Couple oriented counselling improves male partner involvement in sexual and reproductive health of a couple: Evidence from the ANRS PRENAHTEST randomized trial.

BACKGROUND: Male partner involvement (MPI) has been recognized as a priority area to be strengthened in Prevention of Mother to Child Transmission (PMTCT) of HIV. We explored the impact of Couple Oriented Counselling (COC) in MPI in sexual and reproductive health and associated factors. METHOD: From February 2009 to October 2011, pregnant women were enrolled at their first antenatal care visit (ANC-1) and followed up until 6 months after delivery in the Mother and Child Center of the Chantal Biya Foundation within the randomized prenahtest multicentric trial. The MPI index was defined using sexual and reproductive health behaviour variables by using multiple correspondence analysis followed by mixed classification. Men were considered as highly involved if they had shared their HIV test results with their partner, had discussed on HIV or condom used, had contributed financially to ANC, had accompanied their wife to ANC or had practiced safe sex. Factors associated to MPI were investigated by the logistic model with GEE estimation approach. RESULTS: A total of 484 pregnant women were enrolled. The median age of the women was 27 years (IQR: 23-31) and 55.23% had a gestational age greater than 16 weeks at ANC-1. Among them, HIV prevalence was 11.9% (95% CI: 9.0-15.4). The median duration of the women's relationship with their partner was 84 months (IQR: 48-120). MPI index at 6 months after delivery was significantly greater in the COC group than the classical counselling group (14.8% vs 8,82%; p = 0,043; Fig 1). The partners of the women who participated in the COC were more likely to be involved during follow up than others (aOR = 1.45; 95% CI = 1.00-2.10). Partners with no incoming activity (aOR = 2.90; 95% CI = 1.96-4.29), who did not used violence within the couple (aOR = 1.70; 95% CI = 1.07-2.68), and whose partner came early for ANC-1 (aOR = 1.37; 95% CI = 1.00-1.89) were more likely to be involved than others. CONCLUSION: MPI remains low in stable couples and COC improves partner involvement. Our findings also support the need of strengthening outreach towards "stable" couples and addressing barriers. This could go a long way to improve PMTCT outcomes in Cameroon. TRIAL REGISTRATION: PRENAHTEST, NCT01494961. Registered 15 December 2011-Retrospectively registered, https://clinicaltrials.gov/ct2/show/NCT01494961.

One-Pot Conversion of Methane to Light Olefins or Higher Hydrocarbons through H-SAPO-34-Catalyzed in Situ Halogenation.

Methane was converted to light olefins (ethene and propene) or higher hydrocarbons in a continuous flow reactor below 375 °C over H-SAPO-34 catalyst via an in situ halogenation (chlorination/bromination) protocol. The reaction conditions can be efficiently tuned toward selective monohalogenation of methane to methyl halides or their in situ oligomerization to higher hydrocarbons. The presence of C5+ hydrocarbons in the reaction products clearly indicates that by using a properly engineered catalyst under optimized reaction conditions, hydrocarbons in the gasoline range can be produced. This approach has significant potential for feasible application in natural gas refining to gasoline and materials under moderate operational conditions.

Crystal and mol-ecular structure of aflatrem.

The crystal structure of the title compound, C32H39NO4, confirms the absolute configuration of the seven chiral centres in the mol-ecule. The molecule has a 1,1-dimethylprop-2-enyl substituent on the indole nucleus and this nucleus shares one edge with the five-membered ring which is, in turn, connected to a sequence of three edge-shared fused rings. The skeleton is completed by the 7,7-trimethyl-6,8-dioxabi-cyclo-[3.2.1]oct-3-en-2-one group connected to the terminal cyclohexene ring. The two cyclohexane rings adopt chair and half-chair conformations, while in the dioxabi-cyclo-[3.2.1]oct-3-en-2-one unit, the six-membered ring has a half-chair conformation. The indole system of the mol-ecule exhibits a tilt of 2.02 (1)° between its two rings. In the crystal, O-H⋯O hydrogen bonds connect mol-ecules into chains along [010]. Weak N-H⋯π inter-actions connect these chains, forming sheets parallel to (10-1).

