Apoptosis in Buruli ulcer: a clinicopathological study of 45 cases.

AIMS: To investigate the presence and pathogenetic role of apoptosis in Buruli ulcer (BU), a highly destructive skin disease caused by Mycobacterium ulcerans. METHODS AND RESULTS: Forty-five skin biopsies obtained from 30 Beninese patients affected by BU, in different clinical and therapeutic periods, were analysed for the main histopathological features (inflammatory infiltration, necrosis, sclerosis, oedema, granulomas and nerve damage). Immunofluorescent detection of antigens (anti-Bax, anti-caspases-3 and -8), together with deoxyuridine, 5'-triphosphate (dUTP) nick end labelling (TUNEL) assay, were also performed. A significant decrease in inflammatory infiltration (P = 0.0001) was detected between the beginning and end of antibiotic treatment. Neutrophils predominated in the first phase, while lymphocytes and plasma cells were increased at the end of the therapy. An inverse correlation between tissue necrosis and sclerosis was observed (P = 0.001). In 11 cases, inflammatory and regressive changes involved the nerve bundles with axonal degeneration and disruption of nerve fibres. TUNEL assay detected apoptotic bodies within nerve bundles, and these decreased from beginning to end of therapy. Bax, caspase-3 and -8 were down-regulated over the course of antibiotic therapy. CONCLUSIONS: In BU, apoptosis plays a role in promoting and sustaining the destructive changes and is implicated in the neural pathology that is associated with clinically detected anaesthesia.

No indications for HPV involvement in the hypertrophic skin lesions of a Darier disease case without ATP2A2 gene mutations.

Darier disease (DD) is a relatively common genodermatosis characterized by impaired differentiation and abnormal cell-to-cell adhesion. Haploinsufficiency of the ATP2A2 gene product, sarco/endoplasmic reticulum Ca(2+) ATPase isoform 2 (SERCA2), is the underlying cause of most cases. Although DD may have a papillomatous appearance, few and controversial results have been reported about the role of human papillomavirus (HPV) in this disease. The aim of this study was to determine a possible correlation between development of hypertrophic lesions in DD and infection by HPV. We report the case of an 84-year-old woman with a hypertrophic DD variant that has been successfully treated with oral retinoids. HPV analysis for a broad spectrum of cutaneous and mucocutaneous genotypes was performed on surgical specimens obtained from the cutaneous lesions and snap-frozen plucked eyebrows. Genetic analysis of the ATP2A2 gene did not detect any mutations. Epidermal expression of SERCA2b was shown by immunohistochemistry. We describe a patient with DD lacking mutations of the ATP2A2 gene, but with reduced SERCA2b expression in the epidermal keratinocytes. The results obtained by polymerase chain reaction (PCR) genotyping, quantitative real-time PCR, and in situ hybridization indicate that HPV replication was very low and suggest no direct role of the virus in the development of the disease.

Knuckle pads, in an epidermal palmoplantar keratoderma patient with Keratin 9 R163W transgrediens expression.

Epidermolytic PalmoPlantar keratoderma (EPPK) Vörner-type is an autosomal dominantly inherited skin disease, characterized by severe thickening of the palms and soles, caused by mutations in the keratin K9 (KRT9) gene. To date, a number of KRT9 mutations have been detected, most of which affect the highly conserved 1A region of the central alpha-helical domain, important for keratin heterodimerization. The most common mutation is the substitution of the arginine in position 163 with a tryptophan (R163W), which has been reported in North American, European, and Japanese populations. In a small number of cases, EPPK is associated with knuckle pad keratosis, but no correlation between this additional phenotype and a specific mutation has been found. Moreover, K9 is not normally expressed in knuckle skin, raising the question of the pathogenic mechanism leading to this additional phenotype. Here we show that in a family affected by EPPK and knuckle pad keratosis, carrying the R163W substitution, wild type (wt) and mutated K9 are strongly expressed in knuckle pads. These results suggest that the knuckle pad phenotype is due to ectopical expression of K9.

Eradication of basal cell carcinoma of the head and neck using the surgical excision with a new stained margin technique: a preliminary study.

