[Moschella island flap for coverage of thumb defects]. / Homodigitaler Insellappen nach Moschella zur Defektdeckung am Daumen.

OBJECTIVE: The homodigital island flap according to Moschella is used to cover palmar and radiodorsal soft tissue defects of the thumb. INDICATIONS: Soft tissue reconstruction of the thumb with exposed bone and/or tendon if reconstruction of the soft tissue is required. CONTRAINDICATIONS: Extensive scars in the harvesting area of the flap; vascular occlusions of the radial artery. SURGICAL TECHNIQUE: After Doppler sonographic imaging of the origin of the vessel radially at the thumb, the pivot point is determined. The flap is then cut from ulnar to radial, including the dorsoradial artery of the thumb. Removal of the origin of the vessel from the radial artery in the tabatiére and preparation from proximal to distal to the pivot point of the flap at the junction of the artery with the palmar vessels. If necessary, a part of the skin can remain palmar to the pivot point to improve venous drainage. The donor site of the flap can primarily close defect sizes up to 5â€¯× 4 cm. POSTOPERATIVE MANAGEMENT: Until wound healing, soft cotton dressing should protect the vascular pedicle from pressure; immobilisation on a palmar splint may be helpful. RESULTS: In 9 men, sufficient soft tissue reconstruction of thumb defects could be achieved with the described Moschella flap. Eight patients had additional injuries to the fingers and hand.

Early and late-recorded predictors of health-related quality of life of burn patients on long-term follow-up.

BACKGROUND: Unintentional and intentional burn injuries vary across age groups, gender, income, and global region. In high-income countries, the trend over the last several years has been a reduction in burn incidence, burn severity, length of hospital stay, and mortality rate. However, there is a lack of data on predictors of the health-related quality of life (HRQoL) of major burn survivors extending beyond a follow-up period of 10 years. PATIENTS AND METHODS: This single-center cross-sectional study is considering 42 long-term severe burn survivors with deep partial thickness burns and an affected total body surface area (TBSA) of ≥20%. For study eligibility design a minimum follow-up of 10 years was obligatory. Entitled individuals were asked to fill in the generic Short Form 36 (SF-36) questionnaire. The physical (PCS) and mental (MCS) component scores of the SF-36 were used as the primary outcome variables. Putative predictor variables were drawn from medical records. Burn-specific functionality and scar tissue quality were assessed using the Burn Specific Health Scale-Brief (BSHS-B) questionnaire and the Patient and Observer Scar Assessment Scale (POSAS), respectively. Correlation between putative predictor variables and SF-36 norm scores were evaluated by Pearson- and Point-Biserial correlation as well as multivariate linear regression. The SF-36 norm scores were compared to the general German population. RESULTS: Mean follow-up was 14 (±3) years with a minimum and maximum of 10 and 28 years, respectively. Mean age at the time of the incident was 37 (±17) years. The majority of individuals were male (74%). The mean burn size was 39 (±17) % (TBSA) with 76% of the individuals showing a full thickness burn. SF-36 norm scores were not statistically different from the general population. Statistically significant independent predictor variables of the physical summary score were: age at the time of the injury (-0.381), time since injury (-0.466), length of hospital stay (-0.356), limb amputation (-0.318), unemployment (-0.433), work (0.593), hand function (0.601), body image (0.518), affect (0.355), simple abilities (0.602), burns involving the hands (-0.339) and back (-0.343), POSAS patient- (-0.521) and observer scores (-0.483). In multivariate analysis, work (4.315), the POSAS Score (-2.082) and the age at the time of the incident (-0.242) were statistically significant predictors. Statistically significant independent predictor variables of the mental summary score were: duration of mechanical ventilation (-0.459), hand function (0.415), body image (0.502), sexual activity (0.625), social support (0.542), burns involving the back (-0.315) and affect (0.692). In multivariate analysis, affect (13.844) and the length of mechanical ventilation (-0.115) were statistically significant independent predictor variables. CONCLUSION: Ten years after the burn incident, the quality of life was on average comparable to the one in the general population. Multiple variables seem to influence the physical and mental long-term outcome. Herein presented data may support in adapting and designing follow-up strategies tailored to a patient's burn-specific circumstances.

Sternal osteomyelitis: long-term results after pectoralis muscle flap reconstruction.

