Current knowledge in Polypodium leucotomos effect on skin protection.

This article provides an overview of pharmacology, toxicity, pharmacokinetics and clinical data of Polypodium leucotomos L. (PL). PL aerial part has proven to exert antioxidant, photoprotective and immunomodulatory activities; its mechanism of action is complex and includes several activities: (1) PL diminishes the production of reactive oxygen and nitrogen species (ROS, RNS); (2) PL inhibits the photoisomerization of trans-urocanic acid (t-UCA); (3) PL inhibits apoptosis induced by ultraviolet radiation; (4) PL prevents damage to genetic material and (5) PL enhances DNA repair. PL is not mutagenic and does not induce acute or chronic toxicity. Its biological effects have been proved in cell cultures, animal models, murine models and in human beings. Photoprotective activity has been assessed in healthy volunteers as well as in patients suffering from several cutaneous diseases such as vitiligo, psoriasis, idiopathic photodermatosis or melasma. PL results to be an efficient treatment especially for sensitive cutaneous phototypes and adds extra protection when ultraviolet radiation (UVR) exposure cannot be avoided, such as wide or narrow band UVB phototherapy or treatment with psoralens plus UVA exposure radiation.

Topical exposure to exogenous ultraviolet-irradiated urocanic acid enhances Mycobacterium ulcerans infection in a Crl:IAF(HA)-hrBR hairless guinea-pig model of Buruli ulcer disease.

BACKGROUND: Ultraviolet radiation (UVR) pre-exposure enhances Mycobacterium ulcerans infection in the Crl:IAF(HA)-hrBR hairless guinea-pig, possibly via a photoimmunosuppressive mechanism. The trans-cis photoisomerization of epidermal urocanic acid is an important initiator of the web of events leading to photoimmunosuppression. Thus, the hypothesis tested in this paper was that topical pre-exposure to UVR-irradiated urocanic acid mixture containing cis-urocanic acid (UVR-UCA) enhances the ulcerative form of M. ulcerans infection in the Crl:IAF(HA)-hrBR hairless guinea-pig model of human Buruli ulcer disease. METHODS: Groups of six animals were subjected to daily topical treatment with either 0 (vehicle only), 0.1, 0.5 or 1 mg of trans (tUCA) or UVR-UCA (contained a cis : trans urocanic acid isomer ratio of 1 : 9) for three consecutive days. A sham treatment group was also included in the experiment. Three days following their final treatment, the guinea-pigs were intradermally infected in the right dorsal flank with 1.5 x 107 CFU of M. ulcerans in 0.1 ml of phosphate-buffered saline (PBS) and sham infected with 0.1 ml of PBS in the left dorsal flank. The resultant skin lesions were then measured over the next 21 days. At day 21 postinfection, the animals were tested for delayed-type hypersensitivity (DTH) reactivity to M. ulcerans cell fragment antigens (MCF). RESULTS: Distinct, well-demarcated, dermally situated skin nodules were present at infected, but not sham-infected, skin sites by day 3 postinfection, and the lesions progressed to frank ulcers by day 5. Between days 5 and 21, the mean lesion diameters of the UVR-UCA-treated animals were significantly (P<0.001) greater than those of the sham, vehicle only or tUCA-treated groups. UVR-UCA-treated guinea-pigs also had significantly (P<0.001) suppressed DTH responses to MCF compared with the other treatment groups. There were no significant (P>0.4) differences between the lesion sizes and DTH responses of the tUCA, vehicle only or sham treatment groups. These results demonstrate that topical exposure to UVR-UCA promotes M. ulcerans infection and suppresses DTH responses to M. uclerans antigens in infected animals. These results lend credence to the hypothesis that UVR-mediated enhancement of Buruli ulcer disease in the Crl:IAF(HA)-hrBR hairless guinea-pig model occurs via modulation of cis-urocanic acid-susceptible immune pathways.

Urocanic acid isomers are good hydroxyl radical scavengers: a comparative study with structural analogues and with uric acid.

UV-exposure of the epidermis leads to the isomerisation of trans-UCA into cis-UCA as well as to the generation of hydroxyl radicals. This study shows by means of the deoxyribose degradation test that UCA isomers are more powerful hydroxyl radical scavengers than the other 4-(5-)substituted imidazole derivatives, such as histidine, though less powerful than uric acid. UCA, present in relatively high concentrations in the epidermis, may well be a major natural hydroxyl radical scavenger.

[Imidazole derivatives, platelet aggregation inhibitors]. / Dérivés imidazoliques inhibiteurs d'agrégation plaquettaire.

