The surface charges on biodegradable albumin nanoparticles were introduced by covalent coupling different primary amines to examine their influence on phagocytosis by macrophages under in vitro conditions. Albumin particles with a zeta potential close to zero showed a reduced phagocytic uptake in comparison with charged particles, especially nanoparticles with a positive zeta potential. The phagocytic uptake in the present study was examined using an established cell culture model based on primary mouse peritoneal macrophages and a human hematopoietic monocytic cell line (U-937) treated with phorbol-12-myristic-13-acetate to induce cell differentiation. The influence of opsonins on in vitro phagocytosis experiments was characterized using carriers pre-treated with human serum. In the presence of human serum the phagocytic activity of U-937 cells was found to be similar to primary mouse macrophages without serum. In contrast to peritoneal macrophages, U-937 cells showed no phagocytic activity in the absence of serum. In particular, only the C3b- complement deposition on the particle surface seems to promote the phagocytic process. The in vivo distribution of albumin carriers in rats was investigated using magnetic resonance imaging (MRI). No differences in blood circulation times and organ accumulation between different nanoparticle preparations with positive, neutral and negative surface charges could be observed in rats, suggesting that the in vivo fate of albumin nanoparticles is significantly influenced by factors not reflected in the in vitro cell culture models.

A large number of benign melanocytic nevi is the major risk factor for malignant melanoma (MM). In a multicenter case‐control study, the number of common (CN) and clinically atypical (AN) nevi were counted separately at individual sites in 278 melanoma patients and 278 age‐and gender‐matched non‐melanoma controls. Relative risk (RR) adjusted for age and sex was calculated. In men as well as women, the number of CN on the legs was the best predictor of overall melanoma risk. In men, RR for developing MM when ≥1 AN were present on the trunk was 4‐fold (vs. none). In women, presence of AN on the arms increased RR 9.5‐fold. For men and women combined, after adjusting for age and gender, the RR for developing MM on the trunk and on the legs was best predicted by counts of CN at the respective body region. However, high counts of CN on the arms were associated with high melanoma risk on the legs (somewhat lower on the trunk). For AN, no site‐specificity of melanoma risk was found. Our data suggest that nevus counts of the legs are the best predictor of overall melanoma risk if total body nevus counts are not feasible. Although high counts of CN on the trunk and legs are associated with a higher risk of developing MM at the respective site than at another site, our data do not unequivocally support a direct site‐specific melanoma risk. © 1995 Wiley‐Liss, Inc.

Different pigmentary characteristics as well as different parameters of sun exposure have previously been identified as risk factors for developing cutaneous melanoma. The aim of the present study was to identify significant risk factors, determine the related magnitude of their estimated relative risks, and define criteria for the detection of persons at risk. Five hundred thirteen melanoma patients and 498 controls matched for age and sex underwent a whole-body examination for the number and type of melanocytic lesions and were interviewed on ultraviolet exposure and other potential risk factors. The total number of common melanocytic nevi on all body sites represented the most important risk factor in multiple logistic regression analysis with a relative risk of 7.6 for subjects with more than 100 versus no more than 10 melanocytic nevi. Other significant independent risk factors were the number of atypical melanocytic nevi (relative risk, 6.1 for at least 5 melanocytic nevi versus none), the number of actinic lentigines (relative risk, 3.5 for many versus none), hair color, skin type, and reported melanocytic nevus growth. No single parameter of sun exposure was significantly related to melanoma risk in the multivariate analysis. Groups with an estimated relative risk between 1 and 121.0 were distinguished by considering common and atypical melanocytic nevi as well as actinic lentigines as the decisive criteria. In conclusion, even without any information on the case history, whole-body examination and diagnosis of pigmented lesions was found to be an effective strategy for identifying persons at risk of developing melanoma. Furthermore, clinical recognition of at least 5 atypical melanocytic nevi without histologic examination is a key for identifying subjects at high risk.

Several case-control studies identified common and atypical melanocytic nevi as major risk indicators for the development of cutaneous melanoma. The present investigation was planned to detect factors associated with the prevalence of these melanoma risk markers. Whole-body examination findings and interview data of 513 melanoma patients and 498 age- and sex-matched control subjects were analyzed. Existence of more than 50 common melanocytic nevi and the presence of atypical melanocytic nevi were significantly related to age and gender, with significantly elevated relative risk for their prevalence before the age of 60 and in males. Additionally, sunburns before the age of 20 were significantly associated with both more than 50 common melanocytic nevi (relative risk = 1.7) and the presence of atypical melanocytic nevi (relative risk = 1.5). Actinic lentigines were found more frequently with increasing age, and the presence of actinic lentigines was significantly related to a tendency of freckling in adolescence (relative risk = 2.0) and to two or more sunburns after the age of 20 (relative risk = 1.6). In conclusion, sunburns before the age of 20 contribute to the development of multiple melanocytic nevi and atypical melanocytic nevi. In adulthood, this type of sun exposure is associated with the development of actinic lentigines. The relative risk of developing cutaneous melanoma increases in association with the development of these benign melanocytic lesions.