BACKGROUND: Reports regarding long-term follow-up including quality-of-life assessment, pulmonary function, and donor-site morbidity after operative treatment for sternal osteomyelitis are rare. METHODS: Data for 69 consecutive patients were acquired from patients' charts and contact with patients and general practitioners, with special reference to treatment and clinical course. Twenty-four patients were interviewed and physically examined (mean follow-up, 4 years; range, 1 to 9 years). Fifteen of the patients underwent pulmonary function tests, cine magnetic resonance imaging, and pectoralis strength testing using a dynamometer to record butterfly arm compressive movements. Statistical analysis was performed to identify factors influencing wound healing and survival. RESULTS: Mortality rates were 10.1 percent at 30 days, 18.5 percent at 1 year, and 27.0 percent at 5 years (n = 69). In 36.2 percent of the patients, wound-healing difficulties requiring reoperation occurred. Independent of the extent of sternal resection, dynamic pulmonary function values were decreased compared with normal values (n = 15). Dynamometer assessment revealed decreases of 1.5 percent in dynamic maximum strength, 9.7 percent in maximum isometric strength, and 47.2 percent in strength endurance compared with the healthy age-matched control group. Magnetic resonance imaging showed no sign of recurrent osteomyelitis (n = 15). Muscle function was preserved in 93 percent of the patients. Eighty-three percent of the interviewed patients considered their general condition better and 17 percent considered it worse than before the treatment (n = 24). CONCLUSIONS: Pectoralis muscle transfer represents a safe and simple procedure. Although strength loss and pain are considerable, quality of life is improved significantly. Pulmonary function impairment is most likely not exclusively attributable to the muscle transfer or the sternum resection.

The pedicled gastrocnemius muscle flap: a review of 218 cases.

BACKGROUND: Data regarding donor-site morbidity, postoperative clinical course, and functional and aesthetic outcome after gastrocnemius muscle flaps are rare. METHODS: Data regarding 218 consecutive patients treated with gastrocnemius muscle flaps were acquired from patients' charts and from contact with patients, with special reference to treatment and clinical course. Eighty-two were interviewed with a standardized questionnaire, 40 were examined physically, and 34 underwent dynamometric muscle function tests. RESULTS: The authors observed wound-healing difficulties in 7 percent, wound infections in 4 percent, and one flap loss; 4.5 percent of the lateral gastrocnemius patients suffered from postoperative palsy of the peroneal nerve. Eighty-seven percent were not significantly limited walking on even ground, but only 42 percent could run, and 40 percent complained about pain when walking more than 200 m. The average range-of-motion deficit in the ankle joint for flexion and extension was 11 percent and 10 percent, respectively. The maximal plantar flexion force in the ankle joint of the operated leg was 76.2 percent. Strength endurance was reduced approximately 24.4 percent in the operated leg compared with the nonaffected side. CONCLUSIONS: Gastrocnemius muscle transfer represents a safe and simple procedure in the treatment of lower leg defects and in limb preservation. The strength loss and functional impairment (and sensation disorders) are considerable but may not be exclusively attributable to the muscle transfer but rather the result of the preceding trauma, infection, or tumor resection. The donor-site morbidity is well tolerated by the majority of the patients.

Reconstruction of the thoracic wall-long-term follow-up including pulmonary function tests.

PURPOSE: Thoracic wall reconstructions have become a standard procedure for the reconstructive plastic surgeon in the larger hospital setting, but detailed reports about long-term results including pulmonary function and physical examination are rare. MATERIALS AND METHODS: The data of 92 consecutive patients with full thickness chest wall resections were acquired from patient's charts and contact to patients, their relatives or general practitioners, with special reference to treatment and clinical course. At a mean follow-up of 5.5 years, 36 patients were examined physically and interviewed. Twenty-seven of them underwent additional pulmonary function tests. Kaplan-Meier method was used to calculate survival. Regression tests were undertaken to identify factors influencing the outcome. RESULTS: Postoperative complications were observed in 42.4%, but neither mesh implantation nor the size of the defect contributed significantly. The 5-year mortality was worse for patients with recurrent mamma carcinoma (90.6%) than for patients with soft tissue sarcoma (56.3%). No medical history or operation parameter (resection size and localization) besides the general patients' conditions increased mortality. Pulmonary function parameters were only moderately reduced and not significantly affected by the resections' size or its localization. Majority of patients suffer from sensation disorders and motion-dependent pain, which contributed significantly to hypoxemia. Quality-of-life parameters were significantly reduced compared to the healthy control group but similar to the control group with cancer according to the Short Form-36 protocol. We could not detect a relevant decrease in quality of life comparing post- to preoperative values. CONCLUSIONS: Thoracic wall reconstruction provides sufficient thoracic wall stability to maintain pulmonary function, but postoperative pain and sensation disorders are considerable. However, chest wall repair can contribute to palliation and even cure after full-thickness resections.

