Deciphering sex-specific miRNAs as heat-recorders in zebrafish.

In the last decade, a plethora of microRNAs (miRNAs) has been reported in a wide variety of physiological processes, including reproduction, in many aquatic organisms. However, miRNAome alterations occurred by environmental cues due to water temperature increment have not yet been elucidated. With the aim to identify epigenetic regulations mediated by miRNAs in the gonads in a climate change scenario, the animal model zebrafish (Danio rerio) were subjected to high temperatures during sex differentiation, a treatment that results in male-skewed sex ratios in the adulthood. Once the fish reached adulthood, gonads were sequenced by high-throughput technologies and a total of 23 and 1 differentially expressed miRNAs in ovaries and testes, respectively, were identified two months after the heat treatment. Most of these heat-recorder miRNAs were involved in human sex-related cancer and about 400 predicted-target genes were obtained, some with reproduction-related functions. Their synteny in the zebrafish genome was, for more than half of the predicted target genes, in the chromosomes 7, 2, 4, 3 and 11 in the ovaries, chromosome 4 being the place where the sex-associated-region (sar) is localized in wild zebrafish. Further, spatial localization in the gonads of two selected heat-recorder miRNAs (miR-122-5p and miR-146-5p) showed exclusive expression in the ovarian germ cells. The present study expands the catalog of sex-specific miRNAs and deciphers, for the first time, thermosensitive miRNAs in the zebrafish gonads that might be used as potential epimarkers to predict environmental past events.

Topical photodynamic therapy significantly reduces epidermal Langerhans cells during clinical treatment of basal cell carcinoma.

BACKGROUND: Topical photodynamic therapy (PDT) is a widely applied treatment for basal cell carcinoma (BCC). PDT-induced immunosuppression leading to reduced antitumour immune responses may be a factor in treatment failure. OBJECTIVES: To examine the impact of topical PDT on leucocyte trafficking following clinical treatment of BCC. METHODS: Superficial BCCs in eight white caucasian patients were treated with methyl aminolaevulinate (MAL)-PDT. Biopsies for immunohistochemical assessment were taken from BCCs pre-PDT, 1 h and 24 h post-PDT and from untreated healthy skin. RESULTS: Treatment of BCC with MAL-PDT produced a rapid neutrophil infiltration, commencing by 1 h and significantly increased at 24 h post-PDT (P < 0·05 compared with baseline). An associated increase in the number of blood vessels expressing E-selectin was observed at 1 h and 24 h post-PDT (both P < 0·05 compared with baseline). In contrast, the number of epidermal Langerhans cells fell sharply by 1 h post-PDT, and remained significantly reduced at 24 h post-PDT (both P < 0·05 compared with baseline). CONCLUSIONS: Reduction of Langerhans cells during clinical treatment of BCC might potentially impact negatively on antitumour responses through reduced activation of tumour-specific effector cells. Investigation of modified PDT protocols with the aim to minimize immunosuppressive effects while maintaining antitumour efficacy is warranted.

Infliximab restores the balance between pro- and anti-apoptotic proteins in regressing psoriatic lesions.

BACKGROUND: Psoriasis and psoriatic arthritis are treated very efficaciously with infliximab, a chimaeric human-murine antitumour necrosis factor (TNF)-α antibody. As we reported earlier, infliximab, besides its anti-inflammatory properties, induces a caspase-independent programmed cell death of psoriatic keratinocytes. OBJECTIVES: To elucidate this finding further, we investigated the epidermal expression of proteins involved in the mitochondria-dependent (intrinsic) pathway of cell death. METHODS: Quantification of proteins with pro- (p53, AIF, Bax) and anti-apoptotic functions (Bcl-2, Bcl-XL) and of NF-κB was performed by means of immunohistochemistry and digital image analysis of the staining of nonlesional skin and lesional psoriatic skin from patients treated with infliximab at weeks 0, 2 and 6. RESULTS: Serial biopsies from psoriatic plaques of samples taken at days 0, 5, 14 and 21 of therapy demonstrated a significant downregulation of anti-apoptotic proteins Bcl-2, Bcl-XL and NF-κB during treatment and, in parallel, a significant upregulation of pro-apoptotic proteins p53, Bax and AIF. These differences in expression correlated with decreases in epidermal thickness and clinical outcome (Psoriasis Area and Severity Index). At day 21, expression levels of apoptosis-related proteins in lesional skin approximated those found in nonlesional skin. CONCLUSIONS: Our data therefore suggest that TNF-targeting agents may induce the regression of psoriasis at least in part by normalizing the expression of apoptosis-related proteins in lesional keratinocytes.