Evaluating the impact of computer-generated rounding reports on physician workflow in the nursing home: a feasibility time-motion study.

OBJECTIVES: To determine the feasibility and impact of a computer-generated rounding report on physician rounding time and perceived barriers to providing clinical care in the nursing home (NH) setting. SETTING: Three NHs located in Pittsburgh, PA. PARTICIPANTS: Ten attending NH physicians. MEASUREMENTS: Time-motion method to record the time taken to gather data (pre-rounding), to evaluate patients (rounding), and document their findings/develop an assessment and plan (post-rounding). Additionally, surveys were used to determine the physicians' perception of barriers to providing optimal clinical care, as well as physician satisfaction before and after the use of a computer-generated rounding report. RESULTS: Ten physicians were observed during half-day sessions both before and 4 weeks after they were introduced to a computer-generated rounding report. A total of 69 distinct patients were evaluated during the 20 physician observation sessions. Each physician evaluated, on average, four patients before implementation and three patients after implementation. The observations showed a significant increase (P = .03) in the pre-rounding time, and no significant difference in the rounding (P = .09) or post-rounding times (P = .29). Physicians reported that information was more accessible (P = .03) following the implementation of the computer-generated rounding report. Most (80%) physicians stated that they would prefer to use the computer-generated rounding report rather than the paper-based process. CONCLUSIONS: The present study provides preliminary data suggesting that the use of a computer-generated rounding report can decrease some perceived barriers to providing optimal care in the NH. Although the rounding report did not improve rounding time efficiency, most NH physicians would prefer to use the computer-generated report rather than the current paper-based process. Improving the accuracy and harmonization of medication information with the electronic medication administration record and rounding reports, as well as improving facility network speeds might improve the effectiveness of this technology.

Taming of fluoroform: direct nucleophilic trifluoromethylation of Si, B, S, and C centers.

Fluoroform (CF(3)H), a large-volume by-product of the manufacture of Teflon, refrigerants, polyvinylidene fluoride (PVDF), fire-extinguishing agents, and foams, is a potent and stable greenhouse gas that has found little practical use despite the growing importance of trifluoromethyl (CF3) functionality in more structurally elaborate pharmaceuticals, agrochemicals, and materials. Direct nucleophilic trifluoromethylation using CF(3)H has been a challenge. Here, we report on a direct trifluoromethylation protocol using close to stoichiometric amounts of CF(3)H in common organic solvents such as tetrahydrofuran (THF), diethyl ether, and toluene. The methodology is widely applicable to a variety of silicon, boron, and sulfur-based electrophiles, as well as carbon-based electrophiles.

Couple-oriented prenatal HIV counseling for HIV primary prevention: an acceptability study.

BACKGROUND: A large proportion of the 2.5 million new adult HIV infections that occurred worldwide in 2007 were in stable couples. Feasible and acceptable strategies to improve HIV prevention in a conjugal context are scarce. In the preparatory phase of the ANRS 12127 Prenahtest multi-site HIV prevention trial, we assessed the acceptability of couple-oriented post-test HIV counseling (COC) and men's involvement within prenatal care services, among pregnant women, male partners and health care workers in Cameroon, Dominican Republic, Georgia and India. METHODS: Quantitative and qualitative research methods were used: direct observations of health services; in-depth interviews with women, men and health care workers; monitoring of the COC intervention and exit interviews with COC participants. RESULTS: In-depth interviews conducted with 92 key informants across the four sites indicated that men rarely participated in antenatal care (ANC) services, mainly because these are traditionally and programmatically a woman's domain. However men's involvement was reported to be acceptable and needed in order to improve ANC and HIV prevention services. COC was considered by the respondents to be a feasible and acceptable strategy to actively encourage men to participate in prenatal HIV counseling and testing and overall in reproductive health services. CONCLUSIONS: One of the keys to men's involvement within prenatal HIV counseling and testing is the better understanding of couple relationships, attitudes and communication patterns between men and women, in terms of HIV and sexual and reproductive health; this conjugal context should be taken into account in the provision of quality prenatal HIV counseling, which aims at integrated PMTCT and primary prevention of HIV.

WITHDRAWN: Corticosteroids for HELLP syndrome in pregnancy.

