Contribution of melanocortin-1 receptor gene variants to sporadic cutaneous melanoma risk in a population in central Italy: a case-control study

The melanocortin-1 receptor (MC1R) gene is a key determinant of the physiological variation in human skin pigmentation. It is highly polymorphic, and specific MC1R allelic variants have been shown to be low-penetrance melanoma susceptibility alleles. We investigated the contribution of the MC1R genotype to the risk of sporadic cutaneous melanoma in a population in central Italy. One hundred patients with sporadic cutaneous melanoma of any stage and 100 unrelated control individuals were consecutively recruited between 1 September 2000 and 31 December 2001. Information on ethnic background and residential history, phenotypic risk factors for melanoma and ultraviolet exposure habits was collected through a standardized questionnaire and total skin examination. Sequence analysis of the entire coding region of the MC1R gene was performed. A total of 26 MC1R variants, including a novel 123_124insT allele, was identified in our population, with the most frequent allele being V60L. Carriers of high-penetrance 'R' MC1R alleles, that define MC1R variants strongly associated with the red hair colour phenotype, showed a statistically significant increase in melanoma risk [odds ratio (OR), 2.55; 95% confidence interval (CI), 1.19-5.55]. No significant association with melanoma risk was observed for carriers of 'r' variants (OR, 0.90; 95% CI, 0.45-1.82). Amongst individual MC1R variants, the R151C allele was significantly associated with melanoma, with an OR of 2.94 (95% CI, 1.04-8.31). After stratification for clinical and ultraviolet exposure risk factors, the melanoma risk associated with high-penetrance 'R' variants appeared to increase significantly, mainly in the presence of clinically atypical naevi, more than 50 melanocytic naevi, high recreational sun exposure and occupational sun exposure. These results support the contribution of high-penetrance MC1R variant alleles to genetic predisposition to sporadic cutaneous melanoma in a population in central Italy.     

Focal acral hyperkeratosis associated with pitted keratolysis

Focal acral hyperkeratosis is characterized by the same clinical appearance as acrokeratoelastoidosis, but without abnormalities in the elastic fibers. We present the case of a woman with a 10-year case of dermatosis localized on the palms, soles and dorsum of the metacarpophalangeal joints, consisting of multiple polygonal papules and associated hyperhydrosis, clinically compatible with acrokeratoelastoidosis. Her father had a history of the disease. In addition, the patient presented with palmoplantar pitted keratolysis. The histopathological study ruled out elastorrhexis, and the pitted keratolysis was corroborated by the clinical appearance and the presence of coccoid elements in the stratum corneum, evident with a PAS stain. In our opinion, the focal acral hyperkeratosis is not a separate entity from the acrokeratoelastoidosis.     

MAGE-A3 is a frequent tumor antigen of metastasized melanoma

MAGE-3 or MAGE-A3 is one of the best-characterized tumor antigens. Due to its tumor-restricted expression pattern and its recognition by both cytotoxic and helper T cells it constitutes a promising tumor antigen for anticancer immunotherapy, notably of malignant melanoma. Surprisingly, however, only very limited information is available on the frequency and consistency of its expression in metastatic melanoma lesions. We have now investigated the presence of MAGE-A3 mRNA in 316 tumor samples from 147 melanoma patients by RT-PCR. MAGE-A3 mRNA was detectable in 62% of metastases, and expression did not depend on the site of the metastases (skin, lymph node, and internal organs), age, sex, or duration of disease. Southern blot hybridization of the PCR product enhanced sensitivity of detection, and 26% more samples (13/50 samples tested) scored positive, indicating an even higher MAGE-A3 mRNA frequency than determined by simple ethidium bromide gel analysis. In 62 patients, we were able to investigate MAGE-A3 expression in several metastases from the same patient, and unexpectedly, both MAGE-A3-positive and MAGE-A3-negative metastases were found in 32% of these patients (20 of 62). Immunohistochemistry (using mAb 57B) demonstrated that the expression pattern was usually also heterogeneous with positively and negatively stained tumor cells within one metastasis. However, most (90%) of the metastases (47/52) gave a partially positive signal. Taken together, MAGE-A3 is a common and frequent tumor antigen in metastasized melanoma, but its expression is often heterogeneous.     

