesion, and cytoskeleton. METHODS: Confluent cultures of bovine RPE cells were incubated for various times (0 to 24 hours) with alpha-thrombin (0.1 to 100 U/ml) or enzymatically inactive thrombin. Intercellular gaps were quantitated by light microscopy in ten representative fields (magnification X400) as number of gaps per field (gaps/f). RPE cytoskeleton was studied using immunofluorescent staining for vinculin and F-actin. The mechanism of thrombin-induced RPE cell gap formation was studied by preincubation with specific drugs, including a protein kinase inhibitor (staurosporine), protein kinase C inhibitors (H-7 and calphostin C), cyclic adenosine monophosphate (cAMP) inducer (forskolin), and cytoskeleton-disrupting agents (cytochalasin B or colchicine). RESULTS: Intercellular gaps (20 to 80 microns in diameter) were markedly increased in number in thrombin-treated cultures in a dose-dependent and time-dependent manner and were associated with an alteration in the distribution of F-actin and vinculin. Whereas control cultures showed 3.3 +/- 2.4 gaps/f, incubation with 8 U/ml of alpha-thrombin for 3 hours resulted in 44.8 +/- 15.3 gaps/f. These changes were most prominent shortly after the 3-hour coincubation, but the cultures did return to their original confluent state within 24 hours. Cultures treated with an enzymatically inactive thrombin showed fewer intercellular gaps than those treated with enzymatically active thrombin but had significantly more intercellular gaps than control cultures. Thrombin-induced intercellular gap formation was blocked by preincubation with forskolin (14.6 +/- 7.1 gaps/f), staurosporine (10.2 +/- 5.0 gaps/f), or H-7 (24.5 +/- 9.8 gaps/f). CONCLUSIONS: Exposure to an enzymatically active thrombin results in formation of intercellular gaps between cultured RPE cells. Inhibition of this phenomenon by protein kinase inhibitors and by a cAMP inducer suggests that this effect is mediated, at least in part, through protein kinase C- and cAMP-dependent pathways. Thrombin generation associated with intraocular hemorrhage may thus result in direct damage to the RPE monolayer, possibly via the same pathway(s).

Inhibition of rabbit platelet aggregation by 1,4-naphthoquinones.

The effects of four 1,4-naphthoquinone derivatives on the aggregation of rabbit platelets were examined. All the four 1,4-naphthoquinone derivatives inhibited the platelet aggregation of washed rabbit platelets induced by thrombin (0.1 U/ml) and the IC50 is: 2-chloro-3-methyl-1,4-naphthoquinone (CMN), 5 micrograms/ml; 3-methyl-5,8-dihydroxy-1,4-naphthoquinone, 13 micrograms/ml; 5,8-dihydroxy-1,4-naphthoquinone, 18 micrograms/ml; 3-methyl-1,4-naphthoquinone (vitamin K3), 53 micrograms/ml. CMN was the most potent in inhibiting the aggregation and release reaction induced by ADP, arachidonic acid, PAF, ionophore A23187, collagen and thrombin in a dose-dependent manner in washed platelets, platelet-rich-plasma and whole blood. The thromboxane B2 formation caused by collagen and ionophore A23187 was inhibited by CMN. However, the thromboxane B2 formation by arachidonic acid was markedly increased. The platelet inhibitory effect of CMN could not be antagonized either by raising the concentrations of extracellular Ca++ or by wash out. The phosphoinositides breakdown induced by thrombin was inhibited by CMN. Phospholipids (PE, PC, PI) could slightly antagonize the antiplatelet effect of CMN. It is concluded that the inhibitory effect of CMN on rabbit platelet aggregation may be due to the inhibition of phosphoinositides breakdown caused by the inducers.

Determination of eight constituents of hsiao-cheng-chi-tang by high-performance liquid chromatography.