Heterogeneous in vitro effects of doxorubicin on gene expression in primary human liposarcoma cultures.

BACKGROUND: Doxorubicin is considered one of the most potent established chemotherapeutics in the treatment of liposarcoma; however, the response rates usually below 30%, are still disappointing. This study was performed to identify gene expression changes in liposarcoma after doxorubicin treatment. METHODS: Cells of 19 primary human liposarcoma were harvested intraoperatively and brought into cell culture. Cells were incubated with doxorubicin for 24 h, RNA was isolated and differential gene expression was analysed by the microarray technique. RESULTS: A variety of genes involved in apoptosis were up and down regulated in different samples revealing a heterogeneous expression pattern of the 19 primary tumor cell cultures in response to doxorubicin treatment. However, more than 50% of the samples showed up-regulation of pro-apoptotic genes such as TRAIL Receptor2, CDKN1A, GADD45A, FAS, CD40, PAWR, NFKBIA, IER3, PSEN1, RIPK2, and CD44. The anti-apoptotic genes TNFAIP3, PEA15, Bcl2A1, NGFB, and BIRC3 were also up-regulated. The pro-apoptotic CD14, TIA1, and ITGB2 were down-regulated in more than 50% of the tumor cultures after treatment with doxorubicin, as was the antiapoptotic YWHAH. CONCLUSION: Despite a correlation of the number of differentially regulated genes to the tumor grading and to a lesser extent histological subtype, the expression patterns varied strongly; however, especially among high grade tumors the responses of selected apoptosis genes were similar. The predescribed low clinical response rates of low grade liposarcoma to doxorubicin correspond to our results with only little changes on gene expression level and also divergent findings concerning the up- and down-regulation of single genes in the different sarcoma samples.

Trocar injury of the retroperitoneal vessels followed by life-threatening postischemic compartment syndrome of both lower extremities.

BACKGROUND: Lesions of the intra-abdominal organs and vessels caused by trocars and Verres needles are rare but serious complications during laparoscopic surgery. REPORT: We report an unusual case of left common iliac artery and inferior vena cava injury during laparoscopy. This lesion was followed by a bilateral postischemic compartment syndrome of the lower extremities. The patient sustained massive rhabdomyolysis, renal failure, peroneal nerve palsy, and functional loss of the lower extremities. CONCLUSION: Postischemic tissue swelling should be recognized as early as possible because it is a life-threatening condition that necessitates immediate treatment.

A liposome hydrogel with polyvinyl-pyrrolidone iodine in the local treatment of partial-thickness burn wounds.

Local treatment of burn injuries with conventional anti-infective preparations does not provide the moist environment that promotes fast wound healing. In a randomized controlled trial the effects of liposome polyvinyl-pyrrolidone-iodine (PVP-I) hydrogel, a novel formulation of PVP-I in a liposome hydrogel with high water-binding capacity, were investigated in 43 patients with partial-thickness burn wounds in an intraindividual comparison with a conventional silver-sulfadiazine cream. Treatment with liposome PVP-I hydrogel resulted in significantly faster complete healing of the burn wounds compared with silver-sulfadiazine cream (9.9 +/- 4.5 days versus 11.3 +/- 4.9; P < 0.015). The cosmetic result (smoothness, elasticity, appearance) was rated as excellent for 37.0% of study wounds with liposome PVP-I hydrogel compared with 13.0% of wounds treated with silver-sulfadiazine cream. Local tolerability was good; handling and change of dressing were rated as easy. Local treatment with liposome PVP-I hydrogel thus provides fast wound healing with a favorable cosmetic result.

Analysis of neovascularization of PEGT/PBT-copolymer dermis substitutes in balb/c-mice.