BACKGROUND: Hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome is a severe form of pre-eclampsia. Pre-eclampsia is a multi-system disease of pregnancy associated with an increase in blood pressure and increased perinatal and maternal morbidity and mortality. Eighty per cent of women with HELLP syndrome present before term. There are suggestions from observational studies that steroid treatment in HELLP syndrome may improve disordered maternal hematological and biochemical features and perhaps perinatal mortality and morbidity. OBJECTIVES: To summarise the evidence on the effects of corticosteroids on maternal and neonatal mortality and morbidity in women with HELLP syndrome. SEARCH STRATEGY: We searched the Cochrane Pregnancy and Childbirth Group trials register (October 2003). We scanned lists of references from review articles and primary studies. SELECTION CRITERIA: Randomised and quasi-randomised trials evaluating the effects of adjunctive corticosteroids in patients diagnosed with HELLP syndrome were sought. DATA COLLECTION AND ANALYSIS: The two authors independently applied inclusion criteria, assessed trial quality and extracted relevant data. MAIN RESULTS: Of the five studies reviewed (n = 170), three were conducted antepartum and two postpartum. Four of the studies randomised participants to standard therapy or dexamethasone. One study compared dexamethasone with betamethasone. Dexamethasone versus control There were no significant differences in the primary outcomes of maternal mortality and morbidity due to placental abruption, pulmonary oedema and liver hematoma or rupture. Of the secondary maternal outcomes, there was a tendency to a greater platelet count increase over 48 hours, statistically significantly less mean number of hospital stay days (weighted mean difference (WMD) -4.50, 95% confidence interval (CI) -7.13 to -1.87), mean interval (hours) to delivery (41 +/- 15) versus (15 +/- 4.5) (p = 0.0068) in favour of women allocated to dexamethasone.There were no significant differences in perinatal mortality or morbidity due to respiratory distress syndrome, need for ventilatory support, intracerebral hemorrhage, necrotizing enterocolitis and a five minute Apgar less than seven. The mean birthweight was significantly greater in the group allocated to dexamethasone (WMD 247.00, 95% CI 65.41 to 428.59).Dexamethasone versus betamethasone There were no significant differences in all the maternal and perinatal mortality and in primary morbidity outcomes.Women randomised to dexamethasone fared significantly better for: oliguria, mean arterial pressure, mean increase in platelet count, mean increase in urinary output and liver enzyme elevations. AUTHORS' CONCLUSIONS: There is insufficient evidence to determine whether adjunctive steroid use in HELLP syndrome decreases maternal and perinatal mortality, major maternal and perinatal morbidity.

Tissue detection of diphenylchlorin sensitizer (SIM01) by fluorescence and high-performance liquid chromatography.

Cancer is today a major problem of public health. Unfortunately, the current treatments remain still too often impotent or too heavy compared to the gross national product of many countries. The use of PDT in the treatment of the malignant tumours currently raises great hopes. This physicochemical method is based on the combined action of a nontoxic drug given systematically to the patient and of the visible light delivered locally to the tumour using optical fibres. The radiation will activate the significant substance preferentially fixed on cancerous cells and will cause the death of the tumoral cells while releasing from the toxic ridicalizing species which then will deteriorate vital cellular targets. Tissue distribution and elimination kinetics of the SIM01 were analysed in biological samples from mice tissues by spectrofluorometry and HPLC. Measurements were performed 4, 6, 12, 24 and 48h after an intraperitoneal injection for SIM01 doses of 2, 5 and 15mgkg(-1). Elimination seemed to concern essentially gallbladder, liver and stools, where maximum fluorescence reached, respectively, 20,000, 2800 and 15,000cps for 5mgkg(-1), 6h after injection. Among the tissues examined with HPLC, the highest SIM01 levels were found in stools, urine, liver, gallbladder and spleen. Liver, gallbladder, and stool homogenates from drug-treated animals contained an additional peak (16, 7min) detectable only after injection of at least 15mgkg(-1). Our HPLC determinations and in vivo fluorescence detection of SIM01 gave comparable kinetic profiles. These techniques should be considered as complementary rather than exclusive for kinetic profiles determination.

Synthesis, and in vitro and in vivo evaluation of a diphenylchlorin sensitizer for photodynamic therapy.