A high-performance liquid chromatographic method for the simultaneous determination of eight constituents (gallic acid, sennoside B, sennoside A, naringin, hesperidin, honokiol, magnolol and emodin) of the Chinese herbal formula hsiao-cheng-chi-tang was established. Various samples of the formula were separated using a Cosmosil 5C18 column with a linear gradient elution system consisting of acetate buffer as mobile phase. Contents of these marker substances in an unpretreated hsiao-cheng-chi-tang extract could be easily determined within 60 min. The effects of pH, buffer concentration and column selectivity for this method are described.

Capillary electrophoretic determination of the constituents of paeoniae radix.

A method combining the techniques of capillary zone electrophoresis (CZE) and micellar electrokinetic chromatography (MEKC) has been developed to separate a total of eight peony constituents. The CZE method was used to determine the content of paeonol, oxypaeoniflorin, benzoic acid, pentagalloylglucose and gallic acid, and MEKC technique based on sodium cholate was applied to analyze albiflorin, paeoniflorin, benzoyla biflorin, paeonol and oxypaeoniflorin. Linearity around two orders of magnitude of concentration was generally obtained and limits of detection for these compounds were in the range of 2.6-23.7 micrograms/ml. The relative standard deviations of migration times were less than 1.43% (n = 6). Contents of peony constituents in an ethanol-water extract of Paeonia lactiflora Pall. sample could easily be determined by this method.

Determination of glycyrrhizin and glycyrrhetinic acid in traditional Chinese medicinal preparations by capillary electrophoresis.

A simple, rapid, accurate and reproducible capillary electrophoretic method was developed for the assay of glycyrrhizin and glycyrrhetinic acid in traditional Chinese medicinal preparations. The buffer solution used in this method was acetonitrile and 0.02 M sodium dihydrogen-phosphate solution adjusted to pH 7.5 with 0.05 M sodium hydroxide. The linear calibration range was 0.04-2.00 mg/ml (r = 0.9988) for glycyrrhizin and 0.007-0.35 mg/ml (r = 0.9985) for glycyrrhetinic acid and recoveries were 98.1-101.3% for glycyrrhizin and 98.5-101.4% for glycyrrhetinic acid. The relative standard deviations were 1.02% (n = 6) for glycyrrhizin and 0.91% (n = 6) for glycyrrhetinic acid. The content of these two acids in Glycyrrhizae Radix and Glycyrrhizae Radix-containing Chinese medicinal preparations was successfully determined within 10 min.

Capillary electrophoretic determination of the constituents of Artemisiae Capillaris Herba.

Two capillary electrophoretic methods, a micellar electrokinetic electrophoretic (MEKC) one and a capillary zone electrophoretic (CZE) one, were developed for the separation of 12 constituents in Artemisiae Capillaris Herba. Detection at 254 nm with 20 mM sodium dodecyl sulfate and 20 mM sodium borate buffer (pH 9.82) in MEKC or with 25 mM sodium borate and 6.75 mg/ml 2,3,6-tri-O-methyl-beta-cyclodextrin buffer in CZE was found to be the most suitable approach for this analysis. Within 42 min, the MEKC method could successfully separate 12 authentic constituents, whereof chlorogenic acid, however, appeared as a broad and split peak, and capillarisin and chlorogenic acid overlapped partially with other coexisting substances in crude extract of the herb. The CZE method could completely overcome these problems and was used to determine the amounts of capillarisin, chlorogenic acid, scopoletin and caffeic acid in the extract. The effect of buffers on the constituent separation and the validation of the two methods were discussed.

SV40-immortalized and primary cultured human retinal pigment epithelial cells share similar patterns of cytokine-receptor expression and cytokine responsiveness.