Study of the platelet aggregation inhibition has shown the efficiency of compounds with imidazole ring alone or fused. All the compounds resulting of the molecular design starting from these structures has an in vitro activity. We have been able to discuss the correlation existing between activities, toxicities and structures with a particular emphasis to the lipophilicity.

Simultaneous high-performance liquid chromatographic determination of visoltricin, acuminatopyrone and chlamydosporols in Fusarium cultures on maize.

Visoltricin (VIS), acuminatopyrone (ACP), clamydosporol (CL), isochlamydosporol (ICL) and chlamydospordiol (DIOL), recently characterized Fusarium metabolites, were separated on a polymeric RP-18 column eluted with acetonitrile-0.01% ammonia solution (35:65) at 1 ml/min and detected with a diode-array UV detector. The presence of ammonia in the mobile phase improved the shape of the CL and VIS peaks. The use of a polymeric column was required owing to the basic pH of the mobile phase. Maize cultures of several strains of F.tricinctum and F. chlamydosporum were analysed with this procedure after extraction with aqueous methanol, partitioning with methylene chloride and clean-up with a C18 minicolumn. VIS was produced only by F. tricinctum, whereas ACP and chlamydosporols were produced by both Fusarium species.

Visoltricin, a novel biologically active compound produced by Fusarium tricinctum.

The major compound responsible for toxicity to Artemia salina of some Fusarium tricinctum strains has been isolated, and its structure has been elucidated by spectroscopical methods, i.e. UV, IR, MS, 1H-NMR and 13C-NMR. The novel compound, trivially named visoltricin, is the first imidazole derivative produced by Fusarium spp., and its structure has been established as the methyl ester of 3-[1-methyl-4-(3-methyl-2-butenyl)-imidazol-5yl]-2-propenoic acid (molecular formula C13H18N2O2; MW = 234.297). Visoltricin was toxic to A. salina larvae (LD50 = 8.5 x 10(-7) M), and inhibited the growth of six human tumour cell lines (out of 60 lines tested) at concentrations lower than 10(-5) M. Tested on rabbit eye it showed an interesting miotic activity similar to that of pilocarpine, a miotic agent largely used in the therapy of glaucoma. This biological activity could be explained in part by the anticholinesterase properties shown by visoltricin towards both human serum and pure enzymes (EC 3.1.1.7 and EC 3.1.1.8). Kinetics studies showed for visoltricin a mixed-type and reversible inhibition of the EC 3.1.1.7 enzyme with the competitive inhibition constant (Ki) = 1.9 x 10(-4) M.

Characterization of a monoclonal antibody to cis-urocanic acid: detection of cis-urocanic acid in the serum of irradiated mice by immunoassay.

Cis-urocanic acid (cis-UCA), which is formed from the naturally occurring trans-isomer on ultraviolet (UV) irradiation, has been suggested as a photoreceptor for and mediator of the suppressive effects of UV irradiation on systemic immune responses. Trans-UCA is located predominantly in the stratum corneum, and the extent of isomerization to cis-UCA may be analysed by high-performance liquid chromatography (HPLC) of skin extracts. Such an analysis is not suitable for other tissues. In this study a murine monoclonal antibody to cis-UCA was prepared and tested by ELISA using UCA isomers conjugated to protein as antigens. The interaction of the antibody with structural analogues of UCA was assessed by competitive inhibition ELISA which indicated that the antibody had a high specificity for cis-UCA. Screening of sera at various times after UVB irradiation of mice by competitive inhibition ELISA using the monoclonal antibody showed that cis-UCA was present, probably in an unbound form, for at least 2 days after the exposure. Thus, cis-UCA produced in the epidermis following UVB irradiation reaches the serum a few hours later. The implications of this finding for the generation of suppressed immune responses are discussed.

The role of histamine-like receptors in immunosuppression of delayed hypersensitivity induced by cis-urocanic acid.

The cis-isomer of urocanic acid (UCA) has been shown previously to mimic the effect of ultraviolet B (UVB) irradiation in suppressing delayed hypersensitivity (DH) responses to virus in a murine model of herpes simplex virus (HSV) infection. Cimetidine, an H2 receptor antagonist, and terfenadine, an H1 receptor antagonist, abrogated the suppression of DH to HSV induced by cis-UCA, leading to the suggestion that histamine-like receptors may be involved in the mechanism of action of cis-UCA on immune responses. In the present study, cis and trans-isomers of 4 UCA analogues (1- and 2-imidazoyl-acrylic acid), and (2- and 3-pyridyl-acrylic acid) were tested for their ability to suppress DH to HSV in infected mice, and only cis-2-pyridyl-acrylic acid was effective. Second, an H2 and H3 agonist were similarly tested: the former was suppressive and the latter had no effect. Third, an H3 receptor antagonist, thioperamide, did not seem to abrogate the suppression of DH induced by cis-UCA. These results substantiate a role for H1 and H2-like receptors, but probably not H3 receptors, in cis-UCA induced immunosuppression.