Rieger, E.; Soyer, H. P.; Garbe, C.; Buttner, P.; Kofler, R.; Wein, J.; Stocker, U.; Kruger, S.; Roser, M.; Weckbecker, J.; Panizzon, R.; Bahner, F.; Tilgen, W.; Kerl, H.; Orfanos, C. E. Author Information

Roser, M.; Metneki, J.; Weichenthal, M.; Thranet, J.; Breitbartt, E. W. Author Information

A new method for the manufacture of biodegradable albumin-spheres is presented which allows the modification of surface-properties using primary amines. The method is based on an emulsion-technique using glutaraldehyde for cross-linking. Variations in stirring rate and viscosity of the continuous phase are the main factors determining particle sizes in the range of 0.5 to 20 mum. A polymer solution consisting of hydroxypropyl cellulose and chloroform is used as dispersion medium. Contrary to published methods, even nanospheres can be isolated in solid form which are easily resuspended in aqueous media without addition of surfactants. The particles are spherical in shape and possess a smooth surface as demonstrated by scanning electron microscopy. They are reproducibly obtained in good yields (70-90%) with a narrow size distribution. In-vitro degradability was demonstrated by an enzymatic assay. The surface of the spheres can be easily modified by quenching residual aldehyde functionalities with primary amines such as ethanolamine and glycine. This chemical surface-modification is stable in fresh human serum and in aqueous solutions of different pH. These particles offer interesting possibilities for the targeting of colloidal carrier systems, since the surface modification is obtained by covalent attachment of the surface-modifying groups.

Cultured fibroblasts from patients with cutaneous malignant melanoma (CMM) were tested for chromosomal instability by determination of micronuclei (MN) and sister chromatide exchange (SCE). The constitutive as well as the UV-induced level of MN was increased in CMM patients, being most pronounced in the familial cases. There was an increased UV sensitivity in CMM by using SCE, but no spontaneously enhanced SCE value. The enhanced UV sensitivity in CMM patients was in a comparable range as those of XP heterozygotes. We thus conclude that chromosomal instability is a concurrent feature of a genetic predisposition for CMM.

Cultured fibroblasts of 17 first-degree relatives of familial melanoma patients and six first-degree relatives of cutaneous melanoma (CMM) patients with multiple CMM primaries were tested for in vitro sensitivity to UV light. Fibroblasts of nine familial CMM patients with a known UV-sensitivity and 19 healthy probands served as a control. Sister chromatid exchange (SCE) was used as a parameter to detect UV-induced genotoxic damage. We found significantly (p < 0.001) increased UV-induced SCE levels in familial melanoma patients, as well as in first-degree relatives of familial melanoma patients (p < 0.001) after UV-A,B irradiation (375 J/m2), compared to the healthy probands without a family history of CMM. A significant (p < 0.001) increase of UV-induced SCE was also observed in the relatives of CMM patients with multiple CMM primaries. In addition, the spontaneous SCE were significantly increased (p < 0.05) in familial CMM patients. This study shows that increased UV sensitivity is a familial phenomenon. It is consistent with the concept of a genetic predisposition to CMM, which is based on increased UV sensitivity and may help to define groups with an elevated risk of developing cutaneous malignant melanoma.

Spontaneous micronuclei (MN) were determined in 69 fibroblast lines in the first subculture after cryoconservation. 45 cultures (65%) showed micronuclei in the normal range (< 10 MN/500 cells), but 24 (35%) exhibited an elevation up to 60 MN/500 cells. In order to determine whether freezing and thawing is responsible for the enhanced MN level we studied the persistence of elevated MN in 10 cell cultures after freezing and thawing, and found that the MN levels returned to normal after 3 subcultures. In addition, the micronucleus formation before and after freezing was investigated in 2 newly established cell cultures obtained from probands whose cells had a particularly high number of MN. Both cultures had regular MN levels before freezing but a more than doubled number of MN when frozen samples were recultivated. This effect of the freezing procedure was confirmed with 5 separate biopsies obtained from the same donor. Since a freezing-related increase in MN was constantly observed in cells from some individuals but not in others, it might be possible that it is a constitutive trait which causes cultured fibroblasts of some individuals to be hypersensitive to the freezing and thawing procedure. It is also noteworthy that fibroblasts from 8 out of 12 probands with a familial malignant melanoma exhibited this phenomenon.