Retinal pigment epithelial (RPE) cells produce and respond to a variety of cytokines; however, molecular and biochemical studies are restricted by the limited access to large numbers of pure cells and the variability associated with different donor sources. Despite success in establishing primary human RPE (HRPE) cell cultures, the inability to sustain consistent proliferation rates and morphology over several passages remains a concern. This problem was approached by using an immortalized line of simian virus (SV)40 transformed fetal HRPE cells (SVRPE). Cytokine production, receptor expression and responsiveness in the SVRPE cell line was analyzed to determine the usefulness of this model for studying HRPE-cytokine interactions. Using reverse transcriptase polymerase chain reaction (RT-PCR), HRPE and SVRPE cells demonstrated an identical pattern of interleukin-1 receptor (IL-1R), IL-2R (alpha sub-unit), IL-6R, interferon (IFN)-gamma R and tumor necrosis factor-alpha (TNF)R p55 expression. No amplification products for TNFR p75 or granulocyte/macrophage colony stimulating factor (GM-CSF)R were demonstrated in either population. IFN-gamma stimulation induced surface human leukocyte antigen (HLA)-DR in both SVRPE and HRPE, while TNF treatment induced surface expression of intercellular adhesion molecule (ICAM)-1 on SVRPE and upregulated ICAM from basal levels on HRPE. Both cell types showed amplification products for interleukin (IL)-1 beta, IL-6 and transforming growth factor (TGF)-beta 1 using RT-PCR. The bioassays demonstrated that both populations of unstimulated cells constitutively secrete very low levels of TGF-beta and no IL-6.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacillus cereus group strains, their hemolysin BL activity, and their detection in foods using a 16S RNA and hemolysin BL gene-targeted multiplex polymerase chain reaction system.

Hemolysin BL (HBL) is a major virulence factor for Bacillus cereus group strains. It is also a target enterotoxin for the most commonly used B. cereus detection kit, i.e., the B. cereus enterotoxin (diarrheal type) reversed passive latex agglutination (BCET-RPLA) test kit. A survey of the HBL activities and the cytotoxicities to the Chinese hamster ovary (CHO) cells for the B. cereus group strains, however, showed that although only part of the B. cereus group strains are HBL active, all strains show cytotoxicity to the CHO cells. Thus, methods that allow the detection of not only the HBL but also of the B. cereus group strains are important. In this study, by comparison of the gene sequences of the 16S rRNA for B. cereus group and other bacteria strains, we designed primers B16S1 and B16S2 specific to all the B. cereus group strains. In addition, because HBL is a major enterotoxin, we also designed HBL gene-specific polymerase chain reaction (PCR) primers, i.e., Hm1 and Hm2, that generated the same results as those of the hemolysis and BCET-RPLA assays. Primers B16S1/B16S2 and Hm1/Hm2 could be combined into a multiplex PCR system for the simultaneous detection of B. cereus group cells and the possible presence of their HBL enterotoxins. Also, all these PCR systems allowed the detection of n x 10(0) CFU B. cereus cells per g of food sample if an 8-h enrichment step was performed prior to the PCR.

Analysis and processing of Chinese herbal drugs. VIII: The study of sophorae floe.

The quantities of active constituents in commercially available Sophora japonica L. (huai-hua) and its processed samples were analyzed using high performance liquid chromatography. Rutin, quercetin, sophoricoside, sophorabioside and genistein were found. Sophora flower buds (huai-mi) contained a great deal of rutin, while the immature fruits (huai-chiao) contained sophoricoside and sophorabioside. When Sophora flower bud was stir-fried for short periods using low heat, the extracted rate of rutin and quercetin increased slightly, decreasing with longer cooking periods at higher temperatures. When stir-fried with vinegar, it quickly blackened, but when cooked with honey, it changed very little. Immature Sophora fruit, however, exhibits less variation in its active constituents after processing. All huai-hua processed samples give forth a pronounced aroma and could be more easily extracted with water than that of the unprocessed herbs.

Histopathology of polytetrafluoroethylene (Gore-tex) as a scleral buckle in humans.

Several materials have been used for scleral buckling implants in retinal detachment surgery. Of these, polytetrafluoroethylene (PTFE, Gore-tex) has the relative advantage of decreasing infection and extrusion when it is used as a scleral buckle material. In this report, we present the clinical and histopathologic finding of a case where Gore-tex was used as an encircling buckle material for retinal detachment operation.