BACKGROUND: Basal cell carcinomas (BCCs) are common cutaneous neoplasms that mainly affect fair-skinned subjects, in sun-exposed areas of the body. The treatment of choice of BCCs is represented by surgical excision and different techniques are available, in order to allow the complete eradication of the tumor with the best cosmetic results. In this paper, we describe the surgical excision with stained margin technique (SMET) and we report its efficacy for the treatment of BCCs of the head and neck region. METHODS: We retrospectively studied 177 BCCs of the head and neck region treated by SMET: a surgical technique in which each specimen is cut vertically like a bread-loaf in multiple sections of 1 mm of thickness, after marking peripheral margins. RESULTS: We observed an overall recurrence rate of 4.5% after SMET (mean follow-up: 26 months), with higher rate in aggressive subtypes (P=0.04). BCCs located in high-risk sites and those previously undergone to other non-radical therapies required two or more procedures (P=0.008 and P=0.002, respectively), while no correlation was observed between the number of SMET procedures and recurrence rate. CONCLUSIONS: In our experience, since low recurrence rate was obtained by SMET, we suggest that it may be taken into consideration as surgical option for BCCs of the head and neck region.

Mycobacterium ulcerans mycolactone interferes with adhesion, migration and proliferation of primary human keratinocytes and HaCaT cell line.

The pathogenicity of Mycobacterium ulcerans (Buruli ulcer) is closely associated with the secretion of exotoxin mycolactone. The cytotoxicity of mycolactone has been linked to its apoptogenic activity. We explored if low mycolactone concentrations, which are not able to induce apoptosis, can influence other essential activities on two primary human keratinocyte populations, keratinocyte stem cells (KSC) and transit amplifying cells (TAC), and on a human keratinocyte line, HaCaT. We demonstrated that 0.01 and 0.1 ng/ml mycolactone A/B are not able to induce apoptosis in primary human keratinocytes, but interfere with KSC wound repair. Moreover, the same toxin concentrations reduce cell proliferation of KSC and TAC and their ability to adhere to type IV collagen. HaCaT cells are more resistant to the toxin; nevertheless, they show a delayed woud repair when treated with 1 and 10 ng/ml mycolactone A/B. Moreover, these sub-apoptotic concentrations affect their ability to proliferate and adhere to collagen IV. Wound healing is a complex mechanism, which occurs "in vivo" as the outcome of many co-ordinated events. Sub-apoptotic mycolactone concentrations can affect essential mechanisms, which are required to achieve wound repair, such as adhesion, migration and proliferation of human keratinocytes.

[External otitis]. / L'otite esterna.

Otitis externa is one of the most common diseases in ORL practice, during summer; the treatment of otitis externa may be simple and easy or protracted and frustrating, also with fatal outcome. Many local factors may interfere with the normal defences against infections in the external auditory canal. Removing or dissolving the cerumen by water or other instruments eliminates an important barrier to infections: its acids inhibit the growth of bacteria (Staphylococcus aureus and Pseudomonas aeruginosa) and fungi (Aspergillus). Also skin abrasions or irritation, allergic diseases and many systemic condition like anaemia, vitamin deficiency, endocrine disorders (diabetes) and various forms of dermatitis cause a lower resistance to infections in external auditory canal. Even if the prognosis remains benign in the majority of cases, important complications could appear like: malignant otitis externa, facial nerve paralysis, tympanic bone osteomyelitis, pericondrytis. Successful treatment depends on a proper diagnosis and therapy: the most important factor in the treatment is repeated debridement of the external auditory canal by the physician. The use of Castellani' Tintura rubra, hydroalcoholic solution of phenic fuchsin, can be very effective for bacteria and mycotic eradication. Culturing of ear canal infection could be performed on the second or third visit if the otitis externa is not responding to therapy. Complication are not frequent, but malignant otitis externa can be mortal. Dermatological consultation is often necessary for correct diagnosis.

[Study on the appropriateness of utilization of dermatological consultants in emergency unit]. / Appropriatezza delle richieste di consulenza dermatologica in Pronto Soccorso in una Azienda Ospedaliera piemontese.