The role of endothelial cell apoptosis in the effect of etanercept in psoriasis.

BACKGROUND: Psoriasis is a chronic inflammatory skin disease associated with abnormal vascular expansion in the papillary dermis. Tumour necrosis factor (TNF)-α is a proinflammatory cytokine that can induce antiapoptotic proteins and endothelial cell activation factors in psoriasis. OBJECTIVES: The present study investigated the effect of the anti-TNF-α agent etanercept on the expression of endothelial nuclear factor-κB (NF-κB), angiogenic vascular endothelial growth factor (VEGF), endothelial cell marker CD31, antiangiogenic factor thrombospondin-1 (TSP-1), and antiapoptotic factors Bcl-2 and Bcl-xL in psoriasis. METHODS: Sixteen patients with moderate-to-severe psoriasis were included in the study and treated with etanercept 50 mg twice weekly subcutaneously for 12 weeks. Biopsies of lesional skin (baseline, weeks 3, 6 and 10) were obtained and immunohistochemically stained with antibodies for CD31, VEGF, TSP-1, NF-κB, Bcl-2 and Bcl-xL. Double immunofluorescence staining for VEGF and CD31 was evaluated with confocal laser microscopy. The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick-end labelling (TUNEL) assay was applied for apoptosis detection. RESULTS: Etanercept caused a statistically significant time-dependent reduction in the number of dermal blood vessels, the number of CD31+ cells and VEGF in psoriatic lesions, with induction of endothelial cell apoptosis and statistically significant upregulation of TSP-1 in psoriatic vessels. Immunohistochemical analysis showed significant reduction of NF-κB, Bcl-2 and Bcl-xL expression in endothelial cells during treatment. These changes were accompanied by a marked clinical response. CONCLUSIONS: The present findings suggest that treatment with etanercept induces apoptosis, reduces apoptosis-inhibiting factors in psoriatic endothelial cells, and decreases angiogenesis in psoriatic skin.

Comparative analysis of incidence and clinical features of cutaneous malignant melanoma in Crete (Greece) and southern Germany (central Baden-Württemberg).

BACKGROUND: Comparative analysis of the incidence rates and epidemiological features of cutaneous malignant melanoma (CMM) between different ethnic groups exposed to varying environmental factors is critical for consideration of the causes of CMM but can also be utilized in a public health approach to control of the disease. OBJECTIVES: To compare incidence rates and clinical features of CMM in a Greek and a central European population (central Baden-Württemberg, Germany). METHODS: Incident cases of CMM were traced in all hospitals of the island of Crete for the period 1999-2002. Age-standardized incidence rates per 100 000 inhabitants per year for the European Standard Population were calculated based on the Cretan population statistics. A comparison was performed between the Cretan findings and those of southern Germany as registered by the hospital-based Central Malignant Melanoma Registry, which likewise documents more clinical features than normally recorded by population-based cancer registries. RESULTS: Mean incidence rates in Crete for 1999-2002 were 4.01 per 100 000 inhabitants per year for males and 4.05 for females as compared with 10.6 for males and 11.1 for females in southern Germany. There were striking differences in the clinical characteristics of CMMs, with significantly higher tumour thickness in Crete (median 1.4 mm vs. 0.7 mm in southern Germany). Correspondingly, significantly more nodular melanomas were observed in Crete (29%) as compared with southern Germany (11%). CONCLUSIONS: Incidence of CMM in Crete, with about four cases per 100 000 inhabitants per year, is clearly higher than previously estimated, and there is an urgent necessity for earlier recognition of CMM in Crete. However, the incidence of CMM in southern Germany is much higher.

Programmed cell death of keratinocytes in infliximab-treated plaque-type psoriasis.