Frequency doubling technology perimetry in retinal diseases--preliminary report.

The role of frequency doubling technology perimetry (FDT) in glaucoma has been promising in speed and simplicity as well as in its high sensitivity and specificity compared to conventional automatic perimetry. In this study, we evaluate the role of frequency doubling technology perimetry (FDT) in a variety of retinal diseases. FDT using full-threshold C-20 mode was performed in thirty-three patients with a variety of retinal diseases [16 cases of rhegmatogenous retinal detachment (RRD), nine cases of age-related maculopathy (AMD) and eight cases of central serous chorioretinopathy (CSCR)]. The fundus was scored by the extent of disease on each quadrant and macular area (central five degree visual angle). Then the retinal scores were compared with the points depressed to 2% on total deviation plots in FDT C-20 full-threshold mode using Spearman's rank correlation method. The correlation was statistically significant in the group of RRD (R = 0.57, P = 0.01), whereas the correlation was not significant in the group of macular diseases (AMD & CSCR, R = 0.47 & 0.32, P = 0.12 & 0.20). In conclusion, our results suggest that FDT might be a useful and simple technique for functional evaluation in RRD, but not in the macular disease group under this scoring system.

The biometric study in different stages of primary angle-closure glaucoma.

PURPOSE: This study compared the general and ocular biometric characteristics of normal, primary angle closure (PAC), and primary angle-closure glaucoma (PACG) patients to better understand the possible relationship between differences in ocular parameters that might predict risk for PACG in PAC patients. METHODS: One hundred normal, 90 PAC, and 90 PACG eyes were retrospectively reviewed. General characteristics such as age, gender, body height, body weight, blood pressure, pulse, systemic diseases, and education level were recorded. Ocular findings included visual acuity, intraocular pressure, refraction, cup to disc ratio, and ocular biometry. Ocular biometry was obtained by A-scan ultrasonography (Digital A/B scan 5500; Sonomed Inc., Lake Success, NY, USA). The parameters recorded were anterior chamber depth (ACD), lens thickness (LT), axial length (AXL), lens/axial length factor (LAF), and relative lens position (RLP). RESULTS: Although the controls, PAC group, and PACG group were found to be significantly different in age (62.7±9.8; 65.3±7.5; and 66.0±7.4, respectively), there were no gender differences. With regard to ocular parameters, the ACD tended to decrease and the LT and LAF tended to increase from normal to PAC to PACG. The eyes of the PACG group had significantly shallower ACD (P<0.001) and thicker lens (P<0.001) than those of the PAC group. While PAC had similar lens position to the control group, PACG had more anteriorly positioned lens than the PAC group (P<0.001). Logistic regression analysis found a significant association between a decrease in ACD and increased risk of PACG (odds ratio (OR)=3.59 for 0.2 mm decrease in ACD) as well as a significant association between an increase in LT and increased risk of PACG (OR=1.30). CONCLUSIONS: In addition to LT, a shallower ACD owing to a change in RLP may have a role in the progression from PAC to PACG. Owing to the differences of certain biometric characteristics between PAC and PACG, A-scan ultrasonography might potentially be used for the early detection of PACG in PAC eyes.

[The existential phenomenology of family care of psychiatric patients].

This study utilizes the perspectives of phenomenological approach and existential analysis to understand the common meaning and existential status of care through the description of care-giving and care-receiving experiences by both psychiatric patients and their families. Multiple perspectives and methods were used for data cellection, including participant observation and individual and group interviews. Five themes emerged from the hermeneutic data analysis which represent the existential status and types of family care for psychiatric patients: (1) Taken-for-granted care; (2) Nonexistent care; (3) Limited care; (4) Replacemental or transcendental care; (5) Displacemental or exchangeable care. Following the results, several issues are discussed. First, the existential meaning and values of family caregiving for psychiatric patients are explored. Subsequently, indigenous psychiatric mental health care problems in Taiwan are discussed. Finally, health professionals need to pay attention to the mutual existential caring status from self and others as well as the transcendental and exchangeable relationships from both the care-givers' and the care-receivers' perspectives.

