Excised melanocytic lesions in children and adolescents - a 10-year survey.

BACKGROUND: Early recognition and prompt excision is to date the only available strategy for reducing mortality from melanoma. Little is known about the accuracy of melanoma detection in children and adolescents. OBJECTIVES: To assess the accuracy of melanoma detection in a paediatric population. METHODS: From the Department of Dermatology, Medical University of Graz, Austria, we reviewed the dermatopathology reports of naevi and melanomas excised in patients younger than 20 years over a 10-year period (1998-2007). Patients were subdivided into four age groups: 0-4, 5-9, 10-14 and 15-19 years. RESULTS: Accuracy in melanoma detection was tested using the number needed to excise (NNE) value that is obtained by dividing the total number of excised lesions by the number of melanomas. A total of 22564 lesions were reviewed, disclosing 22526 naevi and 38 melanomas, for an overall NNE value of 593.8. Five melanomas were excised in children aged 10-14 years (NNE 1141) and 33 in children aged 15-19 years (NNE 479.8), whereas no melanomas were found among 1026 lesions excised in children younger than 10 years. In children aged 0-4 years, congenital and Spitz/Reed naevi accounted for 34.5% and 20% of lesions, respectively. These percentages decreased progressively when moving to older age groups (P<0.0001). In contrast, the percentage of dermal and compound naevi rose in direct proportion with age, being 3.4% and 20.7%, respectively, in the youngest age group, and 36.7% and 31.9%, respectively, among the oldest patients (P<0.0001). CONCLUSIONS: The overall NNE value in paediatric patients over the 10-year study period was 593.8, meaning that about 594 lesions were excised to find one melanoma. This value is 20 times higher than the rates found in adult patients.

Hyperbaric Oxygen Therapy and Tissue Regeneration: A Literature Survey.

By addressing the mechanisms involved in transcription, signaling, stress reaction, apoptosis and cell-death, cellular structure and cell-to-cell contacts, adhesion, migration as well as inflammation; HBO upregulates processes involved in repair while mechanisms perpetuating tissue damage are downregulated. Many experimental and clinical studies, respectively, cover wound healing, regeneration of neural tissue, of bone and cartilage, muscle, and cardiac tissue as well as intestinal barrier function. Following acute injury or in chronic healing problems HBO modulates proteins or molecules involved in inflammation, apoptosis, cell growth, neuro- and angiogenesis, scaffolding, perfusion, vascularization, and stem-cell mobilization, initiating repair by a variety of mechanisms, some of them based on the modulation of micro-RNAs. HBO affects the oxidative stress response via nuclear factor erythroid 2-related factor 2 (Nrf2) or c-Jun N-terminal peptide and downregulates inflammation by the modulation of high-mobility group protein B1 (HMGB-1), toll-like receptor 4 and 2 (TLR-4, TLR-2), nuclear factor kappa-B (NFκB), hypoxia-inducible factor (HIF-1α) and nitric oxide (NO•). HBO enhances stem-cell homeostasis via Wnt glycoproteins and mammalian target of rapamycin (mTOR) and improves cell repair, growth, and differentiation via the two latter but also by modulation of extracellular-signal regulated kinases (ERK) and the phosphatidylinositol-3-kinase (PI3K)/protein kinase B (AKT) pathway. The HBO-induced downregulation of matrix metalloproteinases-2 and 9 (MMP-2/-9), rho-associated protein kinase (ROCK) and integrins improve healing by tissue remodeling. Interestingly, the action of HBO on single effector proteins or molecules may involve both up- or downregulation, respectively, depending on their initial level. This probably mirrors a generally stabilizing potential of HBO that tends to restore the physiological balance rather than enhancing or counteracting single mechanisms.

In vivo reflectance confocal microscopy: automated diagnostic image analysis of melanocytic skin tumours.

