Growth of bimolecular films of three-tailed amphiphiles.

The three-tailed amphiphile ferric stearate molecule, which forms a bimolecular layer on water surface with molecules in the lower and upper layers in different conformations, has been studied to understand transfer and growth of bimolecular films on the surface of hydrophilic silicon substrates. This bimolecular film forms a two-dimensional lattice on water with a slightly distorted hexagonal lattice where both the in-plane and out-of-plane domain sizes are small. The film also showed larger microscopic rigidity compared to its macroscopic mechanical response. This asymmetric bimolecular layer was found to be preserved when the film is transferred on the substrates at different values of surface pressures ranging from 1 mN/m to near-collapse (55 mN/m). Both the upper and lower layers become denser and interfaces between these layers become sharper with increase in deposition pressure but the growths have different natures. The lower layer of transferred film is dense from 1 mN/m and, except for a steplike increase between 20 and 30 mN/m, changes slowly in density. The density of the upper molecular layer grows continuously with surface pressure.

A prospective assessment of an 'a la carte' regimen of PEG-interferon alpha2b and ribavirin combination in patients with chronic hepatitis C using biochemical markers.

In therapy with standard interferon and ribavirin, five independent risk factors (RF) were predictive of relapse. The aim was to prospectively validate an a la carte regimen of pegylated interferon (PEG-IFN) alpha2b 1.5 microg/kg and ribavirin 11 mg/kg [PEG-IFN-ribavirin (PEG-IFN-R)], taking into account these five risk factors in order to determine whether to continue an additional 24 weeks of treatment in polymerase chain reaction (PCR) negative patients after 24 weeks. Treatment was stopped after 24 weeks in PCR positive patients. The same regimen was continued in PCR negative patients for an additional 24 weeks if patients had two or more RF. FibroTest and ActiTest assessed the impact of treatment on the histological features from baseline to end of follow-up. A total of 96 patients were included; 84 (87.5%) had at least two RF and 12 (12.5%) had no or one RF. A total of 70 patients were sustained virologic response (SVR; 73%), 19 were nonresponders (20%) and seven were relapsers (7%). The SVR in genotypes 2 or 3 was 85% (34/40) vs 64% in other genotypes (36/56; P = 0.02). There was a decrease (P = 0.003) in fibrosis as estimated by FibroTest, from 0.38 +/- 0.03 (mean +/- SE) at baseline to 0.33 +/- 0.03 at the 12-week follow-up, and a decrease in activity as estimated by ActiTest, from 0.49 +/- 0.02 to 0.19 +/- 0.03 (P < 0.0001). Improvement in activity was already significant at 12 weeks, even in virologic nonresponders. This study confirms that an a la carte regimen which takes into account not only genotype but also baseline viral load, fibrosis stage, gender and age, is efficient for the PEG-IFN-R combination. It achieves a 73% SVR and a significant decrease in fibrosis and activity as estimated by biochemical markers.

Dramatic enhancement of capillary wave fluctuations of a decorated water surface.

We have demonstrated by x-ray diffuse scattering that a bimolecular layer of a preformed three-tailed amphiphile, ferric stearate, drastically enhances capillary wave fluctuations on water surface due to a reduction in surface tension to 1 mN/m . The bimolecular layer is composed of molecules in symmetric configuration, on top of molecules in asymmetric configuration with ferric ions in contact with water. Unlike the usual Langmuir monolayers, this layer of molecules does not rupture under compression, but becomes thicker. This behavior mimics folding of a membrane on a liquid surface and is closely related to the cohesive interaction brought by the ferric ions. The low effective tension of this artificial membrane depends on the available area and reduces as the microscopic excess area increases.

Efficacy of selamectin administered topically in the treatment of feline otoacariosis.

The efficacy of a novel avermectin, selamectin (Stronghold, Pfizer), was evaluated against naturally acquired aural infestations of Otodectes cynotis. Selamectin was administered topically in a single spot to the skin of each animal's back at the base of the neck in front of the scapulae at a minimum dosage of 6mgkg(-1). Thirty cats of a cattery of 120 animals with an endemic infestation were treated on days 0 and 30. Including untreated control cats was therefore not possible because of animal welfare consideration. Clinical examination and visualization of mites by otoscopic examination of the external ear canal and microscopic examination of aural debris/exudate were performed twice weekly from days 0 to 30. On day 0 (and day 28 if the ears canals were erythematous), a swab was taken from each canal and sent to bacteriology and mycology units for detection and identification of bacteria (Staphylococcus mainly) and yeast (Malassezia pachydermatis only). Selamectin was safe, parasites were killed before day 3 and eliminated before day 17. Selamectin was 100% effective against natural aural infestations, even if erythema (26-33% of cats) and scratch reflex (23-40% of cats) persisted for 2 weeks after the cats tested negative for mites.