Macular subretinal neovascularization in choroidal tuberculosis.

A 54-year-old Chinese woman with miliary choroidal tuberculosis was followed for more than three years. She had had tuberculous meningitis for about one month before an ophthalmologic examination for blurred vision OU. There were 50 to 60 choroidal tubercles OU which were located mostly at the posterior poles including the macular areas. The meningitis and tubercular lesions resolved with antituberculous medications. In a series of fundus photographs and fluorescein angiograms, a macular subretinal neovascularization was noted in association with the tubercular lesions which resulted in disciform maculopathy. To the best of our knowledge, this case had the largest number of tubercles reported in this century, and the association of macular subretinal neovascularization with choroidal tuberculosis has never been reported.

Enterotoxigenic profiles and polymerase chain reaction detection of Bacillus cereus group cells and B. cereus strains from foods and food-borne outbreaks.

Bacillus cereus is one of the important food pathogens. Since B. cereus group cells, such as B. cereus, B. thuringiensis, B. anthracis and B. mycoides, share many phenotypical properties and a high level of chromosomal sequence similarity, it is interesting to investigate the virulence profiles for B. cereus group cells, including B. cereus strains isolated from foods and samples associated with food-poisoning outbreaks. For this investigation, the presence of enterotoxin genes, such as those of haemolysin BL, B. cereus enterotoxin T and enterotoxin FM, were assayed by polymerase chain reaction (PCR) methods. Meanwhile, their enterotoxin activities were assayed using the BCET-RPLA kit, haemolytic patterns on sheep blood agar and their cytotoxicity to Chinese hamster ovary (CHO) cells. Results showed that there were 12 enterotoxigenic profiles for the 98 B. cereus group strains collected. In addition, if any of the three types of enterotoxins was present in the B. cereus group cells, these cells were shown to be cytotoxic to the CHO cells. Similar enterotoxigenic profiles could be found among strains of B. cereus, B. mycoides and B. thuringiensis. Thus, all B. cereus group strains may be potentially toxigenic and the detection of these cells in foods is important. We thus designed PCR primers, termed Ph1/Ph2, from the sphingomyelinase gene of B. cereus cells. These primers were specific for all B. cereus group strains and could be used for the detection of B. cereus cells contaminated in food samples.

Should asymptomatic atrophic retinal holes be treated prophylactically in pseudophakic eyes after Nd:YAG laser posterior capsulotomy?

BACKGROUND: Retinal detachment (RD) following cataract extraction is a serious and potentially sight-threatening event that will often necessitate further surgical intervention. Of the several risk factors reported in the literature, Neodymium:YAG laser posterior capsulotomy has been reported to increase the risk of RD following cataract surgery up to 4.9-fold. The purpose of this study is to investigate the difference of characteristics in pseudophakic RD with or without Nd:YAG laser posterior capsulotomy and the possible role of atrophic retinal holes in pseudophakic RD after posterior capsulotomy. METHODS: Retrospective review of 78 cases of pseudophakic RD after uncomplicated extracapsular lens extraction and intraocular lens implantation was done. Twenty-one of them received Nd:YAG laser posterior capsulotomy before RD. We measured the numbers, types, and locations of retinal breaks and several other risk factors associated with RD. Analysis was performed with the system of Statistical Products and Services Solutions (SPSS). RESULTS: The ratio of retinal holes only in retinal breaks that caused retinal detachment was significantly higher in the capsulotomy group than the non-capsulotomy group (12/21 vs 14/57, p = 0.05). Only three patients in the capsulotomy group claimed significant symptoms before the detachment. There was no significant difference in mean number of retinal breaks and break location. Other risk factors predisposing to RD did not differ significantly between the two groups (13/21 vs 23/57, p = 0.12), nor did the interval from cataract surgery to RD (31.20 vs 29.44 months, p = 0.48). CONCLUSIONS: This primitive study suggests that asymptomatic atrophic retinal holes, usually not regarded as an absolute risk factor for RD, might lead to RD preferentially after Nd:YAG laser posterior capsulotomy. Early identification and treatment of such breaks might decrease the incidence of pseudophakic RD. We suggest that it is necessary to perform detailed preoperative and postoperative fundus examination and treat prophylactically any identified breaks, including asymptomatic atrophic retinal holes, in pseudophakic eyes after Nd:YAG laser posterior capsulotomy.