BACKGROUND: In vivo reflectance confocal microscopy (RCM) has been shown to be a valuable imaging tool in the diagnosis of melanocytic skin tumours. However, diagnostic image analysis performed by automated systems is to date quite rare. OBJECTIVES: In this study, we investigated the applicability of an automated image analysis system using a machine learning algorithm on diagnostic discrimination of benign and malignant melanocytic skin tumours in RCM. METHODS: Overall, 16,269 RCM tumour images were evaluated. Image analysis was based on features of the wavelet transform. A learning set of 6147 images was used to establish a classification tree algorithm and an independent test set of 10, 122 images was applied to validate the tree model (grouping method 1). Additionally, randomly generated 'new' learning and test sets, tumour images only and different skin layers were evaluated (grouping method 2, 3 and 4). RESULTS: The classification tree analysis correctly classified 93.60% of the melanoma and 90.40% of the nevi images of the learning set. When the classification tree was applied to the independent test set 46.71 ± 19.97% (range 7.81-83.87%) of the tumour images in benign melanocytic skin lesions were classified as 'malignant', in contrast to 55.68 ± 14.58% (range 30.65-83.59%; t-test: P < 0.036) in malignant melanocytic skin lesions (grouping method 1). Further investigations could not improve the results significantly (grouping method 2, 3 and 4). CONCLUSIONS: The automated RCM image analysis procedure holds promise for further investigations. However, to date our system cannot be applied to routine skin tumour screening.

Follow-up of actinic keratoses after shave biopsy by in-vivo reflectance confocal microscopy--a pilot study.

BACKGROUND: Monitoring of treatment efficacy after shave biopsy of actinic keratoses (AK) is often difficult, as clinical and dermoscopic features may not be reliable. OBJECTIVES: We investigated the applicability of in-vivo reflectance confocal microscopy (RCM) for the follow-up of AK after shave biopsy. METHODS: A total of 10 lesions were investigated by RCM before shave biopsy, after 3 and 12 months by two observers in agreement blinded to location, patients and time interval. RESULTS: At baseline all lesions showed typical clinical, dermoscopic and RCM criteria of AK. Three months after shave biopsy, all lesions presented clinically as normal skin (NS), but two lesions showed features suspicious for AK by RCM. After 12 months, one lesion of these two lesions changed into NS in RCM, whereas the other lesion progressed into clinical visible AK. At baseline, the two observers diagnosed 10 of 10 lesions correctly in RCM, after 3 months eight of 10 lesions and after 12 months all lesions were diagnosed correctly. CONCLUSIONS: Our results suggest that RCM might be a useful tool in the follow-up of AK after shave biopsy and might be used in inconclusive clinical and dermoscopic presentations of lesions after surgery or other treatment modalities.

In vivo confocal laser scanning microscopy in the diagnosis of melanocytic skin tumours.

Melanoma of the skin represents one of the greatest challenges in early or preventive detection. Whereas surgical excision in early stages of melanoma development is almost always curative, delayed recognition puts the patient at risk for destructive growth and death from disease once the tumour has progressed to competence for metastasis. The worldwide introduction of dermoscopy has led to improved diagnostic accuracy for melanocytic skin tumours. Whereas dermoscopy has probably reached the method's inherent potential diagnostic accuracy because of the lack of cellular level evaluation, further improvements could be expected by in vivo confocal laser scanning microscopy. In vivo confocal microscopy represents a novel imaging tool that allows the noninvasive examination of skin cancer morphology in real time at a 'quasihistopathological' resolution viewing microanatomical structures and individual cells. Numerous morphological confocal features of melanocytic skin tumours have been described and histopathological correlates of confocal structures have been previously elucidated. Recently, several studies have evaluated the diagnostic accuracy of in vivo confocal microscopy for melanocytic skin tumours, investigating approximately 50,000 tumour images. Remarkably, sensitivity superior to the diagnostic accuracy achieved with dermoscopy could be reached by this imaging modality. These studies represent a significant contribution to the body of research necessary for the evaluation and implementation of in vivo confocal microscopy in clinical practice to avoid many currently unnecessary biopsies. In vivo confocal microscopy probably augurs a sea change in the way we evaluate melanocytic skin tumours in the future and will ultimately move the art of histological diagnosis closer to the bedside.

Reflectance confocal microscopy of facial lentigo maligna and lentigo maligna melanoma: a preliminary study.

