Clonal heterogeneity of melanoma in a paradigmatic case study: future prospects for circulating melanoma cells.

The management of metastatic melanoma is a difficult matter. Nevertheless, the advent of target therapy has significantly improved patient outcome, provided that tumor molecular characteristics become available: the detection of drug-resistant clones can contribute to understanding the reasons for resistance onset, influencing the choice of subsequent therapy. This work aimed to provide a possible explanation for the early resistance to vemurafenib developed by a patient with melanoma, and concurrently to assess the extent, and role, of the tumor clonal heterogeneity. We analyzed tissue samples from different sites and time points: first/second primary, three lymph node metastases, and circulating melanoma cells (CMCs). We first investigated these samples by the routine Sanger sequencing for BRAF, NRAS, and KIT, and then, we focused on specific hotspots by droplet digital PCR. We detected a BRAF V600E mutation by Sanger sequencing in the second primary and distant lymph node metastases, but not in the first primary or sentinel lymph node. Interestingly, by droplet digital PCR, the V600E mutation was also detected in the first primary, and the V600K in the second primary and metastases. Moreover, we identified a rare KIT V569G mutation, appearing to be CMC exclusive. This finding confirms the potential of CMCs as a source of tumor material for genetic analysis, reflecting real-time systemic disease evolution and, most likely, the most aggressive, treatment-resistant clones. In summary, this work underlines the importance of CMCs in the early identification of tumor clones putatively responsible for therapy resistance.

Metallothioneins and the central nervous system: from a deregulation in neurodegenerative diseases to the development of new therapeutic approaches.

Metallothioneins (MT) are a family of proteins actively involved in metal detoxification and storage as well as in prevention of free-radical damage. Changes in the levels of MT have been described in a number of neurodegenerative diseases including Alzheimer's disease, Parkinson's disease, prion protein disease, Binswanger type of subcortical vascular dementia, and amyotrophic lateral sclerosis. This suggests that MT functions might be more complex and vast than what was initially thought. In this review, we summarize the current knowledge on the potential involvement of MT in the mentioned neurodegenerative diseases while also discussing the emerging evidence proposing MT modulation as a feasible therapeutic approach. Enhancing repair mechanisms after neurological damage and/or protection against oxidative stress through a proper modulation of this family of protein might indeed represent an important avenue to cope neurodegeneration.

Effects of a copper-deficient diet on the biochemistry, neural morphology and behavior of aged mice.

Copper dyshomeostasis has been suggested as an aetiological risk factor for some neurodegenerative diseases, such as Alzheimer's disease. However, the precise mechanism at the base of this involvement is still obscure. In this work, we show the effects of a copper-deficient diet in aged CD1 mice and the influence of such a diet on: a) the concentration of various metal ions (aluminium, copper, iron, calcium, zinc) in the main organs and in different brain areas; b) the alteration of metallothioneins I-II and tyrosine hydroxylase immunopositivity in the brain; c) behavioural tests (open field, pole, predatory aggression, and habituation/dishabituation smell tests). Our data suggested that the copper-deficiency was able to produce a sort of "domino effect" which altered the concentration of the other tested metal ions in the main organs as well as in the brain, without, however, significantly affecting the animal behaviour.

Ontogenesis and migration of metallothionein I/II-containing glial cells in the human telencephalon during the second trimester.

Metallothioneins (MT) belong to a widespread family of proteins characterized by a high metal content (mainly Cu(2+) and Zn(2+)) and by the presence of cysteine residues. The expression of metallothionein I-II (MT I/II), glial fibrillary acid protein (GFAP), and vimentin was examined in a series of 16 developing human brains of the second trimester. The brains of a stillborn/newborn individual and two postnatal individuals were studied for comparison. MT I/II-containing cells became consistently and clearly visible only from gestational week 21 onwards. On the other hand, several densely packed GFAP- and vimentin-containing elements were evident in the neuroepithelium at several periventricular locations and in the subventricular zone of all fetuses of the series. GFAP- and vimentin-containing elements also entered the intermediate plate, but only a few elements were evident in the outer layers of the maturing cortex. The relatively late onset of MT I/II expression and their distribution are discussed in relation to the uptake of trace elements during the last trimester of pregnancy, and the role of astrocytes in neuronal guidance and maturation of cortical circuits.

Triage of women with atypical squamous cells of undetermined significance (ASC-US): results of an Italian multicentric study.