Novel ferrocenic artemisinin derivatives: synthesis, in vitro antimalarial activity and affinity of binding with ferroprotoporphyrin IX.

Following our search for novel compounds with high antimalarial activity, a series of artemisinin (QHS) derivatives containing a ferrocenic nucleus was prepared and tested in vitro against Plasmodium falciparum strains. Two new metallocenic derivatives (1 and 3) were found as potent as QHS. All compounds showed a capacity to bind with ferroprotoporphyrin IX. A decrease in the Soret band absorbance of ferroprotoporphyrin IX, resulting from the addition of different drugs concentrations, was shown. The association stoichiometry of compounds to ferroprotoporphyrin IX appears to be 1:2 at equilibrium, with an intermediate 1:1 complexation. These results appear to strengthen the role of adducts between artemisinin derivatives and heme in generation of artemisinin radicals. Such interaction of artemisinin ferrocenyl derivatives with ferroprotoporphyrin IX and its biological significance could form a basis in future drug development.

[Pruritus and the hepatitis C virus. The MULTIVIRC Unit]. / Prurit et virus de l'hépatite C. Le Groupe Multivirc.

OBJECTIVE: Determine the characteristics of pruritus in a selected population of patients with positive hepatitis C virus serology. PATIENTS AND METHODS: In a retrospective study, we re-examined outpatient reports for patients who consulted for pruritus from January 1993 to April 1996 and were followed in a hepatology unit for hepatitis C infection. The following data were collected: age, sex, risk factors, HIV and HBV serologies, duration of pruritus and diagnosis, ALAT, gamma GT and total bilirubin, METAVIR score, HCV RNA PCR, search for cryoglobulins, antiviral and dermatology treatments. There were 1,060 patients followed in the hepatology unit during this period, including 327 with cirrhosis. RESULTS: Twenty-seven patients were retained for study, 16 men and 11 women, mean age 53 years. None of the patients had HIV infection, 7 had a past history of hepatitis B infection (positive for anti-Hbc antibodies). Median duration of pruritus prior to consultation was 3 months (95 p. 100 CI: 3 months-2 years). Pruritus was associated with non-specific lesion in 19 cases (70 p. 100: 95 p. 100 CI: 51-85 p. 100). There were excoriated eczema-like lesions in 11 cases (41 p. 100: 95 p. 100 CI: 15-49 p. 100). Other causes were urticaria in 5 cases (18 p. 100: 95 p. 100 CI: 7-36 p. 100), including 1 case of urticarial vasculitis with cryoglobulinemia, 2 cases with atopic dermatitis, and 1 case of primary biliary cirrhosis. In four cases, lichen planus was associated. Skin biopsies were obtained in 10 patients and showed eczema-like alterations in 9 and urticarial vasculitis in 1. Mean ALAT and gamma GT levels were 2.6 N (95 p. 100 CI: 1.9 N-3.3 N) and 2.2 N (95 p. 100 CI: 1.4 N-3 N) respectively, including 11 cases without cholestasis (normal gamma Gt). PCR was positive in 13 cases out of 15. Cryoglobulinemia was found in 10 cases out of 24. At consultation, 3 patients were given ursolvan, 7 interferon, 1 ursolvan with ribavirin, and 3 an interferon-ribavirin combination. Dermatology treatment associated antihistamine agents, emollients, and corticosteroids. This population of hepatology patients referred for pruritus comprised 2.5 p. 100 of all patients (95 p. 100 CI: 1.7-3.6 p. 100). Among them, 1.8 p. 100 (95 p. 100 CI: 1.1-2.7 p. 100) had eczema-like lesions associated with cutaneous xerosis. DISCUSSION: Pruritus in our patient population was generally associated with non-specific excoriations, prurigo or xerosis in 19 cases (70 p. 100). As only ambulatory patients were retained for analysis, this is not a comprehensive population and the percentage of non-specific pruritus, evaluated at 1.8 p. 100, is probably an underestimation. Cholestasis cannot explain alone these manifestations since 11 patients had normal gamma GT levels. Several etiologies could be involved: a direct effect of HCV, interferon. A prospective study should allow an estimation of frequency, risk factors and possible impact on quality of life.

Effects of cirrhosis, interferon and azathioprine on adverse events in patients with chronic hepatitis C treated with ribavirin.