BACKGROUND: Facial lentigo maligna (LM) and lentigo maligna melanoma (LMM) may be difficult to diagnose clinically and dermoscopically. Reflectance confocal microscopy (RCM) enables the in vivo assessment of equivocal skin lesions at a cellular level. OBJECTIVES: To assess cytomorphological and architectural RCM features of facial LM/LMM. METHODS: Four women and eight men aged 58-88 years presenting with facial skin lesions suspicious of LM/LMM were included. In total, 17 lesion areas were imaged by RCM before biopsy. The histopathological diagnosis of LM was made in 15 areas; the other two were diagnosed as early LMM. RESULTS: A focal increase of atypical melanocytes and nests surrounding adnexal openings, sheets of mainly dendritic melanocytes, cord-like rete ridges at the dermoepidermal junction (DEJ) and an infiltration of adnexal structures by atypical melanocytes were found to be characteristic RCM features of facial LM/LMM. Areas with a focal increase of atypical melanocytes and nests surrounding adnexal openings were observed at the basal layer in three cases. The remaining cases displayed these changes at suprabasal layers above sheets of mainly dendritic melanocytes. Cord-like rete ridges at the DEJ and an infiltration of adnexal structures by atypical melanocytes were observed in all cases. Previously described criteria for RCM diagnosis of melanoma, such as epidermal disarray, pleomorphism of melanocytes and pagetoid spreading of atypical melanocytes, were additionally observed. CONCLUSIONS: We observed a reproducible set of RCM criteria in this case series of facial LM/LMM.

Confocal laser scanning microscopy-guided surgery for neurofibroma.

The neurofibromatoses comprise at least two separate genetic disorders with variable clinical features and an unpredictable course. The most common type, neurofibromatosis 1, is characterized by > or = 6 café-au-lait spots and the occurrence of neurofibromas, which may present as cutaneous, subcutaneous or plexiform lesions. Normally, excision of neurofibromas is only indicated in the presence of neurological symptoms, suspicion of malignancy or for exceptional cosmetic reasons. For a good functional and aesthetic result with the least danger of recurrence, the surgeon's goal is to excise as much tissue as necessary and as little tissue as possible. One of the main issues during the surgical procedure is to distinguish between neurofibroma and surrounding tissue. We report for the first time the use of confocal laser scanning microscopy to differentiate between neurofibroma and healthy skin.

Influence of route of gastric transposition on oxygen supply at cervical oesophagogastric anastomoses.

BACKGROUND: The microcirculation and oxygen supply at the oesophagogastric anastomosis are crucial factors that influence anastomotic healing after oesophagectomy. METHODS: Twenty-nine patients (mean age 61.7 years) underwent gastric transposition via an orthotopic (14) or retrosternal (15) route. Interstitial partial pressure of oxygen (PO2) of the stomach in the anastomotic region was measured during oesophagectomy and in the intensive care unit. Interstitial PO2 values were determined after ligation of the short gastric vessels, after ligation of the left gastric artery, after forming the conduit and after gastric transposition. Postoperative measurements were recorded during endotracheal intubation, while breathing oxygen by mask or through the nose, and while breathing air. RESULTS: Interstitial PO2 levels were significantly higher before ligation of the left gastric artery than after ligation (mean 76.1 (95 per cent confidence interval 54.9 to 103.1) versus 44.9 (24.6 to 77.1) mmHg; P = 0.001). Levels were also higher following orthotopic transposition compared with the retrosternal route (68.2 (44.0 to 118.8) versus 24.6 (10.7 to 39.4) mmHg; P = 0.001) and during each postoperative measurement period. No differences were found between the various oxygen supply systems. CONCLUSION: Oxygen supply at the anastomosis of the gastric conduit reaches higher levels after orthotopic than retrosternal gastric transposition.

Onychomycosis: a new emerging infectious disease in childhood population and adolescents. Report on treatment experience with terbinafine and itraconazole in 36 patients.

