Pietro Santoianni

In vitro effect of 5-aminolaevulinic acid plus visible light on Candida albicans

Photodynamic therapy, currently used as an alternative technique for the treatment of superficial non-melanoma skin cancers, has been employed in vitro to kill different species of microorganisms. Here the development of Candida albicans colonies has been measured after application of 5-aminolaevulinic acid (ALA) plus visible light (VIS) irradiation. C. albicans suspensions (10 colony forming units microl(-1)) have been prepared. For the experiment 30 microl of suspension have been incubated in the dark for 3 h, with increasing concentrations of ALA (125, 250, 300, 350, 400, 450, 500, 550, 600, 750, 1000 mg ml(-1)) and then irradiated with a fixed dose (40 J cm(-2)) of VIS. Immediately after the irradiative session, the C. albicans suspensions were disseminated on dishes containing a Sabouraud agar + CAF medium and cultured in the dark at 27 degree C; after 48 h colony development has been measured. In the same way four controls have been prepared: (i)C. albicans suspensions not treated with ALA-PDT; (ii)C. albicans suspensions incubated with increasing ALA concentrations without VIS; (iii)C. albicans suspensions irradiated with 40 J cm(-2) of VIS without ALA; (iv)C. albicans suspensions irradiated immediately after the addition of increasing concentrations of ALA without the 3 h incubation. Colonies treated with ALA-PDT have been studied with electron microscopy (E.M.). It was found that: (i) none of the controls prepared modified the development of C. albicans colonies; (ii) ALA plus VIS inhibited C. albicans growth in a concentration-dependent way: up to 250 mg ml of ALA concentrations did not affect C. albicans cells, 300 mg ml(-1) induced a 50% reduction in the number of colonies, a complete inhibition started from concentrations of 600 mg ml(-1); (iii) after ALA-PDT E.M. showed modifications of the cell membranes. From the results it is concluded ALA plus VIS light is able to kill C. albicans colonies, at least in vitro. Although other pharmacological approaches are available, further studies could show that PDT is a potential treatment for candidosis.

Drug-induced cutaneous porphyria

I n porphyrias, the control mechanisms of heme synthesis are altered, and more porphyrins and precursors are produced than are converted into heme (Fig 1). This can occur either because of a partial block in heme synthesis as a result of enzymatic deficiency or because of enhanced utilization of heme, which in turn depletes the amount of regulatory heme. The porphyrin metabolic pathway is regulated by the feedback mechanism of the Enal product, heme, and its effects on S-aminolevulinic acid (ALA) synthetase. The porphyrias can traditionally be classified into two general groups, erythropoietic and hepatic, based on the porphyrin or porphyrin precursor content of bone marrow and liver. The hepatic porphyrias include acute intermittent porphyria (AH’), variegate porphyria (VP), and porphyria cutanea tarda (PCT). AIP and VP are clinically characterized by intermittent attacks of abdominal and neuropsychiatric symptoms, which frequently are precipitated by therapeutic doses of commonly used drugs. During acute attacks, these two forms of the disease are associated with excessive urinary excretion of the porphyrin precursors ALA and porphobilinogen (PBG), but differ in the pattern of porphyrins excreted in urine and feces. ALA and PBG or their metabolic products are neurotoxic and responsible for the neurologic symptoms and signs of the acute porphyrias. With possible rare exceptions, AIP is the one form of porphyria in which photosensitivity does not occur. VP may present either cutaneous manifestations of photosensitivity, neurologic aspects of AIP, or both. The massive rise in liver content and excretion of porphyrin pre-

Hyperpigmentation induced by topical 5-aminolaevulinic acid plus visible light.

UNLABELLED Photodynamic therapy (PDT) with 5-aminolaevulinic acid (ALA) is an alternative tool for the treatment of superficial non-melanoma skin cancers. Recently ALA-PDT has been employed with encouraging results also for warts, condylomata and psoriasis. In this study the effects of topical ALA plus irradiation with visible light on intact human skin have been evaluated. Five skin areas (A, B, C, D, and E) on the inner upper part of the arms of five healthy volunteers (skin types III and IV) were treated with (A) ALA 20% in base cream without irradiation, (B) only the vehicle (base cream) without ALA, (C, D and E) ALA cream at the concentrations of 5, 10 and 20%, respectively; all treatments were applied with an occlusive dressing. Four hours after ALA or vehicle application areas B, C, D and E were irradiated with a fixed dose of 40 J/cm(2). ALA penetration through the intact skin was evaluated by in vivo fluorescence determination. The effects on healthy skin were evaluated by clinical and chromometric examinations, light microscopy, immunohistochemistry and electron microscopy. RESULTS (1) in vivo fluorescence demonstrated that ALA is able to penetrate through the intact skin, when applied with occlusive dressing and induces a classical fluorescence peak due to Protoporphyrin IX (PpIX) formation, which is the active photosensitiser. (2) Skin areas receiving ALA plus irradiation showed erythema and swelling just after the irradiative session and hyperpigmentation 48-72 h later. (3) Colourimetric data confirmed significant skin colour changes: values a* (representing the erythematous changes) increased only on the skin areas where ALA+irradiation were applied and during the 48 h after irradiation, thereafter a* began to decrease; values L* (pigmentation) increased during the 2 weeks following treatment. (4) Histopathological, immunohistochemical (S100, HMB-45) and electron microscopic findings showed an absolute increment of the number of melanocytes, which appeared clearly activated. In conclusion the application of ALA cream followed by irradiation is able to induce a pigmentation response in healthy human skin, at least in skin types III and IV. This melanocytic activation could have a potential for the treatment of skin disorders characterised by hypopigmentation.