INTRODUCTION: A fundamental prerequisite for using degradable synthetic biopolymers as composite skin substitutes is the ability to establish vascular tissue. PEGT/PBT block-copolymer matrices have previously been shown as a favorable dermal substitute. In this study, quantitative data on neovascularization of PEGT/PBT block-copolymer matrices are presented. MATERIALS AND METHODS: PEGT/PBT-block-copolymer discs of three different pore diameters (1: < 75 microm, 2: 75-212 microm, 3: 250-300 microm) were implanted into dorsal skinfold chambers of balb/c mice. Histological sections were evaluated 7, 14, and 21 days post implantation by light and scanning electron microscopy. Blood vessel analysis was performed by means of digital image analysis (n = 288) of hematoxylin/eosin stained sections within apical (AOF) and basal (BOF) observation fields of the matrices. RESULTS: Twenty-one days after implantation the density of blood vessels within the BOF of the scaffolds with a pore size of 75-212 and 250-300 microm were 4.6 +/- 0.45 and 5.8 +/- 0.62 (mean +/- S.E.M.; blood vessel profiles (BVF)), respectively. In <75 microm scaffolds, smaller numbers of BVF were found (4.2 +/- 0.39). In contrast, the evaluation within the AOF revealed significantly higher numbers of BVF in 75-212 microm group (3.5 +/- 0.49) and 250-300 microm group (4.5 +/- 0.66) as compared to the < 75 microm group (2.3 +/- 0.48). CONCLUSION: There is evidence that the three-dimensional structure of PEGT/PBT-block-copolymer (pore size structure) influences neovascularization. The porous structures of copolymer matrices with adequate interconnection of pores (pore sizes of 75-212 and 250-300 microm) are characterized by faster ingrowth of vascular tissue.

Response rate of fibrosarcoma cells to cytotoxic drugs on the expression level correlates to the therapeutic response rate of fibrosarcomas and is mediated by regulation of apoptotic pathways.

BACKGROUND: Because of the high resistance rate of fibrosarcomas against cytotoxic agents clinical chemotherapy of these tumors is not established. A better understanding of the diverse modes of tumor cell death following cytotoxic therapies will provide a molecular basis for new chemotherapeutic strategies. In this study we elucidated the response of a fibrosarcoma cell line to clinically used cytostatic agents on the level of gene expression. METHODS: HT1080 fibrosarcoma cells were exposed to the chemotherapeutic agents doxorubicin, actinomycin D or vincristine. Total RNA was isolated and the gene expression patterns were analyzed by microarray analysis. Expression levels for 46 selected candidate genes were validated by quantitative real-time PCR. RESULTS: The analysis of the microarray data resulted in 3.309 (actinomycin D), 1.019 (doxorubicin) and 134 (vincristine) probesets that showed significant expression changes. For the RNA synthesis blocker actinomycin D, 99.4% of all differentially expressed probesets were under-represented. In comparison, probesets down-regulated by doxorubicin comprised only 37.4% of all genes effected by this agent. Closer analysis of the differentially regulated genes revealed that doxorubicin induced cell death of HT1080 fibrosarcoma cells mainly by regulating the abundance of factors mediating the mitochondrial (intrinsic) apoptosis pathway. Furthermore doxorubicin influences other pathways and crosstalk to other pathways (including to the death receptor pathway) at multiple levels. We found increased levels of cytochrome c, APAF-1 and members of the STAT-family (STAT1, STAT3), while Bcl-2 expression was decreased. Caspase-1, -3, -6, -8, and -9 were increased indicating that these proteases are key factors in the execution of doxorubicin mediated apoptosis. CONCLUSION: This study demonstrates that chemotherapy regulates the expression of apoptosis-related factors in fibrosarcoma cells. The number and the specific pattern of the genes depend on the used cytotoxic drug. The response rates on the gene expression level, i.e. the number of genes regulated by the drugs actinomycin D, doxorubicin and vincristine, correlate to the clinical effectiveness of the drugs. Doxorubicin seems to exert its cytotoxic mechanism by regulating genes, which are involved in several different apoptosis regulating pathways. The exact knowledge of the genes affected by the drugs will help to understand the diverse modes of soft tissue sarcoma cell death in response to cytotoxic therapies.

Dedifferentiated liposarcoma with extensive lymphoid component.