The effect of ultraviolet B irradiation, cis-urocanic acid and tumour necrosis factor-alpha on delayed hypersensitivity to herpes simplex virus.

Previously it has been shown that ultraviolet-B (UVB) irradiation or cis-urocanic acid (cis-UCA) treatment of mice before infection with herpes simplex virus (HSV) suppressed the delayed hypersensitivity (DH) response when the mice were subsequently challenged with inactivated virus. In the present study, the time course of the elicitation phase of the DH was examined, and it was found to be biphasic with one peak 1 h following challenge and a second at 24 h. Both UVB irradiation and cis-UCA treatment of mice before infection with HSV significantly suppressed the DH at 1 h as well as at 24 h. The role of tumour necrosis factor-alpha (TNF-alpha) in the suppression was tested by injecting mice intraperitoneally with neutralizing TNF-alpha antibodies 2 h before UVB irradiation or cis-UCA treatment followed by infection with HSV. This had no effect on the suppression of DH to HSV induced by cis-UCA but significantly reduced that generated by UVB exposure. Thus, the mechanism of suppression of DH induced by UVB irradiation or cis-UCA may be different.

Urocanic acid analogues and the suppression of the delayed type hypersensitivity response to Herpes simplex virus.

Ultraviolet irradiated urocanic acid (4-imidazoleacrylic acid) containing a mixture of cis- and trans-isomers has been shown previously to induce suppression of the delayed type hypersensitivity (DTH) response to Herpes simplex virus type 1 (HSV-1) in a murine model of infection. The cis-isomer of urocanic acid was prepared and the cis- and trans-isomers of 2-methylurocanic acid. 2-pyrroleacrylic acid, 2-furanacrylic acid, 2-thiopheneacrylic acid, 3-thiopheneacrylic acid as well as dihydrourocanic acid and histamine. Each was applied at concentrations of 1 and 50 micrograms per mouse to the shaved dorsal skin and the mice were infected subcutaneously with HSV 5 h later. After 8-10 days the DTH response to the virus was measured by an ear swelling test. It was found that cis-urocanic acid was effective in suppressing the DTH response at levels of 1 microgram per mouse or less. The cis- and trans-isomers of 2-furanacrylic acid, 2-pyrroleacrylic acid and 2-thiopheneacrylic acid were also effective, with the cis- form generally being more active than trans, and 2-pyrroleacrylic acid being particularly potent. Cis- and trans-3-thiopheneacrylic acid, on the other hand, were only marginally immunosuppressive while neither isomer of 2-methylurocanic acid had any suppressive ability. Dihydrourocanic acid and histamine were also shown to suppress the DTH response. Thus the structural features necessary for urocanic acid and its analogues to act as mediators of UV-induced immunosuppression could be deduced and implications for their mechanism of action discussed.

Proposal for the mechanism of action of urocanase. Inference from the inhibition by 2-methylurocanate.

Incubation of urocanase with 2-methylurocanate leads, after an initial normal reaction, to a time dependent inactivation of the enzyme. It is suggested that a tautomeric form of the product, 2-methyl-imidazolone propionate, is the actual inhibitor. On the basis of these and of published experimental data a novel mechanism is proposed for the urocanase reaction. The crucial and initial step is the electrophilic addition of enzyme-bound NAD to the 2-position of the imidazole nucleus of urocanate.

Ultraviolet-irradiated urocanic acid suppresses delayed-type hypersensitivity to herpes simplex virus in mice.

Ultraviolet radiation is known to induce a transient defect in epidermal antigen presentation which leads to the generation of antigen-specific suppression of the delayed-type hypersensitivity (DTH) response. The putative receptor in skin for the primary event in UV-suppression is urocanic acid (UCA) which may then interact locally, or systemically, with antigen presenting cells or initiate a cascade of events resulting in suppression. We present the first direct evidence that UCA, when irradiated with a dose (96 mJ/cm2) of UVB radiation known to suppress the DTH response to herpes simplex virus, type 1 (HSV-1) in mice, can induce suppression following epidermal application or s.c. injection of the irradiated substance. This suppression is transferable with nylon wool-passed spleen cells.

Convenient synthesis of alkyl esters of urocanic acid.

Ethyl, n-dodecyl, and n-hexadecyl esters of urocanic acid (4-imidazoleacrylic acid) were prepared from 4-imidazolecarboxaldehyde in satisfactory yields via the Wittig reaction.