Tyrosinemia Type II in Two Cases Previously Reported as Richner-Hanhart Syndrome

Two sibs with palmo-plantar keratosis and dendritic corneal opacities, previously described as suffering from Richner-Hanhart syndrome by other authors, about 25 years ago, showed increased plasma and urine tyrosine levels. Their neurological and mental features were within normal limits. A comprehensive review of the literature showed a total of 47 cases of fully documented tyrosinemia type II; 8 more patients also had the clinical features of the disease, but aminoacidemia had never been observed. The importance of early diagnosis is stressed, since a low tyrosine-phenylalanine diet dramatically improves the symptoms and may prevent mental retardation.

Influence of cinnarizine on patch test reactions

Tromantadine hydrochloride is an effective antiviral drug derived from amantadine. Its antiviral action presumably depends on an alteration in the enzyme systems responsible for the cellular penetration of the herpes virus (1). It is often prescribed for herpes simplex, and is used frequently, applied to acute inflammatory lesions. Some authors have observed a contact sensitivity to the tromantadine alone (2-4), others to the tromantadine and serol base (5), and yet others to tromantadine and amantadine (6) and to some paragroup substances (7). A 17-year-old boy with recurrent labial herpes simplex developed an allergic contact dermatitis after applying an ointment containing tromantadine hydrochloride (Viruserol). After stopping it and using topical anti-eczematous therapy, he was promptly cured. Patch tests to the ICDRG standard series were negative; a test to the ointment was strongly positive (3 + ). Additional tests were carried out with: tromantadine hydrochloride 0.5% and 1 % pet., paraben mix 1 %, sorbic acid 5%, adragant gum 2.5%, glycerine 16%, amantadine hydrochloride 1%, starch-water 3%; the patient showed a positive reaction to tromantadine hydrochloride alone. References

Hailey-Hailey disease successfully treated with vitamin D oral supplementation

Hailey‐Hailey disease (HHD) also known as familial benign chronic pemphigus is a rare autosomal dominant genodermatosis. HHD treatment is often not satisfactory and hence, various modalities of treatment have been tried. We describe the case of a 37‐year‐old woman with a 2 years history of macerated erythematous plaques along with erosions, fissures, and crusts located on axillae and submammary areas, successfully treated with only oral supplementation of vitamin D (800 I.U./die) for 3 months. We reported this case to suggest that oral vitamin D may be enumerated in the various treatments proposed for HHD so far due to its rapid efficacy on skin lesions and symptoms.

Topici dermatologici (e sistemi di veicolazione intradermica)

Allo scopo di ottenere un effetto sulla funzionalita di epidermide, derma e annessi, possono essere trasportati, in condizioni diverse della pelle, farmaci e altre sostanze attive, evitando una possibile influenza su altre strutture dell’organismo.

Fluorescence imaging for diagnosis of skin diseases

To the Editor: Recently, fluorescence, a natural phenomenon of individual chemical cotnpounds that show characteristic absorption and emission spectra, has been used to differentiate normal and neoplastic tissues in vivo. The detection of tumours via laser-induced fluorescence of exogenous compounds accumulating into them is at present a well-established technique. Nevertheless, diagnostic methods based on the detection of natural fluorescence (autofluorescence) would be, in principle, more suitable for their fast and non-invasive application. We did a study to distinguish between malignant and benign skin lesions in vivo by their autofluorescence characteristics. Skin areas of 61 patients with clinically diagnosed non-neoplastic and neoplastic skin pathologies (different types of nevus cell nevus, angiomas, fibromas, seborrheic keratosis, actinic keratosis, basal cell epitheliomas, squamous cell epitheliomas, superficial spreading melanoma) were observed. The clinical diagnosis was confirmed by histopathological examination for all observed pathologies. The excitation was performed by a 200 W Hg lamp (ORIEL 66007) with pass-band filters centered at either 365 or 313 nm (ORIEL 56531 and 56511) and the fluorescence emitted was collected through cut-on filters at either 460 or 610 nm respectively (Corion LL450 or LL400). The fluorescence was imaged onto an intensified videocamera RCA TC 1030 and the images were elaborated with an IBAS II Kontron System (Fig. 1). The best defined images of all skin lesions were examined with excitation at 365 nm and observation at wavelengths above 460 nm. Our results show that only actinic keratosis, the most common epithelial precancerous lesion, denotes a significant endogenous fluorescence. It is known that histopathologically actinic keratosis is characterized by epidermal cells, varying in size and shape, sometimes multinucleated, with prominent hyperand parakeratosis. In most of the 15 cases of actinic keratosis examined, we observed an intense fluorescence spot in correspondence with areas of previously clinically diagnosed actinic keratosis (Fig. 2). In some cases of chronically photoexposed skin, we detected a number of spots of fluorescence denoting subclinical actinic keratosis. Among the epithelial tumours only squamous cell epitheliomas exhibit strong fluorescence emission. No fluorescence is observed for the other skin pathologies investigated. Leffel et al. showed that the excitation of skin areas in vivo by helium-cadmium laser (325 nm) yields characteristic tissue autofluorescence spectra that are not related to age, pigmentation, or skin thickness. On the other hand the fluorescence was affected by sun-exposure, showing a different spectral distribution between axillae and temple or other solar exposed areas, maybe due to differences in the elastin biochemical nature (e.g. desmosine content) present in these different skin areas. Lohman and Paul examined melanomas excited in vivo by radiation of the spectral line at 366 nm of a 100 W high-pressure Hg lamp filtered with a BP 366/11 filter. They detected a very similar fluorescence behaviour for all melanomas observed: all spectra are peaked at 475 nm and are more intense at the edge of the tumour. At this location, a shoulder at 445 nm was also evident. Since there are strong indications that the fluorescence band with a maximum at 475 nm is due to NADH (nicotinamide adenine dinu-