BACKGROUND: Tumour necrosis factor (TNF)-alpha blockade using infliximab, a chimeric anti-TNF-alpha antibody, is an effective treatment for plaque-type psoriasis, inducing remission in about 80% of patients. OBJECTIVES: To examine infliximab-induced programmed cell death (PCD) of keratinocytes in psoriatic plaques on serial skin biopsy samples. METHODS: Five patients with moderate to severe plaque-type psoriasis received infliximab infusions intravenously (5 mg kg(-1)) at weeks 0, 2 and 6. Biopsies of nonlesional and lesional skin (days 0, 5, 14 and 21) were obtained. Conventional microscopy was used to examine the morphology of the psoriatic keratinocytes. In situ detection of apoptosis was performed by electron microscopy and by immunohistochemical staining with anti-p53 and anti-caspase-3 antibodies. Results Infusion of infliximab induced a clinical response in all five patients with psoriasis, with a mean Psoriasis Area and Severity Index improvement of 24.8% already at day 5. This was accompanied by significant histopathological changes in the skin biopsy samples after infliximab treatment. Light and electron microscopic evaluation revealed apoptosis-like morphological changes in lesional keratinocytes, i.e. nuclear condensation, chromatin fragmentation and cytoplasmic vesiculation, visible already after the first infusion. These damaged keratinocytes stained positively for p53, but not for active caspase-3. CONCLUSIONS: The effects of infliximab in psoriasis extend beyond merely anti-inflammatory actions, and may include caspase-independent PCD of lesional keratinocytes. The PCD of keratinocytes may be an important mechanism that could explain at least in part the rapid and sustained therapeutic effect of infliximab in psoriasis.

Cutaneous alternariosis revealing acute myeloid leukaemia in an adult patient.

We report a case of cutaneous alternariosis in a 69-year-old male patient. During hospitalization for treatment of the skin disorder, acute myeloid leukaemia was diagnosed. He received multiple chemotherapeutic agents but the leukaemia remained refractory to therapy and the patient died. The clinical picture, diagnosis and treatment of cutaneous alternariosis will be discussed and a review of the literature regarding patients with haematological diseases will be given.

In vivo fluorescence kinetics and photodynamic therapy in condylomata acuminata.

BACKGROUND: Topical application of 5-aminolaevulinic acid (ALA) to condylomata acuminata leads to accumulation of protoporphyrin IX (PpIX); therefore ALA-induced photodynamic therapy (ALA-PDT) appears to be a potential treatment. OBJECTIVES: To investigate in vivo the PpIX fluorescence time course after topical application of ALA in order to determine the optimal time for irradiation, and to assess the efficacy of subsequently performed ALA-PDT. METHODS: Fluorescence kinetics was studied in 12 male patients with condylomata acuminata. Confirmation of diagnosis was established with conventional histology and polymerase chain reaction. Lesions were treated with 20% ALA and irradiated at the optimal time with a dose of 70 J cm-2 or 100 J cm-2 light. An additional session with 100 J cm-2 was administered 1 week later to lesions that persisted. RESULTS: The in vivo study of fluorescence kinetics indicated that the optimal time for irradiation varied among patients from 6 to 11 h. The overall cure rate was 72.9%, 12 months after treatment. CONCLUSIONS: Topical ALA-PDT is a potentially effective treatment for condylomata acuminata.

Early development of multiple epithelial neoplasms in Netherton syndrome.

We report a case of Netherton syndrome manifested as congenital ichthyosiform erythroderma, trichorrhexis invaginata and atopy, who in early adulthood developed multiple, aggressive epithelial neoplasms in sun-exposed areas of the skin, in areas with papillomatous skin hyperplasia and at the left parotid region. The occurrence of cutaneous neoplasia has been reported in syndromes with congenital ichthyosis and suggests that the underlying genetic defects may cause the development of cancer in prone patients.

In vivo detection of human papilloma virus-induced lesions of anogenital area after application of acetic acid: a novel and accurate approach to a trivial method.