This paper reports the synthesis of a new diphenylchlorin photosensitizer, 2,3-dihydro-5,15-di(3,5-dihydroxyphenyl)porphyrin (SIM01). The photodynamic properties, cell uptake and localization of SIM01 were compared with those of structurally related meso-tetra(hydroxyphenyl)chlorin (m-THPC). In vitro studies were conducted on rat glioma cells (C6) and human adenocarcinoma (HT-29), and in vivo studies on human colon adenocarcinoma cells (HT-29) and human prostate adenocarcinoma cells (PC3). Both dyes showed an absorption maximum at around 650 nm, with a molar extinction coefficient of 13017 M(-1) cm(-1) for SIM01 and 22718 M(-1) cm(-1) for m-THPC. Their capacity to generate singlet oxygen was identical, but differences in partition coefficients indicated that SIM01 was slightly more hydrophilic. In vitro, SIM01 was slightly more phototoxic than m-THPC for C6 cells (4.8 vs. 6.8 microg ml(-1)). However, phototoxicities were nearly identical for HT29 cells (0.45 microg ml(-1) for 5 h incubation followed by 300 mW, 20 J cm(-2)). Pharmacokinetics in vivo in mice, as determined by fibre spectrofluorimetry, showed that the SIM01 fluorescence signal in the tumor was maximal between 6 and 12 h after injection, as compared to 72 h for m-THPC. With a 2 mg kg(-1) dye dose and laser irradiation at 300 J cm(-2) (650 nm, 300 mW), the optimal PDT response occurred when the interval between injection and irradiation was 6 h for SIM01 and 24 h for m-THPC. For SIM01 with 5 mg kg(-1) injection, the optimal PDT response occurred with a 12 h delay and with the same irradiation parameters as described above, in this case the tumor response showing 40% growth. Considering the tumor volume doubling time, the value was 6.5 days in the control group and increased to 13.5 days with SIM01. Thus, SIM01 may be a powerful sensitizer characterized by strong in vitro and in vivo phototoxicity and faster tissue uptake and elimination than m-THPC.

PDT effects of m-THPC and ALA, phototoxicity and apoptosis.

The purpose of this study was to estimate the efficacy of an endogenous sensitizer (delta-aminolevulinic acid (or ALA) induced protoporphyrin IX (or PpIX)) and an exogenous sensitizer (meta(tetrahydroxyphenyl)chlorin or m-THPC) on two different cell lines, rat colon adenocarcinoma PROb cells and murine melanoma B16A45 (B16) cells, in apoptosis production. After sensitizer incubation, cells were irradiated with an argon dye laser. LD(50) with m-THPC was 2.8 microg/ml and 4.7 microg/ml under irradiation of 25 J/cm(2) respectively for PROb and B16 cells. With ALA, LD(50) was 150 microg/ml and 175 microg/ml under 25 J/cm(2) respectively for PROb and B16 cells. Apoptosis induction by m-THPC or ALA-PDT was detected by DNA gel electrophoresis and quantified using an ELISA assay 24 h after PDT. The maximal apoptosis enrichment factor (MAEF) was reached for 6 microg/ml m-THPC at 10 J/cm(2) for PROb and B16 cells and for 50 microg/ml ALA at 25 J/cm(2) for PROb or B16 cells. Both m-THPC and PpIX are efficient photosensitizers and apoptosis inducers. However, MAEF is obtained by sensitizer or laser doses inducing very different phototoxic effects: MAEF was obtained after m-THPC-PDT with LD(78) for PROb cells and LD(30) for B16 cells and after ALA-PDT with LD(22) for PROb cells and LD(18) for B16 cells. However the overall m-THPC/PDT apoptotic induction (under the curve surface analysis) was not different whatever the cell line for 10 and 25 J/cm(2). On the contrary, ALA-PpIX/PDT apoptotic induction was twice for 25 J/cm(2) as compared to 50 J/cm(2) (p < 0.01) for both the PROb and B16 cells. These results indicate that the apoptosis rate in PDT cell killing varies considerably according to cell type and sensitizer.

Effects of BAPTA-AM, Forskolin, DSF and Z.VAD.fmk on PDT-induced apoptosis and m-THPC phototoxicity on B16 cells.