OBJECTIVES: To compare the performance of immediate colposcopy, repeat Pap test and HPV test as triage options for women diagnosed as having atypical squamous cells of undetermined significance (ASC-US) while attending organised screening for cervical carcinoma in five centres of the Veneto region. METHODS: Women consecutively diagnosed as having ASC-US were included in a prospective study, and underwent colposcopy and collection of cervico-vaginal cells for conventional Pap test and HPV test (Hybrid Capture 2, High-risk probe set, Digene). Repetition of all three tests was scheduled for 12 months later. DNA was subsequently extracted from residual cells of positive samples, and analysed by polymerase chain reaction with several primers for typing of HPV sequences. Sensitivity, specificity and positive predictive value (PPV) of the different triage options for histology-confirmed cervical intraepithelial neoplasia, grade 2 or worse (CIN2+) were calculated among all women and by age (under and above 35 years). RESULTS: Seven hundred forty-nine women 25-64 years old (median age 42 years) were enrolled in the study. Pap smears at enrolment were read as ASC-US or more severe in 211 (29.4%) cases, colposcopy disclosed an atypical transformation zone in 254 (34.2%) women, and HPV test was positive in 181 (24.2%). High-grade cervical lesions developed in 29/749 (3.9%) women. HPV typing was possible in 163 (90%) of the samples, and carcinogenic types were present in 123. CONCLUSIONS: HPV test showed the best performance; overall, it had the highest sensitivity (92.3%), specificity (78.6%) and PPV (14.9%).

Oxidative metabolism of dopamine: a colour reaction from human midbrain analysed by mass spectrometry.

In order to identify the protein responsible for a dopamine peroxidizing activity, previously described in human normal and parkinsonian substantia nigra by our group, we developed non-denaturing polyacrylamide gel electrophoresis conditions, mimicking the characteristic colour in vitro reaction, resulting from cyclic oxidation of dopamine (DA). After separating protein mixtures from human normal midbrain homogenates on two sets of identical native gels, one gel set was subjected to specific activity staining by using DA and hydrogen peroxide. An activity red/orange band appeared in midbrain tissue lanes, similarly to the lane where commercial horseradish peroxidase (HRP) was present as control of peroxidative activity. The second set of gels, stained with Coomassie Blue, showed other, not enzymatically active protein bands. Mass spectrometry analysis of the bands containing the activity and the corresponding Coomassie Blue bands revealed the presence of proteins that may play a role in neurodegenerative disease, highlighting a possible functional link among dopamine/dopaminochrome redox cycle and protein metabolism.

Accumulation of copper and other metal ions, and metallothionein I/II expression in the bovine brain as a function of aging.

Accumulation of metal ions in the brain contributes to heighten oxidative stress and neuronal damage as evidenced in aging and neurodegenerative diseases, both in humans and in animals. In the present paper we report the analysis of Cu, Zn and Mn in the brain of two series of respectively young (8-16 months) and adult (9-12 years) bovines. Our data indicate that the concentrations of Cu varied of one order of magnitude between 1.67 and 15.7microg/g wet tissue; the levels of Zn varied between 6.13 and 17.07microg/g wet tissue and the values of Mn resulted between 0.19 and 1.24microg/g wet tissue. We found relevant age-dependent differences in the distribution of Cu and Zn, whose concentrations were markedly higher in older animals. By contrast, Mn seemed to redistribute in the different cerebral areas rather than drastically change with age. Tissues from bovine brain were also analysed immunohistochemically for the presence and distribution of metallothionein I/II and also for the expression of glial fibrillary acidic protein. Metallothionein I/II immunoreactive elements included ependymal cells lining the lateral ventricles and neural cells in middle layer of the cerebellar cortex. No age differences were evident between calves and adult. The presence of liquor-contacting metallothionein I/II in cells confirms that their functions in the central nervous system are not yet completely established.

Association of a presenilin 1 S170F mutation with a novel Alzheimer disease molecular phenotype.

OBJECTIVE: To report an ataxic variant of Alzheimer disease expressing a novel molecular phenotype. DESIGN: Description of a novel phenotype associated with a presenilin 1 mutation. SETTING: The subject was an outpatient who was diagnosed at the local referral center. PATIENT: A 28-year-old man presented with psychiatric symptoms and cerebellar signs, followed by cognitive dysfunction. Severe beta-amyloid (Abeta) deposition was accompanied by neurofibrillary tangles and cell loss in the cerebral cortex and by Purkinje cell dendrite loss in the cerebellum. A presenilin 1 gene (PSEN1) S170F mutation was detected. MAIN OUTCOME MEASURES: We analyzed the processing of Abeta precursor protein in vitro as well as the Abeta species in brain tissue. RESULTS: The PSEN1 S170F mutation induced a 3-fold increase of both secreted Abeta(42) and Abeta(40) species and a 60% increase of secreted Abeta precursor protein in transfected cells. Soluble and insoluble fractions isolated from brain tissue showed a prevalence of N-terminally truncated Abeta species ending at both residues 40 and 42. CONCLUSION: These findings define a new Alzheimer disease molecular phenotype and support the concept that the phenotypic variability associated with PSEN1 mutations may be dictated by the Abeta aggregates' composition.