BACKGROUND: Onychomycosis is a rare disease in children with an estimated prevalence ranging from 0% to 2.6%. Thus far, only limited experience with itraconazole and terbinafine treatment in children with onychomycosis is available in the literature. AIM OF THE STUDY: Evaluation of treatment experience with itraconazole or terbinafine in childhood onychomycosis. SUBJECTS: Thirty-six children and adolescents (aged 4-17 years, 18 males and 18 females) with clinical and mycologically proven onychomycosis were enrolled in the present study. METHODS AND OUTCOME: In 27 of 36 patients, the causative agent (Trichophyton rubrum in 26 cases and Trichophyton interdigitale in one patient) could be identified by means of cultivation. Nineteen patients were treated with itraconazole 200 mg once daily for 12 weeks, and 17 patients were treated with terbinafine for 12 weeks in a dosage according to their body weight, respectively. Clinical cure was achieved within 1 to 5 months after discontinuation in all patients treated with itraconazole and in all but two patients after cessation of terbinafine treatment. Neither in the itraconazole nor in the terbinafine group were serious adverse events reported. Clinical cure was achieved within 1 to 5 months after discontinuation in all patients treated with itraconazole and in all but two patients after cessation of terbinafine treatment. Neither in the itraconazole nor in the terbinafine group were serious adverse events reported. CONCLUSION: To our experience, a mycological and clinical cure appears in children in a shorter time after treatment discontinuation (average 2-5 months) compared with adults. Itraconazole and terbinafine seem to be safe and effective in childhood onychomycosis; therefore, these antifungals seem to be potential alternatives to griseofulvin.

Automatic identification of diagnostic significant regions in confocal laser scanning microscopy of melanocytic skin tumors.

OBJECTIVES: Confocal laser scanning microscopy (CLSM) is used for quick medical checkups. The aim of this study is to check the discrimination power of texture features for the automatic identification of diagnostic significant regions in CLSM views of skin lesions. METHODS: In tissue counter analysis (TCA) the images are dissected in equal square elements, where different classes of features are calculated out. Features defined in the spatial domain are based on histogram (grey level distribution) and co-occurrence matrix (grey level combinations). The features defined in the frequency domain are based on spectral properties of the wavelet Daubechie 4 transform (texture exploration at different scales) and the Fourier transform (global texture properties are localized in the spectrum). Hundred cases of benign common nevi and malignant melanoma were used as the study set. Classification was done with CART (Classification and Regression Trees) analysis which splits the set of square elements into homogenous terminal nodes and generates a set of splitting rules. RESULTS: Features based on the wavelet transform provide the best results with 96.0% of correctly classified elements from benign common nevi and 97.0% from malignant melanoma. The classification results are relocated to the images by use of the splitting rules as diagnostic aid. The discriminated square elements are highlighted in the images, showing tissue with features in good accordance with typical diagnostic CLSM features. CONCLUSION: Square elements with more than 80% of discrimination power enable the identification of diagnostic highly significant parts in confocal microscopic views of malignant melanoma.

[Safety and efficacy of interferon alfa-2b in the adjuvant treatment of uveal melanoma]. / Verträglichkeit und Wirksamkeit einer adjuvanten Interferon-alfa-2b-Behandlung beim Aderhautmelanom.

PURPOSE: In this manuscript, the safety and efficacy of adjuvant interferon alfa 2b treatment of uveal melanoma is described. PATIENTS AND METHODS: A total of 39 patients (23 male and 16 female, mean age 56.5 years, range 35-78 years) with uveal melanoma were treated with interferon alfa 2b, 3 million units three times a week subcutaneously for 1 year after therapy of the primary tumor. In all patients age, gender, primary melanoma data, therapeutic interventions, treatment side effects and outcome were documented. RESULTS: Of the 39 patients, 31 (80%) finished the treatment as scheduled after 1 year. In 18 patients (46%) the initial dose had to be reduced due to leucopenia, thrombopenia, cardiac symptoms, elevated of liver function or vertigo (WHO grade I-III). In eight patients, therapy had to be withdrawn because of serious side effects (five patients) and the appearance of metastases (three patients). Neither a univariate approach nor a multivariate approach could show a protective effect of interferon treatment on survival. CONCLUSIONS: Adjuvant treatment of uveal melanoma with interferon alfa should be abandoned until the question of dose and administration for cutaneous melanoma is solved.