As many types of cells exposed to photodynamic therapy (PDT) appear to undergo apoptosis, various apoptosis inhibitors have already been used in studies of PDT-induced apoptosis. Although these inhibitors decrease apoptosis, their real effect on the phototoxic efficacy of photosensitisers is unclear. The good phototoxicity of m-THPC was confirmed on murine melanoma B16-A45 cells. Toxicity and phototoxicity studies were then carried out using four apoptosis inhibitors: BAPTA-AM, Forskolin, DSF, and Z.VAD.fmk. Although all inhibitors tested blocked PDT-induced apoptosis, none produced a significant modification of the phototoxic effect of m-THPC on B16 cells. It has been suggested that apoptosis and necrosis share common initiation pathways and that the final outcome is determined by the presence of an active caspase. This implies that apoptosis inhibition reorients cells to necrosis, i.e. those cells sufficiently damaged by PDT appear to be killed, regardless of the mechanism involved.

Enhancement of delta aminolevulinic acid-photodynamic therapy in vivo by decreasing tumor pH with glucose and amiloride.

OBJECTIVES/HYPOTHESIS: Delta aminolevulinic acid (ALA)-induced protoporphyrin IX (PpIX) is a fluorescent sensitizer that permits detection and treatment of squamous cell carcinoma of the oral cavity. An exogenously induced decrease in tissue pH was evaluated for its effect in enhancing cellular uptake of ALA and facilitating its transformation into PpIX. STUDY DESIGN: Mice grafted with HT29 colonic cancers had been given glucose and amiloride to modify the pH of tissues. Influence of pH changes has been evaluated on ALA-induced PPIX fluorescence by optic fiber spectrofluorimetry as well as on tumor growth. RESULTS: The pH in HT 29 tumor decreased from 7.1 to 6.67 (P < .05) after intraperitoneal injection of glucose and amiloride. The PpIX fluorescence ratios in tumor or muscle before, between, and 2 hours after glucose and amiloride injection were not higher than control ratios. Aminolevulinic acid-photodynamic therapy was more efficient on HT 29 tumor-bearing mice when the pH value was decreased with glucose and amiloride, showing a difference in the tumor growth index ratio from the 1st to 14th day of 22% between amiloride-glucose aminolevulinic acid-photodynamic therapy and aminolevulinic acid-photodynamic therapy alone (P < .05). CONCLUSIONS: Glucose and amiloride did not change PpIX fluorescence in HT 29 tumor after intraperitoneal injection of aminolevulinic acid but enhanced aminolevulinic acid-photodynamic therapy efficacy. This was probably a result of mechanisms other than an increase in aminolevulinic acid cellular penetration and PpIX production, such as susceptibility to free radical toxicity or alteration of cellular repair enzymes under acidotic conditions. If a decrease of pH induces a more efficient photodynamic therapy as suggested by our results, an easier way to obtain this decrease than glucose and amiloride would be necessary for clinical applications.

Use of alkaline Comet assay to assess DNA repair after m-THPC-PDT.

Photodynamic therapy (PDT) with Photofrin has already been authorized for certain applications in Japan, the USA and France, and powerful second-generation sensitizers such as meta-(tetrahydroxyphenyl) chlorin (m-THPC) are now being considered for approval. Although sensitizers are likely to localize within the cytoplasm or the plasma membrane, nuclear membrane can be damaged at an early stage of photodynamic reaction, resulting in DNA lesions. Thus, it is of critical importance to assess the safety of m-THPC-PDT, which would be used mainly against early well-differentiated cancers. In this context, m-THPC toxicity and phototoxicity were studied by a colorimetric MTT assay on C6 cells to determine the LD50 (2.5 microg/ml m-THPC for 10 J/cm2 irradiation and 1 microg/ml for 25 J/cm2 irradiation) and PDT doses inducing around 25% cell death. Single-cell electrophoresis (a Comet assay with Tail Moment calculation) was used to evaluate DNA damage and repair in murine glioblastoma C6 cells after LD25 or higher doses for assays of PDT. These results were correlated with m-THPC nuclear distribution by confocal microspectrofluorimetry. m-THPC failed to induce significant changes in the Tail Moment of C6 cells in the absence of light, whereas m-THPC-PDT induced DNA damage immediately after irradiation. The Tail Moment increase was not linear (curve slope being 43 for 0-1 microg/ml m-THPC and 117 for 1-3 microg/ml), but the mean value increased with the light dose (0, 10 or 25 J/cm2) and incubation time (every hour from 1 to 4 h) for an incubation with m-THPC 1 microg/ml. However, cultured murine glioblastoma cells were capable of significant DNA repair after 4 h, and no residual DNA damage was evident after 24-h post-treatment incubation at 37 degrees C. An increase in the light dose appeared to be less genotoxic than an increase in the m-THPC dose for similar toxicities. Our results indicate that m-THPC PDT appears to be a safe treatment since DNA repair seemed to not be impaired and DNA damage occurred only with lethal PDT doses. However, the Comet assay cannot give us the certainty that no mutation, photoadducts or oxidative damage have been developed so this point would be verified with another mutagenicity assay.