Induction of polyploidy by histone deacetylase inhibitor: a pathway for antitumor effects

Histone deacetylase (HDAC) inhibitors can induce various transformed cells to undergo growth arrest and/or death. Suberoylanilide hydroxamic acid (SAHA) is an HDAC inhibitor which is in phase I/II clinical trials and has shown antitumor activity in hematologic and solid tumors at doses well tolerated by patients. HDAC is the target for SAHA, but the mechanisms of the consequent induced death of transformed cells are not completely understood. In this study, we report that SAHA induced polyploidy in human colon cancer cell line HCT116 and human breast cancer cell lines, MCF-7, MDA-MB-231, and MBA-MD-468, but not in normal human embryonic fibroblast SW-38 and normal mouse embryonic fibroblasts. The polyploid cells lost the capacity for proliferation and committed to senescence. The induction of polyploidy was more marked in HCT116 p21WAF1-/- or HCT116 p53-/- cells than in wild-type HCT116. The development of senescence of SAHA-induced polyploidy cells was similar in all colon cell lines. The present findings indicate that the HDAC inhibitor could exert antitumor effects by inducing polyploidy, and this effect is more marked in transformed cells with nonfunctioning p21WAF1 or p53 genes.     

Analysis of a functional BTNL2 polymorphism in type 1 diabetes, rheumatoid arthritis, and systemic lupus erythematosus

The aim of this study was to test whether the functional variant rs2076530 of the BTNL2 gene confers susceptibility to the autoimmune diseases type 1 diabetes (T1D), rheumatoid arthritis (RA), and systemic lupus erythematosus (SLE). Our study populations consisted of 326 patients with T1D and 351 healthy subjects, 808 patients with RA and 1137 healthy controls, and 372 patients with SLE and 280 healthy controls. Genotyping of the BTNL2 gene rs2076530 polymorphism was performed by real-time polymerase chain reaction technology, using the TaqMan 5′-allele discrimination assay. We observed statistically significant differences in the distribution of BTNL2rs2076530 alleles between patients with T1D, RA, and SLE and healthy controls (p = 0.0035, 0.000003, and 0.00002, respectively), but in two divergent ways: the G allele was associated with T1D and RA, and the A allele was associated with SLE. However, the polymorphism exhibited strong linkage disequilibrium with HLA DQB1–DRB1 haplotypes previously identified as predisposing to the diseases. When the BTNL2 polymorphism was tested conditional on HLA DQB1–DRB1haplotypes, the BTNL2 effect was no longer significant in all three study populations. The BTNL2 rs2076530 polymorphism is associated with T1D, RA, and SLE because of its strong linkage disequalibrium with predisposing HLA DQB1–DRB1 haplotypes in Caucasian populations.     

Seroreactivity to and avidity for recombinant antigens in toxoplasmosis

To improve serodiagnostic methods for the diagnosis of acute toxoplasmosis during pregnancy, a new test system has been developed and evaluated based on the use of recombinant antigens. Five recombinant Toxoplasma gondii antigens (ROP1, MAG1, SAG1, GRA7, and GRA8) were cloned in Escherichia coli, purified, and applied directly onto nitrocellulose membranes in a line assay (recomLine Toxoplasma). A panel of 102 sera from 25 pregnant women with supposed recent toxoplasmosis and from two symptomatic children was compared to a panel of 71 sera from individuals with past infection. Both panels were analyzed using a recombinant line assay for immunoglobulin G (IgG), IgM, and IgA antibodies and a reference enzyme-linked immunosorbent assay. Within the IgM-positive samples, antibodies against ROP1 were predominant regardless of the infection state. In IgG analysis a characteristic antibody pattern was found for very recent infections. This pattern changed to a different one during the time course of infection: antibodies against GRA7 and GRA8 were characteristic for very early IgG, whereas antibodies against SAG1 and MAG1 appeared significantly later. These results were further confirmed by determination of the IgG antibody avidity for every single recombinant antigen. In the time course of infection, IgG antibodies against the early recognized antigens matured significantly earlier than those directed against the later antigens did. The IgA patterns did not give reliable information about the infection time points. The data revealed that the recombinant line assay provides valuable information on the actual state of infection, especially during the early infection time points.     