An unusual variant of dedifferentiated liposarcoma with extensive lymphocytic component is described. A 71-year-old patient suffered from a relapse of an atypical lipomatous tumor/well-differentiated liposarcoma with early micronodular (low-grade) dedifferentiation, which had been resected 4 years before. The relapse revealed features of a dedifferentiated liposarcoma with spindle-cell, partly pleomorphic dedifferentiation and osseous metaplasia. Clearly separated from the spindle-cell areas, an extensive homogeneously dense lymphoid (lymphocytic) tumor-component was evident, with relative abrupt transition to the well-differentiated liposarcoma component. Using immunohistochemistry and PCR, the lymphoid ("lymphoma-like") infiltrate proved to be a polyclonal lymphocytic proliferation. Fluorescence in situ hybridization (FISII) analysis revealed no signs of MDM2- and CDK4-gene amplification in the lymphoid areas, although within this mononuclear lymphoid population, large polymorphic nuclei displayed an amplified number of MDM2/CDK4 gene copies, indicating the presence of truly dedifferentiated tumor cells within the lymphoid component. The results favor a reactive lymphocytic infiltration versus a neoplastic one, which might be caused for example by chemoattractive agents. An extensive lymphoid "overgrowth" must be considered within the spectrum of unusual variants and in the differential diagnosis of dedifferentiated liposarcoma.

Preoperative oral supplementation with long-chain Omega-3 fatty acids beneficially alters phospholipid fatty acid patterns in liver, gut mucosa, and tumor tissue.

BACKGROUND: The uptake of omega-3 polyunsaturated fatty acids (PUFAs) into the liver, gut mucosa, and tumor tissue and plasma levels after preoperative administration of supplemented enteral nutrition was investigated in patients with malignancies of the upper gastrointestinal tract. The objective of the study was to evaluate the incorporation of preoperatively administrated PUFAs, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) into cell phospholipids. METHODS: Patients undergoing major gastrointestinal surgery (n = 40) were prospectively randomized to receive a PUFA-supplemented liquid oral diet 5 days preoperatively or an isocaloric control diet. The planned diet intake was 1000 mL/d providing 3.7 g of PUFA. The diet was given in addition to the usual hospital diet. The phospholipid fractions in plasma were analyzed on the day of surgery. Tissue samples of liver, gut mucosa (small intestine), and tumor were taken during surgery and homogenized. EPA and DHA content was analyzed using liquid gas chromatography. RESULTS: Both patient groups (PUFA group: n = 20; control group: n = 20) were similar in age, weight, and surgical procedures. As compared with the control group, the PUFA group had significantly increased levels of EPA in liver tissue (0.4 vs 1.3 weight %), gut mucosa (0.3 vs 1.0 weight %), and tumor tissue (0.3 vs 0.8 weight %). Also, the DHA levels in the PUFA group were significantly higher than the control group: liver tissue (4.1 vs 7.5 weight %), gut mucosa (2.1 vs 3.7 weight %) and tumor tissue (1.9 vs 4.2 weight %). CONCLUSIONS: This study suggests that administration of PUFA-enriched diets leads to increased incorporation of EPA and DHA not only in liver and gut mucosa tissue, but also in tumor tissue in patients with solid gastrointestinal tumors. Thus, preoperative administration of oral PUFA-enriched diets could have an impact on the postoperative inflammatory response after major abdominal surgery.

Inhibition of early steps in the lentiviral replication cycle by cathelicidin host defense peptides.

BACKGROUND: The antibacterial activity of host defense peptides (HDP) is largely mediated by permeabilization of bacterial membranes. The lipid membrane of enveloped viruses might also be a target of antimicrobial peptides. Therefore, we screened a panel of naturally occurring HDPs representing different classes for inhibition of early, Env-independent steps in the HIV replication cycle. A lentiviral vector-based screening assay was used to determine the inhibitory effect of HDPs on early steps in the replication cycle and on cell metabolism. RESULTS: Human LL37 and porcine Protegrin-1 specifically reduced lentiviral vector infectivity, whereas the reduction of luciferase activities observed at high concentrations of the other HDPs is primarily due to modulation of cellular activity and/ or cytotoxicity rather than antiviral activity. A retroviral vector was inhibited by LL37 and Protegrin-1 to similar extent, while no specific inhibition of adenoviral vector mediated gene transfer was observed. Specific inhibitory effects of Protegrin-1 were confirmed for wild type HIV-1. CONCLUSION: Although Protegrin-1 apparently inhibits an early step in the HIV-replication cycle, cytotoxic effects might limit its use as an antiviral agent unless the specificity for the virus can be improved.