Cellular distribution and phototoxicity of benzoporphyrin derivative and Photofrin.

Photodynamic therapy (PDT) induces cell-membrane damage and alterations in cancer-cell adhesiveness, an important parameter in cancer metastasis. These alterations result from cell sensitivity to photosensitizers and the distribution of photosensitizers in cells. The efficacy of photosensitizers depends on their close proximity to targets and thus on their pharmacokinetics at the cellular level. We studied the cellular distribution of photosensitizers with a confocal microspectrofluorimeter by analysing the fluorescence emitted by benzoporphyrin derivative-monoacid ring A (BPD-MA) and Photofrin relative to their cell sensitivity. Two cancer cell lines of colonic origin, but with different metastatic properties, were used: PROb (progressive) and REGb (regressive). For BPD-MA (1.75 microg/ml), maximal fluorescence intensity (8,300 cts) was reached after 2 h for PROb and after 1 h (4,900 cts) for REGb. For Photofrin (10 microg/ml), maximal fluorescence intensity (467 cts) was reached after 5 h for PROb and after 3 h (404 cts) for REGb. Intracellular studies revealed stronger cytoplasmic than nuclear fluorescence for both BPD and Photofrin. Both of the sensitizers induced a dose-dependent phototoxicity; LD50 with BPD-MA was 93.3 ng/ml for PROb and 71.1 ng/ml for REGb, under an irradiation of 10 J/cm2. With Photofrin, LD50 was 1,270 ng/ml for PROb and 1,200 ng/ml for REGb under an irradiation of 25 J/cm2. The photosensitizer effect within PROb and REGb cancer cells was assessed by incorporation kinetics and toxicity-phototoxicity tests. The intracellular concentration of the photosensitive agent was one important factor in the effectiveness of PDT, but not the only one contributing to the photodynamic effect. In conclusion, this study showed that there was a clear difference between sensitizer uptake and phototoxicity, even in cancer cells of the same origin. This could induce cell-killing heterogeneity in clinics.

Heterogeneity of delta-aminolevulinic acid-induced protoporphyrin IX fluorescence in human glioma cells and leukemic lymphocytes.

Delta-aminolevulinic acid (ALA)-PDT efficacy is particularly dependent on the quality of protoporphyrin IX (PpIX)-induced synthesis. The purpose of this study was to determine the ability of cells from two human cancer types to synthesise PpIX after ALA administration. Biopsies of glioma cells have been obtained from patients with glioblastomas that have or have not been given ALA IV (ex vivo incubation). Peripheral blood lymphocytes, obtained from leukemic patients, have also been ALA-incubated in vitro. In glioma cells, fluorescence heterogeneity was extensive either in ALA infused patients or in ex vivo ALA incubated cells. Mean intensities after 3 h were 110 cts (range 0-340) and 1000 cts (range 0-3600). Similar results were found in leukemic lymphocytes where cell fluorescence varied from 0 to 480 cts with a percentage of fluorescent cells varying with time and from one patient to another. Furthermore, PpIX was not detectable in two patients with CLL. These observations suggest that a marked heterogeneity of ALA uptake and/or PpIX synthesis exists in a given human cancer cell population particularly after systemic administration. Improvements for ALA transformation into PpIX are strongly recommended to ensure the efficacy of ALA/PpIX-PDT.