The acute coronary syndrome--pre-hospital diagnostic quality.

BACKGROUND AND OBJECTIVE: In the Austrian emergency medical service (EMS), emergency medical technician-staffed and physician-staffed vehicles are in operation. Patients with suspected acute coronary syndromes (ACS) are treated in the pre-hospital phase and transported to the hospital by an emergency physician (EP). This study evaluates the diagnostic performance of EPs in ACS and the impact of this emergency system on the outcome of ACS in an urban area. DESIGN: Retrospective case control study. METHODS: All protocol sheets from the emergency physicians were searched for the diagnosis of ACS. The database of the emergency department (ED) was searched for patients with ACS as an admission diagnosis or ACS as discharge diagnosis. For patients admitted to an intensive care unit (ICU), the medical history from the ICU was reviewed. According to the diagnosis and the aggressiveness of therapy, patients were divided in five categories of severity at each stage of care (pre-hospital category, ED category, ICU category). RESULTS: A total of 3585 patients was analysed. Only 17.8% of the patients with ACS as the admission diagnosis and 20.3% of the patients with ACS as the discharge diagnosis were transported by an EP. 46.8% of the ACS diagnosis by EPs were confirmed in hospital. Patients transported by EPs showed a higher all-cause mortality in hospital (1.6% vs. 0.6%; p=0.011). There was no significant correlation between the pre-hospital category of patients treated by EPs and the ED category. When a 12-lead-electrocardiogram was recorded, the correlation improved slightly (rho: 0.139; p=0.006). CONCLUSIONS: The percentage of ACS patients transported to hospital by an EP is very low, and EPs seem to be "over-aware" in the diagnosis of ACS.

Ultraviolet irradiation induces acute changes in melanocytic nevi.

Ultraviolet (UV) light represents one of the factors that might play a role in the initiation and promotion of malignant transformation of human melanocytes. To determine the short-term effects of UV irradiation on melanocytic nevi in vivo, we investigated one half of symmetric melanocytic nevi after a single UV exposure with double the patient's minimal erythema dose. This half was compared with the nonirradiated, shielded half of the same nevus. The different parts were examined histologically for differences and immunohistochemically for the presence of HMB-45 antigen and proliferating cell nuclear antigen. The features were assessed quantitatively by image analysis. One week after the single UV irradiation, we observed a significant increase of suprabasally located melanocytes and a markedly enhanced expression of HMB-45, whereas proliferative activity of the cells was unchanged. In nevi that were excised 2 or 3 weeks after irradiation, no significant differences were observed between the irradiated and the nonirradiated part. The results indicate that a single UV irradiation may induce transient melanocytic activation with morphologic and histologic changes. Although these data do not formally assess resemblance to melanoma, these changes may be similar to those of melanoma in situ.

Langerhans cell density in epithelial skin tumors correlates with epithelial differentiation but not with the peritumoral infiltrate.

We investigated the intraepithelial density of Langerhans cells in 17 epithelial skin tumors by immunohistologic and morphometric methods. There was a significant difference between seborrheic keratosis (Langerhans cell density 431 +/- 31/mm2; normal epidermis: 378 +/- 20/mm2), basal cell carcinoma (28 +/- 6/mm2), and squamous cell carcinoma (100 +/- 21/mm2). No correlation was found between the Langerhans cell density and the number of intraepithelial T lymphocytes or the extent of the peritumoral inflammatory infiltrate. A significant inverse correlation was demonstrated between mean nuclear area of the epithelial tissue and the Langerhans cell density (r = -0.7; p less than 0.05). These data indicate that the number of Langerhans cells does not influence the extent of the antitumoral immune response. The correlation with the level of epithelial differentiation may be due to different homing conditions.

Computer simulations of histologic patterns in melanoma using a cellular automaton provide correlations with prognosis.