Human papilloma virus infection is increasing at an alarming rate. The ability of the virus to establish a subclinical infection and its association with malignancy of the lower genital tract make the statistics even more worrisome. Topical application of acetic acid solution provokes temporal alterations of the light-scattering properties of human papilloma virus-induced lesions of anogenital area. For the in vivo study of the phenomenon, an imaging system has been employed, which performs time-lapse imaging and enables the calculation and display of the kinetics of the provoked alterations in any point within the examined area. Confirmation of diagnosis has been established with conventional histology and polymerase chain reaction. It has been shown that the method provides early detection and staging of skin alteration or transformation due to human papilloma virus infection and enables mapping of the infected area.

In vivo fluorescence kinetics and photodynamic therapy efficacy of delta-aminolevulinic acid-induced porphyrins in basal cell carcinomas and actinic keratoses; implications for optimization of photodynamic therapy.

Photodynamic therapy (PDT) with topical d-aminolevulinic acid (ALA) has become a therapeutic option of growing interest for superficial non-melanoma precancerous and malignant lesions. After application of ALA, in situ conversion to endogenous porphyrins is accomplished in a gradual manner. Therefore, the determination of fluorescence kinetics and spatial distribution in vivo versus time is a crucial point for the success of ALA-PDT. Seventeen basal cell carcinomas (BCC) and 20 actinic keratoses (AK) were enrolled in this study. In 5 BCC and 4 AK, in vivo fluorescence kinetics were performed over 24 hrs and for the remaining lesions between 2 and 7 hrs after ALA application. In vivo spatial and quantitative detection of the fluorescence intensity versus time showed considerable variations among tumors of the same type, so light irradiation was performed according to patient individualities. Both BCC and AK showed maximal median fluorescence intensity at 4-6.5 hrs post-application. In the present study, a high cure rate was proven after topical ALA-PDT (70.6% in BCC and 85% in AK). The results of fluorescence studies suggest that optimum irradiation time for BCC is approximately 3.5-5 hrs and for AK 5 hrs after ALA application, when relative maximal fluorescence intensity in correlation with fluorescence selectivity on the lesion, is obtained.

Cutaneous alternariosis in a patient with idiopathic pulmonary fibrosis.

A 78-year-old farmer presented with symptomless skin lesions for evaluation. Two years prior, he had developed idiopathic pulmonary fibrosis (IPF) and had been treated thereafter with oral prednisolone 20 mg/day and occasionally with colchicine 1 mg/day. On examination, erythematoviolaceous, slightly infiltrated plaques, measuring approximately 5 x 9 cm, rubbery in consistency, intermingled with pustules, sometimes eroded, with distinctive borders, were noted on the dorsum of both hands and on the extensor surface of both forearms. The lesions had developed over a 20-day period. The skin of these areas was atrophic or eroded with multiple ecchymoses (Fig. 1). The abnormal laboratory findings included an elevated white blood cell count of 17,100/mm3, with 79% neutrophils, 16% lymphocytes, and 5% monocytes, C-reactive protein of 33.15 mg/dL (normal, <0.8 mg/dL), and immunoglobulin G of 598 mg/dL (normal, 701-1545 mg/dL). Other blood and urine tests performed were within normal limits. The diagnosis of IPF was reconfirmed through radiology, high-resolution computed tomography, and spirometry, as well as bronchoscopy and bronchoalveolar lavage fluid analysis. Coexistence of presumptive pulmonary alternariosis was excluded. Hematoxylin and eosin stained sections of the excised cutaneous specimen showed focal ulceration of the epidermis adjacent to a mainly intradermal abscess cavity. Within the latter, remnants of a partly destroyed hair follicle were seen amongst degenerating polymorphonuclear leukocytes, as well as many histiocytes and a few Langhans-type multinucleated giant cells. Minute collections of polymorphonuclear leukocytes were seen in the adjacent epidermis. Periodic acid-Schiff (PAS) and Gomori's silver methenamine stains showed a multitude of broad branching fungal hyphae and large spores within the aforementioned cavity, both free and within the cytoplasm of giant cells (Fig. 2). Immunohistochemistry was performed by means of the alkaline phosphatase anti-alkaline phosphatase (APAAP) method. Sections showed that the infiltrate consisted of an almost equal number of B and T lymphocytes, whereas histiocytes and the few giant cells were labeled with anti-CD68 antibodies. Skin smears and biopsy specimens taken twice from all lesions were used for mycologic examination. Wet mounts revealed numerous, brownish, septate hyphae and ovoid Skin smears and biopsy specimens taken twice from all lesions were used for mycologic examination. Wet mounts revealed numerous, brownish, septate hyphae and ovoid structures. Biopsy material was plated on Sabourand's dextrose agar with cloramphenicol (0.05 mg/mL). After 7 days at 27 degrees C, dark, gray-white colonies with a dark brown underside appeared. Microscopic examination of the colonies revealed hyphae with typical conidia having transverse and longitudinal septa. Based on macroscopic and microscopic examination, the isolates were identified as Alternaria alternata (Fig. 3). Treatment with prednisolone was reduced to 10 mg/day and the patient received oral itraconazole (200 mg/day). This resulted in progressive improvement of alternariosis, and the lesions healed completely within 3 months, when treatment was interrupted. Two years later, there is no evidence of recurrence.