The aim of this study is to evaluate the rate of appropriate use of dermatologist consultants, in order to identify its determinants and to define its economic impact. We analysed 1750 dermatological procedures performed in the emergency room of the Azienda Ospedaliera "Maggiore della Carità" in Novara. The 87,64% of the procedures were inappropriate. We also observed low rates of diagnostic agreement between emergency unit and dermatology unit; 40.88% of discordant diagnoses were due to severe mistakes. The rate of inappropriate dermatological procedures was related to the clinical diagnosis and to patient's access time. The economical analysis was performed using the Activity Based Costing methodology. We measured an average expenditure of euro 7 for procedure (total expenditure euro 9.850). As possible determinants of inappropriateness, we identified the lack of "filter action" by general practitioners, the incorrect behaviour of patients and the professional uncertainty phenomena.

Clinical considerations on Buruli ulcer employing two molecular tests for the detection of Mycobacterium ulcerans in 100 skin biopsies.

OBJECTIVES: Buruli ulcer (BU) is an infected cutaneous lesion, the etiological agent of which is Mycobacterium ulcerans. Diagnosis is confirmed by the identification of acid-fast bacilli and culture. In clinically suspicious forms with negative bacteriological or Ziehl-Neelsen (ZN) findings, molecular tests are used. This study compared the concordance of nested polymerase chain reaction (PCR) (targeting IS2404) and PCR (targeting IS2606) in different clinical situations. METHODS: A total of 57 samples were sourced from 39 BU patients. Control samples (n = 43) were obtained from non-BU ulcers in 38 patients. Samples were divided into two pieces and submitted to, respectively, histological examination and ZN staining, and PCR. Subsamples submitted to PCR were divided and submitted to nested PCR IS2404 and PCR IS2606, respectively. RESULTS: Of the 57 BU biopsies, positive results were obtained by nested PCR in 18 (31.6%) and by IS2606 PCR in 37 (64.9%) cases. Sequencing of the positive samples confirmed the specificity of amplicons in all nested PCR samples and in 26 of 37 (70.2%) samples positive to IS2606. Hence, nested PCR was more specific (100% vs. 93%) and less sensitive (32% vs. 46%) than IS2606 PCR. In the BU samples, nested PCR was negative in 15 instances, and IS2606 PCR was negative in 11 instances in which ZN histology had been positive (false negatives). Both PCRs were positive in six ZN-negative smears. CONCLUSIONS: We considered 57 samples from 39 BU patients in various clinical stages and at different times after the beginning of therapy. These provided positive results in 18 cases with IS2404 nested PCR and in 37 cases with PCR IS2606; only 26 of the latter remained positive subsequent to sequencing. Hence, even if IS2404 PCR is considered more specific, in subjects who appear to fail to respond to therapy, it is advisable to also carry out IS2606 PCR. A possible interpretation of the discordance between the two techniques due to unavoidable technical errors as well as to different sensitivity of the two tests at M. ulcerans DNA low concentration (i.e. in recent infection and in well-treated cases) is discussed.

A Mycobacterium ulcerans toxin, mycolactone, induces apoptosis in primary human keratinocytes and in HaCaT cells.

The pathogenicity of Mycobacterium ulcerans (Buruli ulcer) depends on cytotoxic effect of its exotoxin mycolactone. Since epidermis represents a barrier against infectious agents and balanced apoptosis is essential in epidermal homeostasis, we explored if mycolactone A/B induces apoptosis on two human keratinocyte populations, stem cells (KSC) and transit amplifying cells (TAC), and on human keratinocyte line, HaCaT. Treatment of TAC with 1 and 10 ng/ml mycolactone-induced 60 and 90% apoptosis. KSC were more resistant than TAC: 50 and 75% of cells underwent apoptosis after 10 and 100 ng/ml toxin-treatment. Higher doses (1000 ng/ml) induced about 30% apoptosis on HaCaT. In contrast, mycolactone A/B was devoid of toxicity neither on human hepatoma HuH7 nor on human embryonic kidney HEK 293 T cell lines. In conclusion, mycolactone induces apoptosis in human keratinocytes, thus contributing to Buruli ulcer lesions development.

Serum cytokine profile during Mycobacterium ulcerans infection (Buruli ulcer).