Computer simulations have been used frequently in the life sciences to investigate the mechanisms of morphologic pattern formation. The cellular automaton program SMN5 is designed to simulate tumor growth and to estimate biologic properties by comparing real tumor patterns with computer-simulated reference patterns. This method was applied to 195 cases of primary melanoma of the skin. S-100-stained sections were evaluated by image analysis and compared statistically to a reference set of 4000 simulated patterns. Estimates of tumor cell proliferation, motility, cell loss, cohesion, stroma destruction, and intercellular signals (autocrine and paracrine factors affecting growth, motility, and cell loss) were calculated. Twelve of 18 estimated parameters correlated significantly with tumor progression, as indicated by vertical tumor thickness (linear regression analysis: p < or = 0.05), and 13 of 18 parameters carried prognostic significance (log rank test: p < or = 0.05). Poor prognosis was associated particularly with a pronounced increase in the estimates of proliferation, tumor cell motility, and stromal degradation. Poor prognosis was also associated with a decrease in the estimates of cell loss, tumor cell cohesion, and paracrine growth factor dependence. In multivariate analysis using Cox's proportional hazard model, stromal degradation and motility showed prognostic information in addition to conventional prognostic parameters. The study shows that analytical comparison of real tumors with computer-simulated patterns of a cellular automaton facilitates a functional interpretation of tumor morphology, which carries prognostic significance in cutaneous melanoma.

Epidermal Langerhans cells in myelodysplastic syndromes are abnormal.

The myelodysplastic syndromes (MDS) represent clonal disorders of the hematopoietic stem cell that are associated with quantitative and qualitative disturbances of the peripheral blood cells and a high risk for the transition to overt leukemia. As epidermal Langerhans cells (LC) are bone-marrow-derived cells, we were interested to see whether they are altered in patients with MDS. Epidermal sheets were prepared from biopsies taken from the thighs of nine patients with MDS and five control persons and processed for immunoperoxidase staining of CD1a antigens. The density and morphology of CD1a+ cells (i.e., LC) was evaluated by visual assessment as well as automatic image analysis. The density of LC was reduced in seven of nine patients (range, 30-75% of normal), whereas the morphology of LC appeared to be altered in all MDS patients in that the LC displayed large and bizarre cell bodies with only a few and often abnormally long dendrites. The HLA-DR expression by LC was not altered, as shown by double immunofluorescence staining of CD1a and HLA-DR antigens. Ultrastructurally, LC again appeared enlarged and often presented with bizarre nuclei, yet displayed no other abnormalities. Our findings suggest that LC are abnormal in MDS and might even indicate a more wide-spread involvement of the dendritic cell lineage in this syndrome.

Nuclear size and shape parameters correlate with proliferative activity in cutaneous melanocytic tumors.

Proliferative activity and morphometric data have previously been shown to be related with the degree of malignancy in melanocytic skin tumors. In the present study, the proliferative activity, as defined by Ki 67 monoclonal antibody (reactive with all actively cycling cells), has been determined by immunohistologic and morphometric methods in cutaneous melanocytic tumors. Quantitative morphologic features of Ki 67-positive and Ki 67-negative nuclei were separately assessed using computer-assisted image analysis. Comparing morphometric features and proliferative activity, the most significant correlation was found between mean nuclear volume and the percentage of Ki 67-positive nuclei in each lesion (linear regression analysis: r = 0.73; p = less than 0.05). On multidimensional discriminant analysis, tumors with high proliferative activity (more than 5 X 10(3) Ki 67-positive cells per mm3 tumor tissue) were detected at a specificity of 92% and a sensitivity of 75%. Within one lesion, Ki 67-positive nuclei showed an increase in nuclear volume (Wilcoxon test: p = less than 0.05), a more spheroid shape (p = less than 0.05), and a wider dispersion of nuclear volume values (Siegel-Tukey test: p = less than 0.05) than negative nuclei. Discriminant analysis on the basis of nuclear volume density functions facilitated an estimation of the proliferative state (resting or cycling) of a given nucleus. The results are consistent with increased cellular synthetic activity in highly proliferating lesions and particularly in actively cycling cells. The association of proliferative activity and quantitative nuclear features may be helpful in the interpretation of morphometric studies concerning melanocytic skin tumors.

Entry into afferent lymphatics and maturation in situ of migrating murine cutaneous dendritic cells.