In vitro fluorescence, toxicity and phototoxicity induced by delta-aminolevulinic acid (ALA) or ALA-esters.

Synthesis of delta-aminolevulinic acid (ALA) derivatives is a promising way to improve the therapeutic properties of ALA, particularly cell uptake or homogeneity of protoporphyrin IX (PpIX) synthesis. The fluorescence emission kinetics and phototoxic properties of ALA-n-pentyl ester (E1) and R,S-ALA-2-(hydroxymethyl) tetrahydrofuranyl ester (E2) were compared with those of ALA and assessed on C6 glioma cells. ALA (100 micrograms/mL), E1 and E2 (10 micrograms/mL) induced similar PpIX-fluorescence kinetics (maximum between 5 and 7 h incubation), fluorescence being limited to the cytoplasm. The 50% lethal dose occurred after 6 h with 45, 4 and 8 micrograms/mL of ALA, E1 and E2, respectively. ALA, E1 and E2 induced no dark toxicity when drugs were removed after 5 min of incubation. However, light (25 J/cm2) applied 6 h after 5 min incubation with 168 micrograms/mL of each compound induced 85% survival with ALA, 27% with E1 and 41% with E2. Increasing the incubation time with ALA, E1 and E2 before washing increased the phototoxicity, but E1 and E2 remained more efficient than ALA, regardless of incubation time. ALA-esters were more efficient than ALA in inducing phototoxicity after short incubation times, probably through an increase of the amount of PpIX synthesized by C6 cells.

Protoporphyrin IX fluorescence kinetics in C6 glioblastoma cells after delta-aminolevulinic acid incubation: effect of a protoporphyrinogen oxidase inhibitor.

PpIX synthesis after incubation with delta-aminolevulinic acid (ALA) is highly variable from one cell to another within a single cell population and in human glioblastomas in vivo. To improve PpIX synthesis, we attempted to modify the PpIX synthesis pathway in a C6 glioma cell model. To perform this experiment we used confocal microspectrofluorometry to analyse the effects of a highly purified form of sulfentrazone (FP846) on the kinetics of PpIX synthesis after ALA administration to living C6 cells. Our results show that PpIX fluorescence was maximal (seven-fold higher than basal values) 3 to 4 hrs. after the beginning of incubation with ALA. FP846 depressed this increase in fluorescence nearly to basal levels not only in C6 cells but also in HT29 and HepG2 cells. Fluorescence spectra shape were not affected by FP846, except for intensity. ALA/PpIX-induced photocytoxicity was perfectly correlated with fluorescence intensity recorded in cell cytoplasm. ALA alone (100 microg/ml) did not induce a significant decrease in cell survival, but irradiation of 25 J/cm2 leading to an overall cell death of 60%. FP846 added together with ALA suppressed ALA/PpIX-induced phototoxicity. The fact that the FP846-induced decrease in PpIX synthesis was not the same in animal and plant cells suggests that the porphyrin metabolic pathway differs due to the relative amounts of substrate or the effect of inhibitor and that another chemical would be needed alone or in combination with FP846 to improve PpIX synthesis.

Development of a "continuous-flow adhesion cell" for the assessment of hydrogel adhesion.

The purpose of this study was to develop an in vitro perfusion technique or "continuous-flow adhesion cell" model to predict the in vivo performances of different mucoadhesive drug delivery systems based on hydrogels. Two studies were performed, either using a rabbit small intestine or a polyethylene surface; the adhesion of four gels--two poly(acrylic acid)s (PAAs) (carbomer [CM] and polycarbophil [PC]), an ethyleneoxide-propyleneoxide block copolymer (Poloxamer 407 [PM]), and a polysaccharide (scleroglucane [SG])--were evaluated. In this respect, scleroglucane was used as a control. The adhesiveness of the different gels for both supports is in accordance with that described in the literature, that is, polycarbophil adhered more strongly than carbomer, which itself adhered more strongly than poloxamer. This study proved that the gels adhere more strongly to the polyethylene tube than to the rabbit small intestine, thus indicating that evidence for adhesion properties does not need any presence of mucus. Therefore, our in vitro model could be a good method, more precise and more simple than an ex vivo technique, to predict the bioadhesion of gelified devices.