Correlation between genetic and biological aspects in primary non-metastatic breast cancers and corresponding synchronous axillary lymph node metastasis.

This study investigated the changes, if any, in the level of expression of a well defined panel of cell proliferation, differentiation and apoptosis markers between the primary breast tumor and the corresponding synchronous lymph node metastasis from a population of patients with a comparable disease status, in terms of clinical features, and natural history.Ninety pure invasive ductal carcinomas with 10 or more axillary lymph nodes involved and without evidence of distant metastasis were included in this study. Primary tumor and corresponding metastatic lymph node tissue specimens were evaluated for the expression of Cyclin B1, MMP1 metalloproteinase, ICAM-1, RARbeta, Ki67, ER, PgR, p53, bcl-2 and c-erbB2 by immunohistochemistry using standard methods. The bivariate Pearson correlation analysis demonstrated a close relationship between primary and matching corresponding metastatic node. A high grade of correlation has been maintained even when staining results where categorized as positive/negative according to each one marker cut-off level of staining expression. We report the most extensive immunohistochemical analysis of biological determinants in a well defined population of patients with invasive ductal carcinomas and involvement of 10 or more axillary nodes and no distant metastasis. We found a close correlation between the primary tumor and corresponding metastatic node in terms of the expression of all 10 of the markers investigated in this study. The not complete concordance observed could be explained by the gene expression modulation by extrinsic factors and by the microenvironment in which the cancer cells reside.

The effect of Zn(II) and streptozotocin administration in the mouse brain.

Streptozotocin is a natural antibiotic produced by Streptomyces achromogenes able to induce diabetes in experimental animals. Among various toxic properties, streptozotocin is a potent source for reactive oxygen species. In this paper, we report the biological response of brain, upon treatment with streptozotocin in terms of metal ions dismetabolism and metallothionein expression. In addition, important information on the preventive effect of zinc in eliciting the pharmacological effect of the drug are reported, in relation to the effective role of the metal ions in inducing metallothionein synthesis. In the brain, streptozotocin treatment affects mostly the hippocampus and cerebellum as shown by a high GAFP and MT-I-II immunopositivity of glial cells. The Zn pre-treatment reduces significantly, as a general effect, the occurrence of hyperglycaemic status. At the brain level, the observed astrocytosis is strongly reduced. The high inducibility of MT represents a rapid and convenient response able to prevent the deleterious effects consequent to the oxidative stress. All together these results support the efficacy of the Zn treatment in order to prevent streptozotocin effects, including brain tissues.

Metallothionein-I-II expression in young and adult bovine pineal gland.

Aging is characterized, among other features, by an increased concentration of metal ions in the brain that may contribute to a greater increase in free radicals production. The present paper reports data regarding the concentration of some relevant metal ions (Cu, Fe, Mn, Zn), as well as the immunopositivity of metallothionein-I-II and GFAP in the bovine pineal gland with respect to animal aging. The pineal gland of young bovines displays several immunoreactive metallothionein-I-II positive elements in the parenchyma, whose number decreases with age. We also report that a well defined group of neurons bordering the third ventricle and located close to the subcommissural organ shows an intense metallothionein-I-II immunopositivity. The presence of metallothionein-I-II was confirmed by means of liquid chromatography coupled to tandem mass spectrometry. In particular, it proved possible to identify the amino acid sequences of the unique tryptic peptide not containing cysteine and two pepsin fragments containing cysteines. In conclusion, our data suggest the presence of a metallothionein-I-II expressing system in the pineal gland and ventricle-adjacent areas of the bovine epithalamus might possibly be related to the anti-aging effects of melatonin.

Effects of chronic treatment with sodium tetrachloroaurate(III) in mice and membrane models.