An important property of dendritic cells (DC), which contributes crucially to their strong immunogenic function, is their capacity to migrate from sites of antigen capture to the draining lymphoid organs. Here we studied in detail the migratory pathway and the differentiation of DC during migration in a skin organ culture model and, for comparison, in the conventional contact hypersensitivity system. We report several observations on the capacity of cutaneous DC to migrate in mouse ear skin. (i) Upon application of contact allergens in vivo the density of Langerhans cells in epidermal sheets decreased, as determined by immunostaining for major histocompatibility complex class II, ADPase, F4/80, CD11b, CD32, NLDC-145/DEC-205, and the cytoskeleton protein vimentin. Evaluation was performed by computer assisted morphometry. (ii) Chemically related nonsensitizing or tolerizing compounds left the density of Langerhans cells unchanged. (iii) Immunohistochemical double-staining of dermal sheets from skin organ cultures for major histocompatibility complex class II and CD54 excluded blood vessels as a cutaneous pathway of DC migration. (iv) Electron microscopy of organ cultures revealed dermal accumulations of DC (including Birbeck granule containing Langerhans cells) within typical lymphatic vessels. (v) Populations of migrating DC in organ cultures upregulated markers of maturity (the antigen recognized by monoclonal antibody 2A1, CD86), but retained indicators of immaturity (invariant chain, residual antigen processing function). These data provide additional evidence that during both the induction of contact hypersensitivity and in skin organ culture, Langerhans cells physically leave the epidermis. Both Langerhans cells and dermal DC enter lymphatic vessels. DC mature while they migrate through the skin.

Quantitative evaluation of melanoma cell invasion in three-dimensional confrontation cultures in vitro using automated image analysis.

Tumor invasion is a crucial feature of tumor growth in vivo. Confrontation cultures of multicellular melanoma spheroids and embryonic chick heart fragments provide a model for invasive growth in vitro. We have developed an image analysis method, which facilitates the objective measurement of tumor cell invasion in this model. Cryostat sections of confrontation cultures were immunohistochemically stained with an antiserum directed against the stromal component for automated recognition of the stroma tissue. The slides were automatically processed by a grey level based computerized image analysis system. On Spearman's rank correlation test, 25 out of 39 parameters correlated with the reference value of invasion, which was derived from the subjective evaluation of five independent observers. Two parameters combining the stroma margin and the total amount of stroma tissue completely reproduced the judgement of the morphologists in our test set. The quantitative evaluation of tumor invasion in vitro by automated image analysis may be helpful in pharmacologic and pathogenetic studies of tumor growth.

Topical treatment with liposomes containing T4 endonuclease V protects human skin in vivo from ultraviolet-induced upregulation of interleukin-10 and tumor necrosis factor-alpha.

Exposing human skin to ultraviolet radiation causes DNA damage, sunburn, immune alterations, and eventually, skin cancer. We wished to determine whether liposomes containing a DNA repair enzyme could prevent any of the acute effects of irradiation when applied after ultraviolet exposure. Fifteen human patients with a prior history of skin cancer were exposed to two minimal erythema doses of ultraviolet radiation on their buttock skin. Liposomes containing T4 endonuclease V or heat-inactivated enzyme were applied immediately and at 2, 4, and 5 h after ultraviolet irradiation. Transmission electron microscopy after anti-T4 endonuclease V-staining and immunogold labeling on biopsies taken at 6 h after ultraviolet exposure revealed that the enzyme was present within cells in the skin. Immunohistochemical DNA damage studies suggested a trend toward improved DNA repair at the active T4 endonuclease V liposome-treated test sites. Although the active T4 endonuclease V liposomes did not significantly affect the ultraviolet-induced erythema response and microscopic sunburn cell formation, they nearly completely prevented ultraviolet-induced upregulation of interleukin-10 and tumor necrosis factor-alpha RNA message and of interleukin-10 protein. These studies demonstrate that liposomes can be used for topical intracellular delivery of small proteins to human skin and suggest that liposomes containing DNA repair enzymes may provide a new avenue for photoprotection against some forms of ultraviolet-induced skin damage.