Effects of photodynamic therapy on adhesion molecules and metastasis.

Photodynamic therapy (PDT) induces among numerous cell targets membrane damage and alteration in cancer cell adhesiveness, an important parameter in cancer metastasis. We have previously shown that hematoporphyrin derivative (HPD)-PDT decreases cancer cell adhesiveness to endothelial cells in vitro and that it reduces the metastatic potential of cells injected into rats. The present study analyzes the influence of PDT in vivo on the metastatic potential of cancers cells and in vitro on the expression of molecules involved in adhesion and in the metastatic process. Photofrin and benzoporphyrin derivative monoacid ring A (BPD) have been evaluated on two colon cancer cell lines obtained from the same cancer [progressive (PROb) and regressive (REGb)] with different metastatic properties. Studies of BPD and Photofrin toxicity and phototoxicity are performed by colorimetric MTT assay on PROb and REGb cells to determine the PDT doses inducing around 25% cell death. Flow cytometry is then used to determine adhesion-molecule expression at the cell surface. ICAM-I, MHC-I, CD44V6 and its lectins (àHt1.3, PNA, SNA and UEA) are studied using cells treated either with BPD (50 ng/ml, 457 nm light, 10 J/cm2) or Photofrin (0.5 microgram/ml, 514 nm light, 25 J/cm2). Changes of metastatic patterns of PROb cells have been assessed by the subcutaneous injection of non-lethally treated BPD or Photofrin cells and counting lung metastases. First, we confirm the metastatic potential reduction induced by PDT with respectively a 71 or 96% decrease of the mean number of metastases (as compared with controls) for PROb cells treated with 50 ng/ml BPD and 10 or 20 J/cm2 irradiation. Concerning Photofrin-PDT-treated cells, we find respectively a 90 or 97% decrease (as compared with controls) of the mean number of metastases for PROb cells treated with 0.5 microgram/ml Photofrin and 25 or 50 J/cm2 irradiation. Then, we observe that CD44V6, its lectins (àHt1.3, PNA, SNA) and MHC-I are significantly decreased (compared with the other molecules tested) in PROb and REGb cells after both BPD and Photofrin PDT treatment. These modifications in adhesion-molecule expression, particularly of CD44V6, can thus account only for part of the decrease in the metastatic potential of PDT-treated cancer cells. Changes in adhesion-molecule expression induced by PDT are only transient, implying that the rate of metastatic reduction is probably not linked simply to these changes.

In vitro and in vivo spectrofluorometry of a water-soluble meta-(tetrahydroxyphenyl)chlorin (m-THPC) derivative.

The pharmacokinetics of a water-soluble derivative obtained from meta-(tetrahydroxyphenyl)chlorin (m-THPC) was evaluated in in vitro and in vivo studies. Cytoplasm fluorescence was measured in two cell models (L1210 and HT29) using a flow cytometer and a confocal microspectrofluorometer. Cells were incubated with the compound at several doses (0-150 micrograms ml-1 for flow cytometry) and for several time periods (0-6 h for microspectrofluorometry). For in vivo studies, nude mice were grafted with human adenocarcinoma 15 days before intraperitoneal injection of polyethylene glycol-m-THPC (PEG-m-THPC). Fluorescence was recorded through an optical fibre spectrofluorometer using the 660 nm peak for detection. In in vitro studies, the fluorescence was found to be proportional to the dose. Maximum fluorescence was recorded in L1210 cells earlier and more intensely than in HT29 cells (3 h at 202 +/- 14 counts s-1 and 5 h at 43 +/- 2.15 counts s-1 respectively). Concerning in vivo studies, maximum tumour fluorescence was observed 24 h after injection (3568 +/- 178 counts s-1). Selectivity was expressed by the calculated tumour-to-skin and tumour-to-muscle ratios. The time taken to observe the maximum ratios (2.95 +/- 0.16 for tumour-to-skin and 6.61 +/- 0.3 for tumour-to-muscle) was almost the same as the time taken to observe the maximum fluorescence in the tumour. Studies are in progress to correlate these results with photodynamic effects.