To determine adverse events of ribavirin in the treatment of chronic hepatitis C, 41 patients (18 with cirrhosis), treated with ribavirin at an initial dose of 600-1200mg day(-1), were analysed retrospectively (six patients were treated twice because adverse effects during the first treatment necessitated cessation of ribavirin). Indications for ribavirin included a contraindication (n = 15) an intolerance (n = 11) or a non-response (n = 15) to interferon (IFN). Ribavirin was combined with IFN 3 million units (MU) three times weekly for 15 patients and with azathioprine for six patients (five of whom were transplant patients). No cirrhotics and only one patient treated with ribavirin + IFN received azathioprine. The mean duration of treatment was 5 months (range 1-18 months). Sixteen of 47 treatments (34%) with ribavirin were stopped: four because of vomiting (8.5%), two for psychiatric disorder, one for dry cough, one for an unrelated cause, and eight (at 1-2 months) because of a fall in the level of haemoglobin (Hb) of 4.6 g dl(-1) (range 2.7-5.9 g dl[-1]); however, according to the rules of international protocol, we would have expected only four treatments (two in patients receiving azathioprine) with Hb < 8.5 g dl(-1) to be stopped. The decrease in Hb level occurred more slowly in patients treated with IFN plus ribavirin than in patients treated with ribavirin alone and was of lower clinical significance in patients with cirrhosis than in patients without cirrhosis. After exclusion of patients receiving azathioprine, there was no significant difference in the fall of Hb level between cirrhotic and non-cirrhotic patients and between patients treated with IFN plus ribavirin and patients treated with ribavirin alone. Interestingly, the platelet count of patients treated with IFN plus ribavirin fell less than in patients treated with IFN alone. The most important and expected adverse event associated with ribavirin was haemolysis. Anaemia < 8.5 g dl(-1), requiring cessation of ribavirin therapy, was present in 9% of patients and was worsened by azathioprine. Abdominal discomfort and dry cough were other, potentially important, clinical adverse events found in our study.

Extension of the 4-week safety study for detecting immune system impairment appears not necessary: example of cyclosporin A in rats.

A 4-week study was conducted to shed light on the question of whether compounds impairing immune homeostasis may escape the standard safety testing. Wistar rats were orally treated with cyclosporin A at dosages of 0 (control: olive oil), 1, 5 or 25 mg/kg/day. Ten rats/sex/group (study segment 1) were not immunized while six other rats/sex/group (study segment 2) were immunized 4 days before killing to perform a plaque forming cell (PFC) assay. All rats were subjected to routine safety evaluations (OECD guideline 407) and determination of IgM and IgG serum levels. Other immune parameters were evaluated using cells from spleen and mesenteric lymph nodes (segment 1). Effects on safety parameters were similar for immunized and non-immunized rats. A slight decrease of body weight gain (males, 25 mg/kg) accompanied slight clinical chemical and histomorphologic evidence of renal tubulotoxicity. Changes in safety parameters indicative of immune system alterations were: increased thymic corticomedullary ratio (> or = 5 mg/kg) and 25 mg/kg) minimal lymphopenia, low thymus weight, thymic cortical lymphocytolysis and low lymphoid cellularity of spleen and lymph nodes. They were associated with (males at > or = 1 mg/kg) dose-related decreases of T-cell receptor+ and CD4+ cells and increases of CD8+ cells, and decreased PFC (> or = 5 mg/kg) and lymphoproliferative responses to mitogens and alloantigens (25 mg/kg). There were no changes in natural killer activity. The conventional assay identified the drug as a potential immunomodulator. Specific immune assays (phenotyping, PFC) improved the threshold of detection. These results did not support the incorporation of specific immune tests in the standard 4-week study protocol.

Immunotoxicological investigation using pharmaceutical drugs. In vitro evaluation of immune effects using rodent or human immune cells.

In order to evaluate the relevance of in vitro methods for immunotoxicity assessment, the effects of pharmaceutical drugs on lymphoproliferative and cytotoxic functions of mouse splenocytes and human peripheral blood mononuclear cells (hPBMC) were studied. A comparison of sensitivity of immune cells from different origins to an in vitro exposure to different xenobiotics was performed using non-immunosuppressive (cimetidine and furosemide) and immunosuppressive (azathioprine (AZA), cyclosporine A (CSA), and dexamethasone (DEX)) drugs. For CSA, sensitivity of both rat and mouse splenocytes following in vitro exposure was compared to the one of hPBMC. Immune function tests included lymphoproliferative response to mitogenic lectins (concanavalin A (Con A) and phytohemagglutinin (PHA-P)) or to allogeneic cells (mixed leukocyte response (MLR)) and cytotoxicity assays (cytotoxic-T lymphocyte (CTL) and natural killer (NK) cell-mediated cytolysis). Additionally, to evaluate how well in vitro assays represent the in vivo situation, a comparison of the effect of cyclosporine A on the same immune function tests following in vivo or in vitro exposure was performed. The data obtained show numerous similarities in the effects observed following in vitro exposure of rodent or human cells to the drugs and a very similar sensitivity of rat and mouse cells to CSA in vitro. Discrepancies between human and rodent cells such as lymphoproliferative response to PHA-P following exposure to DEX or sensitivity of CTL-mediated cytolysis to CSA do exist. In vitro assays were very representative of the in vivo situation, both in the rat and in the mouse, following CSA exposure, except for NK cell activity in the rat. These data show the usefulness of in vitro systems for immunotoxicity assessment. They allow direct comparison of rodent and human systems, and could be representative, for drugs altering specifically the immune system like CSA does, of the in vivo situation.