Assessment of nasal mucosa blood supply by quantitative endoscopic imaging of the back-scattered light.

In this article we present a method for the objective assessment and monitoring of tissue blood supply using a specially developed endoscopic imaging colorimeter that enables quantitative color modeling of the back-scattered light during endoscopic examination. Tissue blood volume changes in the nasal mucosa, induced by xylometazoline hydrochloride nasal spray, were evaluated with this method. It was found that quantitative imaging provides sensitive, reproducible, and reliable means for the monitoring and mapping of tissue blood supply and is easy to use routinely. The results showed that saturation decreases with time, being the most sensitive color parameter to the vasoconstriction procedure. It appears that objective indexes for optical tissue characterization and analysis may be promising in the understanding of the pathophysiology of tissue changes and in the objective evaluation of their response to different therapeutic schemes.

Epidemiologic observations on the natural course of pemphigus vulgaris.

BACKGROUND: During the last 20 years, few prospective studies on the natural course of pemphigus vulgaris (PV) have been performed. METHODS: Various correlations of disease duration, clinical activity fluctuations, serology, and coexistence with other disease states were analyzed with regard to their impact upon the natural history of pemphigus. Thirty-seven consecutive PV patients were involved in this 1-year retrospective follow-up study. RESULTS: The disease activity decreases with time, but when exacerbated it is of unpredictable intensity; skin involvement is equally distributed between the sexes; relapses occur mostly during the first 2 years after disease onset, and can be marginally predicted by autoantibody titers. CONCLUSIONS: Notice should be taken of the relative frequencies of PV associated with neoplasia, ionizing radiation, and familial occurrence.

The finite element method as a research and teaching tool in the analysis of local skin flaps.

BACKGROUND: The finite element analysis (FEA) is a recently introduced method in biomechanics that permits modeling of complex structures considering them as an aggregate of small elements. Skin flaps are highly suggested to be amenable to the continuum mechanic laws that underly the development of FEA. OBJECTIVE: A combination of "large deformation analysis," based on FEA with the criteria for skin flap selection, was attempted. METHODS: Serial defects were experimentally created on piglet skin stripes, which were consequently covered through designing appropriate flaps. Skin samples were modeled after the development of a computer FEA program and they were scanned by incorporating their photographs. RESULTS AND CONCLUSION: On the graphic interfaces the flap movement, the closure of the defect, and the whole deformation were found to match with the skin stripe postincisional alterations. This work permits the prediction and offers planning guides for different skin reconstructions.

Photodynamic treatment of skin malignancies with aminolevulinic acid. Emphasis on anatomical observations and in vivo erythema visual assessment.

BACKGROUND: Photodynamic therapy with delta-aminolevulinic acid is a promising alternative treatment for superficial skin malignancies. OBJECTIVE: Further clinical experience, study of tissue alterations leading to recovery, and correlation/prediction of the therapeutic response through in vivo skin color changes as represented by erythema development. METHODS: The therapeutic procedure, sequential histology and histochemistry, and the development of a remote machine vision system to measure, map, and monitor the erythema development. RESULTS/CONCLUSIONS: A high cure response rate with adequate follow-up was shown. A significant correlation of the clinical-histologic response of tumors subjected to treatment with the erythema measurements implies that erythema inspection and quantitative analysis offer a reliable predictor of the therapeutic outcome and a clue for optimization of this treatment modality.