In vivo visualization of platelet/endothelium cell interaction in muscle flaps.

Increasing evidence underlines the substantial pathophysiological impact of platelets on the development of ischemia/reperfusion injury (I/R) in flaps. Methods for studying dynamic platelet mechanisms in flaps in vivo are not available. The aim of this study was to develop a model enabling quantitative analysis of platelet kinetics and platelet-endothelium cell interaction within the microcirculation of muscle flaps in vivo. Balb/c mice (n = 16) were anesthetized, and an epigastric muscle flap was prepared. Autologous platelets were separated from blood donor animals (n = 16) and labeled ex vivo by means of rhodamine-6-G. After I/R (90 minutes' clamping, 10 minutes' reperfusion), the platelets were administered intra-arterially (i.a.). Microhemodynamics and kinetics of platelets were investigated by intravital fluorescence microscopy. I/R of muscle flaps induced disturbances in microcirculation. The number of rolling platelets, as well as platelets adhering to the inner vessel wall of venules, was increased in the ischemia group. Using intravital fluorescence microscopy, platelet kinetics were analyzed directly in flap microcirculation in vivo for the first time. Since platelet/endothelial cell interaction is a key event in the pathophysiology after microsurgical procedures, this model will help to understand basic molecular mechanisms of platelet behavior during I/R.

Neovascularization of poly(ether ester) block-copolymer scaffolds in vivo: long-term investigations using intravital fluorescent microscopy.

Poly(ether ester) block-copolymer scaffolds of different pore size were implanted into the dorsal skinfold chamber of balb/c mice. Using intravital fluorescent microscopy, the temporal course of neovascularization into these scaffolds was quantitatively analyzed. Three scaffold groups (diameter, 5 mm; 220-260 thickness, microm; n = 30) were implanted. Different pore sizes were evaluated: small (20-75 microm), medium (75-212 microm) and large pores (250-300 microm). Measurements were performed on days 8, 12, 16, and 20 in the surrounding normal tissue, in the border zone, and in the center of the scaffold. Standard microcirculatory parameters were assessed (plasma leakage, vessel diameter, red blood cell velocity, and functional vessel density). The large-pored scaffolds showed significantly higher functional vessel density in the border zone and in the center (days 8 and 12) compared with the scaffold with the small and medium-sized pores. These data correlated with a larger vessel diameter and a higher red blood cell velocity in the large-pored scaffold group. Interestingly, during the evaluation period the microcirculatory parameters on the edge of the scaffolds returned to values similar to those found in the surrounding tissue. In the center of the scaffold, however, neovascularization was still active 20 days after implantation. Plasma leakage and vessel diameter were higher in the center of the scaffold. Red blood cell velocity and functional vessel density were 50% lower than in the surrounding tissue. In conclusion, the dorsal skinfold chamber model in mice allows long-term study of blood vessel growth and remodeling in porous biomedical materials. The rate of vessel ingrowth into poly(ether ester) block-copolymer scaffolds is influenced by pore size and was highest in the scaffold with the largest pores. The data generated with this model contribute to knowledge about the development of functional vessels and tissue ingrowth into biomaterials.

Pigmented (melanotic) neurofibroma. Report of an unusual case with immunohistochemical, ultrastructural and cytogenetic analyses.

In the spectrum of neurofibromas, pigmented tumors are rare variants usually showing only faint, macroscopically obvious pigmentation. We report a case of a huge pigmented neurofibroma with extended, macroscopically striking pigmentation in a patient with stigmata of neurofibromatosis. The immunohistochemical and ultrastructural findings support a melanotic line of differentiation besides schwann cell differentiation and indicate a phenotypic neoplastic spectrum between tumorous schwann cells and melanocytes. Using comparative genomic hybridization, striking chromosomal aberrations were not detected. High level amplifications of the known chromosomal regions, including genes of major enzymes responsible for melanin synthesis, appear to be unlikely. However, smaller chromosomal defects might have been overlooked by the limited resolution of this screening method. Therefore, other mechanisms up-regulating melanogenesis, such as mutations in regulatory genes, have to be considered.