A comparative study on commercial samples of the roots of Paeonia vitchii and P. lactiflora.

A total of 37 commercial samples of paeoniae radix (Paeonia lactiflora Pall. = P. albiflora Pall, and P. vitchii Lynch), were collected from Taiwan's herbal market. The contents of eight constituents (gallic acid, oxypaeoniflorin, albiflorin, paeoniflorin, benzoic acid, pentagalloylglucose, paeonol, and benzyoylalbiflorin) in these samples were determined by high-performance liquid chromatography. It was found that constituent contents in P. lactiflora samples were generally higher than in P. vitchii samples except for the albiflorin/oxypaeoniflorin ratio and paeonol content. The major compounds of P. lactiflora were paeoniflorin, albiflorin, and pentagalloylglucose, while those in P. vitchii were paeoniflorin, pentagalloylglucose, and oxypaeoniflorin. The peak-area ratio of albiflorin/oxypaeoniflorin was higher than unity in P. lactiflora samples but lower than unity in P. vitchii, and the ratio can be used to differentiate the origin of Paeonia species clearly. In addition, the former had higher contents in the core wood but the latter in the cortex.

A reporter gene assay for the detection of phytoestrogens in traditional Chinese medicine.

Bupleurum & Peony Formula (Jia Wei Xiao Yao San) is a herbal formula which possesses a clinical history for the treatment of menopausal syndrome and menstrual irregularity. The present investigation reports the ability to monitor the formula's phytoestrogen content that will allow for the implementation of a standardization protocol that is based on a quantifiable biological response. Utilizing an oestrogen-sensitive chimeric receptor/reporter gene element which has been stably transfected into HeLa cells, the botanical formula was shown to induce the expression of the reporter gene, luciferase, in a dose dependent manner. Pretreatment of the HeLa cells with the botanical formula produced a 5-fold increase in bioluminescence compared with the control. Additionally, our studies showed that the response of the cells, when challenged by the botanical formula, was oestrogen specific. Pretreatment of the cells with tamoxifen effectively blocked the activation of the chimeric oestrogen receptor by the botanical formula. The cell line provides a sensitive assay that can easily detect the presence of phytoestrogens in complex botanical formulas.

Ocular manifestations of tuberculosis.

OBJECTIVE: To present the clinical and histopathologic findings in five cases of tuberculosis (TB) with various ocular manifestations. DESIGN: Observational case series. METHODS: Retrospective review of clinical findings, course, and treatment of five patients. Diagnostic techniques, including biomicroscopic, histopathologic, and molecular biologic test results, are presented. MAIN OUTCOME MEASURES: Visual acuity, slit-lamp biomicroscopy, indirect ophthalmoscopy, and fluorescein angiography results. RESULTS: The ocular manifestations of TB in our patients included panophthalmitis, endophthalmitis, posterior uveitis with choroidal tubercles, keratitis, and a lid mass. Mycobacterium tuberculosis was identified in four cases in ocular specimens using acid-fast bacilli microscopy and in three cases by culture. Rapid diagnosis using polymerase chain reaction was obtained in one case. Extraocular foci of TB were identified in three cases with an intraocular infection at presentation. No patients had the human immunodeficiency virus (HIV) and none were immunocompromised. Two eyes could not be saved using antituberculous treatment because of delayed diagnosis and treatment. CONCLUSIONS: In this age of the HIV pandemic, TB is becoming more common. Because it is curable, heightened awareness and better understanding of the disease's ocular manifestations should be of concern to all ophthalmologists.