BACKGROUND: Buruli Ulcer (BU) is a severe cutaneous and subcutaneous disease due to Mycobacterium ulcerans infection, mainly distributed in sub-Saharan Africa and tropical areas. The role of T helper (TH) cytokines in the development and clinical course of the disease has been previously studied by investigating the in vitro immune response of lymphocytes from affected patients and immunohistochemical analyses of bioptic samples. METHODS: TH cytokine levels (IFNγ, TNF-α, IL-2, IL-10, IL-4, IL-5, IL-17) were evaluated in serum of 34 Beninese subjects by cytofluorimetric and immunoenzymatic assays: 16 patients affected with active BU, 4 patients who had healed after specific therapy, and 14 matched controls. RESULTS: Levels of IFNγ were higher in patients with late BU (>2 months from onset) and healed patients than in controls, and in ulcerative than in pre-ulcerative patients. Analysis of 4 patients with "late" disease evaluated both at the beginning of antibiotic therapy and 6 months later showed that IFNγ levels were always lower in the second evaluation. By contrast, no differences were found in levels of the other cytokines. CONCLUSIONS: IFNγ production is low in early BU, and increases in late BU and healing, suggesting a role of this cytokine in infection clearance. Moreover, evaluation of IFNγ serum levels may be a useful tool to monitor the immune response during the BU course.

High beta-HPV DNA loads and strong seroreactivity are present in epidermodysplasia verruciformis.

Epidermodysplasia verruciformis (EV) is a rare disease, characterized by cutaneous warts and associated with a strong predisposition to beta-genus human papillomavirus (HPV). Earlier studies reported high copy numbers of HPV-DNA in nearly all skin tumors from EV patients, but neither HPV replication status in non-lesional skin nor anti-HPV seroreactivity in these patients have been reported yet. We therefore performed a comprehensive viral load analysis for the more common beta-HPV types on skin samples and plucked eyebrow hairs from four EV patients treated at our dermatology department. The results clearly demonstrate that they carry a multiplicity (up to eighteen types) of beta-HPV genotypes in both skin sites. Worthy of note, a high intrapatient concordance for specific types between hair bulbs and skin biopsies was observed and the same beta-PV profile was maintained over time. Viral load analysis revealed a load range between less than one HPV-DNA copy per 100 cells to more than 400 HPV-DNA copies per cell in both eyebrow hairs and skin proliferative lesions. Evaluation of seroreactivity to beta-HPV types in the four EV patients revealed that antibodies against the 16 beta-HPV were significantly more prevalent and showed higher titers than in the controls.

Ultrasonography for the monitoring of subcutaneous damage in Mycobacterium ulcerans infection (Buruli ulcer).

We used ultrasonography to evaluate the nature and the extent of subcutaneous damage provoked by Mycobacterium ulcerans (M. ulcerans) and to investigate the possible involvement of the tributary lymph nodes during the various stages of progression of Buruli ulcer. Nineteen patients affected by M. ulcerans infection in Benin, West Africa, were studied. Ultrasonography was performed on all subjects, except one, at the site of nonulcerated lesions and/or at perilesional site. The tributary lymph nodes were also studied in six patients. Ultrasound (US) evaluation was carried out using a 10 MHz linear probe and all lesions were compared with the homologous unaffected controlateral site. The ultrasonography showed relevant alterations at the dermo-hypodermic level, in agreement with histological specimens. In the active forms of the disease, these alterations are characterized by significant oedematous imbibition of the adipose tissue and necrosis (adiponecrosis) that leads to varying irregularities in the echogenicity of the hypodermis, which is generally thicker. In agreement with the clinical examination, the lymph nodes in six patients evaluated, despite their possible histological involvement with necrotic phenomena described in literature in M. ulcerans infection, did not display significant alterations visible by ultrasonography. The US scanning we have performed is the first use of this technique for M. ulcerans infection. We have shown that it can reveal the subcutaneous depth and the peripheral extent of the pathological process and it is particularly useful for monitoring the efficacy of or resistance to antibiotic treatment, especially in extensive ulcero-oedomatose forms. Such monitoring offers also a useful guide to the surgeon allowing the reduction or postponement of the removal of the large cutaneous areas that were carried out until recently.