Genetically enhanced sensitivity to ultraviolet (UV) radiation may play an important role in the development of cutaneous malignant melanoma (CMM). This was studied in cultured fibroblasts of 26 CMM patients and controls by micronucleus (MN) test and sister chromatid exchange (SCE) after UV irradiation (375 J/m2). Sister chromatid exchange and MN formation were used as parameters to detect the UV-induced genotoxic damage in the individual cell strains. We found that the UV-induced level of MN was significantly increased in CMM patients (p = 0.0005), being most pronounced in the familial cases (p = 0.0001). Ultraviolet-induced SCE was also elevated in CMM patients (p = 0.001), but there was no difference between familial and nonfamilial cases. The present findings indicate that genetic predisposition contributes to the development of CMM in a subset of CMM patients and may be due to an enhanced susceptibility to UV light.

The clinical diagnosis of malignant melanoma (MM) is based on the subjective evaluation of objective measurable parameters (criteria). The accuracy of melanoma diagnosis by dermatologists is only 75%. Particularly difficult is the diagnosis of precursors or early stages of MM. Therefore, we have studied on the one hand the intra- and interindividual reproducibility of the clinical diagnosis of pigmented lesions, and on the other hand the clinico-histopathological correlation. In addition, we have conducted a preliminary investigation designed to evaluate whether image analysis (objective and reproducible) could be used as an auxiliary instrument to differentiate between benign and malignant melanocytic lesions. In the clinical study, the intraindividual reproducibility of the combination of criteria was 69%. The interindividual reproducibility of single criteria even exhibited a range of up to 36%. Histologically "atypical/dysplastic" melanocytic lesions were considered to require excision as frequently as histologically regular melanocytic lesions. Using image analysis (single threshold segmentation, standard deviation of intensity distribution, ratio of area to circumference, Fourier analysis), we could show that it may be possible to differentiate between benign and malignant melanocytic lesions. Therefore, image analysis may be very helpful in determining the dignity of melanocytic lesions.

The certainty of a strong genetic predisposition to malignant melanoma was first established over 35 years ago. Since it has been shown that constitutive chromosomal instability is significantly correlated with the familial occurrence of cancer, we have studied spontaneous micronucleus rates in fibroblast cultures from 44 melanoma patients, 44 healthy probands and 78 patients with bronchial carcinoma. Here we report a significantly (p = less than 0.0005) increased spontaneous chromosomal instability in patients with cutaneous malignant melanoma compared to healthy controls and other tumor patients (bronchial carcinoma).

Fibroblast cultures of seven patients with xeroderma pigmentosum (XP), 19 healthy sibs or parents of XP patients (XP-heterozygotes), and 24 healthy normal controls were studied for chromosome instability induced by ultraviolet rays (UV). We used a UV source that contained predominantly UV-A and UV-B at an intensity of 500 J/m2 and evaluated the induction of micronuclei (MN) and sister chromatid exchange (SCE). the XP homozygotes had a UV sensitivity that was clearly above that of all heterozygotes and normal controls. Heterozygotes had an increased rate of UV-induced MN (4.76 +/- 1.96 vs. 1.82 +/- 2.05, p less than 0.0001) and increased UV induction of SCE (13.21 +/- 3.49 vs. 9.01 +/- 1.25, p less than 0.001), as compared to normal controls. These data support epidemiologic findings that suggest that XP heterozygotes are particularly cancer prone. In addition, the determination of the UV sensitivity in vitro as described may be used for genetic counseling of asymptomatic relatives of XP patients.

In vitro detection of micronuclei was used to determine spontaneous chromosomal instability in cultured fibroblasts of 28 healthy normal controls, 28 individuals with nonfamilial cutaneous malignant melanoma (CMM) and 14 people with familial occurrence of CMM. Lymphocytes from 40 healthy controls, 40 CMM patients and 6 individuals with familial CMM were also compared for spontaneous expression of micronuclei in vitro. In the familial cases micronucleus frequency was found to be higher, both in cultured fibroblasts (P<0.005) and in lymphocytes (P<0.001) as compared to normal controls. There was no significant difference between nonfamilial cases and normal controls. We conclude that chromosomal instability may contribute to a genetic risk for development of cutaneous malignant melanoma.