Immune parameters are affected differently after cyclosporine A exposure in Fischer 344 rats and B6C3F1 mice: implications for immunotoxicology.

Knowledge of interspecies differences, commonly evaluated in other disciplines such as carcinogenesis, is a prerequisite for an appropriate assessment of immunotoxicological risks. The purpose of this study was to assess interspecies differences following exposure of Fischer 344 rats and B6C3F1 mice to cyclosporine A. These animals were exposed daily to cyclosporine A by oral gavage at 0, 5, 10, 25 mg/kg/day for 14 consecutive days. The results showed that splenocytes lymphoproliferation in response to concanavalin A or phytohemagglutinin, and IgM antibody-forming cells to sheep red blood cells, were affected in both species. Cytotoxic T-lymphocyte activity and mixed lymphocyte response were significantly inhibited in the rat following cyclosporine A exposure while they remained unaffected in the mouse. In contrast, natural killer cell activity was significantly depressed in the B6C3F1 mouse but not in the Fischer 344 rat. The discrepancies between the two species in cytotoxic T-lymphocyte activity and mixed lymphocyte response assays could partially be explained by the constantly higher blood level of cyclosporine A in the rat than in the mouse. When these tests were performed using rat and mouse splenocytes exposed to cyclosporin A in vitro (10(-9) to 10(-5) M) it was possible to correlate in vivo and in vitro data for concanavalin A- and phytohemagglutinin-induced lymphoproliferation and for cytotoxic T-lymphocyte activity but not for mixed lymphocyte response. Natural killer activity was 10-fold more sensitive in mice than in rats in vitro but these results did not clarify the in vivo difference. In conclusion, these results emphasize that the utilization of more than one species should be considered when assessing immunotoxicity.

Immunotoxicological investigation using pharmaceutical drugs: in vivo evaluation of immune effects.

Traditional methods for toxicological assessment have indicated that the immune system is a frequent target of toxic insult following subchronic or chronic exposure to xenobiotics. However, most of the xenobiotics evaluated in standardized protocols were environmental chemicals and correlation with available clinical data was not possible. The purpose of this work was to evaluate the potential immunosuppressive effects of pharmaceutical drugs using a standardized protocol developed for immunotoxicological assessment. Two groups of pharmaceutical drugs were utilized: (a) drugs without known immunosuppressive effect linked to their utilization in human therapy (cimetidine, furosemide, indomethacin, amoxicillin, and procainamide) and (b) immunosuppressive drugs (azathioprine, cyclosporine A, and dexamethasone). Ex vivo tests using B6C3F1 mice were performed after a 28-day repeat dose regimen and assessed: (a) immunopathology, (b) cell-mediated immunity, (c) humoral immunity, and (d) nonspecific immunity. Host resistance to Listeria monocytogenes was also assessed following exposure to immunosuppressive drugs. The results showed that (a) immunopathology and immune function assays were necessary to detect all immunotoxicants and (b) the effects observed with nonimmunotoxic drugs were sometimes statistically significant but the biological significance of these effects is unlikely.

Effects of a primary immune response to T-cell dependent antigen on serotonin metabolism in frontal cortex: in vivo microdialysis study in freely moving Fischer 344 rat.

Antigenic challenge is known to influence brain catecholamine turnover, e.g. hypothalamic norepinephrine activity, but little is known about effects on the activity of serotoninergic neurons, i.e. the release of the neurotransmitter at nerve terminals. In the present study, we first investigated the changes of central serotonin (5-HT) metabolism in Fischer 344 male rats at 2, 3, 4 and 5 days following i.v. immunization with sheep red blood cell (SRBC). Major decreases in 5-HT levels were evident in the hypothalamus (Hy) and cortex (Cx) at a time which corresponded to the late phase of the production of specific antibodies to SRBC measured with a plaque-forming cell assay (PFC). A pretreatment with an immunosuppressive drug, cyclosporin A (CsA; 12.5 mg/kg by gavage for 7 days) prevented the decreases in cortical 5-HT levels. Concomitantly, a 2-fold increase in the basal 5-HT release at frontocortical nerve terminals was observed by using in vivo microdialysis in awake rats on Day 3 following SRBC inoculation. This effect was totally suppressed by CsA. Our data suggest that the decrease in brain 5-HT levels that occurs after antigen administration may reflect a specific short-lasting CsA-dependent-release of 5-HT at frontocortical nerve terminals at a time (Day 3 or 4) when the splenic immune response is maximal.