Gold is a nonessential element with a variety of applications in medicine. A few gold(I) compounds are used in the clinics for treatment of rheumatoid arthritis and of discoid lupus. Some novel gold(III) compounds are under evaluation as anticancer agents. It is known that gold compounds generally produce toxic effects on the kidneys and characteristic lesions in the brain. However, information concerning the neurotoxicity of gold derivatives in humans as well as in experimental toxicology is rather scarce. For this reason we tried to shed some further light on this aspect of gold neurotoxicity by chronic treatment of mice with sodium tetrachloroaurate(III) in order to observe possible biophysical and morphological alterations that may occur in the brain. Chronic gold treatment resulted in a markedly decreased expression of metallothioneins and of glial fibrillary acidic protein in astrocytes of different brain areas. To examine its effects on cell membranes, interactions of sodium tetrachloroaurate(III) with molecular models were also evaluated. The models consisted in bilayers built-up of classes of phospholipids located in the outer and inner monolayers of biological membranes. Structural perturbation of cell membrane models was observed only at concentrations 10(5) times higher than those detected in the brains of animals after three months' treatment. These results show that toxic effects on animal brain upon treatment with sodium tetrachloroaurate develop with difficulty and may be observed only at high doses.

Effects of steroid hormones on the Zn, Cu and MTI/II levels in the mouse brain.

The effects of some steroid hormones (corticosterone, hydrocortisone, testosterone and estrone) on the Zn, Cu metabolism and metallothioneins levels in the mouse brain were studied. To administrate the hormones, aqueous suspensions and olive oil solutions injected subcutaneously were used alternatively. The quantification of metals and metallothioneins concentrations in brain homogenates revealed significant alterations of both metal ions and protein expression levels, yet the subcutaneous oil injection increased per se the tissue metallothionein expression and metal content. We have also defined by immunohistochemistry the area-specific distribution of metallothioneins isoforms-I/II and of glial fibrillar acid protein. Upon treatment, corpus callosum, mesencephalon, pons, hippocampus and cerebellum were found to be the areas that increase the protein expression levels, whereas all other brain areas were marginally affected or were unaffected in terms of immunopositive metallothionein reaction. The metallothionein-I/II expression was compared with the immunopositivity of glial fibrillar acid protein and the results are discussed within the framework of the physiological role of corticosteroids and the potential therapeutical importance of sexual hormones.

Zn and Cu alteration in connection with astrocyte metallothionein I/II overexpression in the mouse brain upon physical stress.

The distribution of metallothioneins I/II in the mouse brain and their specific area distribution upon physical stress were studied. To induce physical stress, groups of mice were subjected to total darkness for different periods (2 weeks, 1 month, and 2 months). The concentration of metallothioneins, evaluated by immunohistochemistry, as well as area-specific protein expression, were found in the following quantitative order: corpus striatum, cerebellum, mesencephalon, hippocampus with fornix, parts of thalamus, and pons. All other brain areas were marginally affected, or even unaffected, in terms of immunopositive metallothionein reaction. Metallothionein I/II expression was compared with the immunopositivity of glial fibrillary acidic protein (GFAP). It is noteworthy that metallothioneins and GFAP are expressed in different types of astrocytes.

Transferrin C2 variant does confer a risk for Alzheimer's disease in caucasians.

Several gene mutations are associated with an increased risk of Alzheimer's disease. Previous studies reported higher transferrin C2 allele frequencies in Alzheimer's disease compared with normal controls. However, potential interactions between transferrin C2 and APOE (epsilon 4), have not been extensively investigated and have been the subject of controversial reports from several laboratories. We have carried out a case-control study on the association between Alzheimer's disease and transferrin C2 and APOE epsilon 4 alleles. epsilon 4 allele was associated with a four fold increase in the risk of disease, and transferrin C2 allele was significantly associated with Alzheimer's disease only in epsilon 4 negative subjects. These results suggest that apoE and transferrin may be part of a complex mechanism in the pathogenesis of Alzheimer's disease.

The effect of chronic aluminum(III) administration on the nervous system of aged rats: clues to understand its suggested role in Alzheimer's disease.