Cryopreservation of human fetal organs

Summary The effects of low temperature preservation on morphology, viability and differentiation capacity of different human fetal organs were studied using transmission (TEM) and scanning (SEM) electron microscopy, in vitro cultivation as well as xenogeneic transplantation. For this purpose fragments of lung, kidney, small intestine, thyroid, brain, liver and spleen from 10 human fetuses (aged 9 to 14 weeks of gestation) were frozen by a three step cooling procedure. After 3 to 12 months the specimens were thawed rapidly and processed for TEM and/or in vitro cultivation and/or transplantation into nude mice. TEM studies on frozen lung, kidney and intestine revealed generally a well preserved ultrastructure whereas liver and spleen fragments appeared highly necrotic. From three fetuses, frozen intestine and lung specimens were used for the establishment of monolayer cultures. Following trypsinization, both epithelial and mesenchymal cells formed a continous layer on the bottom of plastic bottles. During further subpassages the number of epithelial cells decreased resulting in the formation of pure fibroblast cultures. Frozen brain tissue from one fetus was also successfully cultivated forming cell clusters and fiber bundles of variable size at the surface of glass cover slips. Following subcutaneous implantation into nude mice, the vast majority of fragments from lung, kidney, intestine and thyroid was found to growth in the recipients. The growth of xenografts was accompanied by persistence (kidney, intestine) or even increase (lung, thyroid) in cellular differentiation studied by TEM or autoradiography. Transplanted liver and spleen fragments, however, regularly regressed forming solid scars in the subcutaneous tissue of nude mice.In summary, the three-step cooling procedure is an effective method for storage of different human fetal organs preserving morphology, viability and differentiation capacity of the tissues. Thawed specimens may be used for several experiments including in vivo and in vitro studies.In memoriam Prof. Dr. med. G. Töndury, 15.3.1985     

DNA damage induced by a quinoxaline 1,4-di-N-oxide derivative (hypoxic selective agent) in Caco-2 cells evaluated by the comet assay

The DNA damage induced by 7-chloro-3-[[(N,N-dimethylamino)propyl]amino]-2-quinoxalinecarbonitrile 1,4-di-N-oxide hydrochloride (Q-85 HCl) in Caco-2 cells under hypoxic and well-oxygenated conditions has been studied by using the comet assay. This compound has shown a good in vitro profile of high selective toxicity in hypoxia, but its mechanism of action is unknown. The DNA damage has been evaluated by performing the comet assay after a 2-h treatment with Q-85 HCl (0.1, 0.2, 0.4 microM in hypoxia; 20, 40 microM in well-oxygenated conditions). The number of cells in apoptosis has also been assessed by flow cytometry analysis of Annexin V-FITC staining. The capability of the cells to repair the DNA damage and the proliferation rate was evaluated at different times after the treatment (24-168 h). Under hypoxic conditions, a clear dose-dependent increase in the number of nuclei with a comet was observed (comet score: 132 +/- 13, 343 +/- 30 and 399 +/- 1; control comet score: 42 +/- 14). Under well-oxygenated conditions, the number of nuclei with comet increased significantly with respect to the control (comet score: 273 +/- 14 and 312 +/- 9; control comet score: 27 +/- 4). Cells in apoptosis were not detected by the comet assay nor by flow cytometry. The recovery from DNA damage was time- and concentration-dependent in hypoxia (cells treated with the highest concentration still showed DNA damage after 72 h) and rather time-dependent in well-oxygenated conditions (DNA was completely repaired after 24 h). In conclusion, Q-85 HCl acts by DNA damage and not only the reduced intermediate is genotoxic but also some other derivatives and Q-85 HCl itself may be acting.     

The effect of physical and psychosocial loads on the trapezius muscle activity during computer keying tasks and rest periods

The overall aim was to investigate the effect of psychosocial loads on trapezius muscle activity during computer keying work and during short and long breaks. In 12 female subjects, surface electromyography (EMG) was recorded bilaterally from the upper trapezius muscle during a standardized one hand keying task—interspaced with short (30 s) and long (4 min) breaks—in sessions with and without a combination of cognitive and emotional stressors. Adding psychosocial loads to the same physical work did not increase the activity of the trapezius muscle on either the keying or the control side, both of which remained at median and static EMG activity levels of around 5% and 2.5% of the maximal voluntary electrical activity (EMG_max), respectively. The difference between the keying and the control side was significant; and further the control side activity was significantly increased above resting level. During both short and long breaks, exposure to psychosocial loads also did not increase the activity of the trapezius muscle either on the side of the keying or the control hand. Of note is that during long breaks the muscle activity of the keying side as well as that of the control side remained at the same level as during the short breaks, which was increased above resting level. This was to be seen from the static and the median EMG activity levels as well as gap times, the overall mean values being: 0.4%EMG_max, 1.1%EMG_max, and 50% in gap time, respectively. In conclusion: psychosocial loads are not solely responsible for increased non-postural muscle activity; and increasing the duration of breaks does not per se cause muscle relaxation.