The effect of chronic aluminum intake has been investigated in the brain of aged male Wistar rats to assess the potential role of the accumulation of this metal ion on the development of neurodegenerative features observed in Alzheimer's disease. AlCl3 x 6 H2O (2g/L) was administered to experimental animals for 6 months in the drinking water. The total content of Al (microg/g fresh tissue) was measured by inductively coupled plasma atomic emission spectrometry (ICP-AES), while the content of Cu, Zn and Mn was determined by flame AAS in the prosencephalon + mesencephalon, pons-medulla and cerebellum of control and Al(III)-treated animals. The area occupied by mossy fibres in the CA3 field of the hippocampus was estimated by a computer-assisted morphometric method following Timm's preferential staining. In Al(III)-treated rats the concentration of Cu, Zn and Mn did not increase significantly (p < 0.5) in prosencephalon + mesencephalon, nor in pons-medulla (p < 0.5) except for Cu (p < 0.05) in pons-medulla. In the cerebellum the only significant increase was seen for Zn (p < 0.01) while no change was observed for Cu and Mn. The area occupied by the mossy fibres in the hippocampal CA3 field was significantly increased (+32%) in aged Al(III)-treated rats. Since Cu, Zn and Mn are essential components of the cytosolic and mitochondrial superoxide dismutases, it is possible that the increased content of these ions in aged Al(III)-treated rats represents an increased amount of genetic expression of these antioxidant enzymes. Considering that the positivity to Timm's reaction is based on the presence of free or loosely bound Zn2+ ions within synaptic terminals and that Zn2+ ions are reported to be accumulated by hippocampal neurons when tissue injury occurs, the increased area of the mossy fibres in CA3 field of Al(III)-treated rats could indicate increased hippocampal damage in these animals. Taken together, the present findings indicate that the aging CNS is particularly susceptible to Al(III) toxic effects which may increase the cell load of oxidative stress and may contribute, as an aggravating factor, to the development of neurodegenerative events as observed in Alzheimer's disease.

Increased dopamine peroxidation in postmortem Parkinsonian brain.

We have previously reported the presence, in human midbrain, of an enzymatic activity which catalyzes the formation of dopaminochrome from dopamine (DA) and hydrogen peroxide. Here, we report, for the first time, an increased DA peroxidizing activity in the midbrain and basal ganglia of autoptic Parkinsonian brains. The crude activity was determined spectrophotometrically in extracts of paraffin-embedded slices obtained from autopsied brain. No addition of substrate was necessary since endogenous substrates such as DA and hydrogen peroxide were present in the samples. In Parkinson's patients' midbrain, this activity was substantially increased compared to normal midbrain. Moreover, the DA peroxidizing activity, which was absent in basal ganglia of normal people, was detectable in all our Parkinson's patients. These observations suggest that a peroxidizing pathway of DA may be present in human brain. The increased peroxidizing activity in Parkinson's patients generates the toxic compound dopaminochrome which may play a role in the pathogenesis of this disease.

Astrocytosis, microgliosis, metallothionein-I-II and amyloid expression in high cholesterol-fed rabbits.

Cholesterol is considered a risk factor in vascular dementia as well as in Alzheimer's disease. Several biochemical, epidemiological and genetic aspects established a correlation between cholesterol concentration and Alzheimer's disease. Microglia activation, astrocytosis with metallothionein-I-II overexpression, amyloid beta intraneuronal accumulation and a rare formation of amyloid beta extracellular positive deposits were the major immunohistochemical features observed in the brain of high cholesterol-fed animals. The relevance on the cholesterol metabolism in Alzheimer's disease pathogenesis is also discussed.

Metallothionein I-II immunocytochemical reactivity in Binswanger's encephalopathy.

Binswanger's disease is a subacute form of hypertensive encephalopathy characterized by patchy-confluent myelin loss of the deep hemispheric white matter, associated with marked regressive changes of the oligodendrocytes and variable astroglial reaction. To understand the distribution and the specificity of astrocyte pathology in Binswanger's disease we quantified reactive and degenerating astrocytes in different regions of the deep and subcortical white matter and of the cerebral cortex. Sections of frontal, temporal, parietal, and occipital lobes of 12 histologically proven cases of Binswanger's disease were immunostained with antibodies to glial fibrillary protein (GFAP) and to metallothionein I and II (MT-I-II), markers which specifically identify normal and reactive astrocytes. Control tissues were from 6 elderly patients without neurological diseases. The brains of Binswanger's disease were characterized by few and lightly immunostained astrocytes in the deep white matter, but normal and reactive astrocytes, strongly immunoreactive for GFAP and MT-I-II, were prominent in the subcortical white matter and the cerebral cortex. However, the relative distribution of GFAP positive and MT-I-II positive astrocytes was significantly different between the cerebral cortex and the subcortical white matter, the MT-I-II positive astrocytes being more frequent in the cerebral cortex, and the GFAP positive astrocytes in the subcortical white matter (p < 0.02). The GFAP and MT-I-II expressions in subsets of reactive astrocytes in the cortico-subcortical layers together with regressive astroglial changes in the deep white matter suggest that the dynamic plasticity of astroglia is topographically and biochemically differentiated in vascular dementia of Binswanger type.