Whole genome methylation profiles as independent markers of survival in stage iiic melanoma patients

ABSTRACT: BACKGROUND: The clinical course of cutaneous melanoma (CM) can differ significantly for patients with identical stages of disease, defined clinico-pathologically, and no molecular markers differentiate patients with such a diverse prognosis. This study aimed to define the prognostic value of whole genome DNA methylation profiles in stage III CM. METHODS: Genome-wide methylation profiles were evaluated by the Illumina Human Methylation 27 BeadChip assay in short-term neoplastic cell cultures from 45 stage IIIC CM patients. Unsupervised K-means partitioning clustering was exploited to sort patients into 2 groups based on their methylation profiles. Methylation patterns related to the discovered groups were determined using the nearest shrunken centroid classification algorithm. The impact of genome-wide methylation patterns on overall survival (OS) was assessed using Cox regression and Kaplan-Meier analyses. RESULTS: Unsupervised K-means partitioning by whole genome methylation profiles identified classes with significantly different OS in stage IIIC CM patients. Patients with a "favorable" methylation profile had increased OS (P = 0.001, log-rank = 10.2) by Kaplan-Meier analysis. Median OS of stage IIIC patients with a "favorable" vs. "unfavorable" methylation profile were 31.5 and 10.4 months, respectively. The 5 year OS for stage IIIC patients with a "favorable" methylation profile was 41.2% as compared to 0% for patients with an "unfavorable" methylation profile. Among the variables examined by multivariate Cox regression analysis, classification defined by methylation profile was the only predictor of OS (Hazard Ratio = 2.41, for "unfavorable" methylation profile; 95% Confidence Interval: 1.02-5.70; P = 0.045). A 17 gene methylation signature able to correctly assign prognosis (overall error rate = 0) in stage IIIC patients on the basis of distinct methylation-defined groups was also identified. CONCLUSIONS: A discrete whole-genome methylation signature has been identified as molecular marker of prognosis for stage IIIC CM patients. Its use in daily practice is foreseeable, and promises to refine the comprehensive clinical management of stage III CM patients.     

Perception of effort reflects central motor command during movement execution

It is thought that perception of effort during physical tasks is the conscious awareness of the central motor command sent to the active muscles. The aim of this study was to directly test this hypothesis by experimentally varying perception of effort and measuring movement-related cortical potential (MRCP). Sixteen healthy, recreationally active men made unilateral dynamic elbow flexions to lift a light (20% one repetition maximum, 1RM) and a heavier (35% 1RM) weight with a fatigued arm and a nonfatigued arm while rating of perceived effort (RPE), biceps brachii electromyogram (EMG), and MRCP were recorded. RPE, EMG amplitude, and MRCP amplitude at Cz during weight raising increased with weight and with muscle fatigue. There was a significant correlation between RPE and MRCP amplitude at the vertex during the weight raising epoch. This study provides direct neurophysiological evidence that perception of effort correlates with central motor command during movement execution.     

Ferroportin is a monomer in vivo in mice

Ferroportin (FPN) is the main iron export protein in mammals. The actual structure of FPN in vivo and the pathogenesis of ferroportin-related disease are unknown. We aimed at studying the structure and biochemical properties of FPN in mouse tissues that are key for iron homeostasis during various iron manipulations in vivo. We performed glycosylation and oligomerization studies in spleen and liver extracts from mice fed a standard, iron-deprived or iron-enriched diet for 5 months. Purification by affinity chromatography and sucrose gradient show that FPN is not part of a large multiprotein complex. Dietary manipulations did not affect the monomeric status of the native or denatured protein. The glycosylation studies showed that ferroportin is digested by peptide: N-glycosidase F but not by endoglycosidase H. The same results were obtained using protein extracts from iron-deficient or iron-loaded mice. In conclusion, our studies indicate that mouse FPN, regardless of the tissue iron status, is glycosylated but not enriched in mannose residues, and that exists mainly in monomeric form. The latter finding may have important implications for understanding the pathogenesis of the disease due to ferroportin mutations.     

The lack of Pneumococcal surface protein C (PspC) increases the susceptibility of Streptococcus pneumoniae to the killing by microglia

Microglial cells, the resident phagocytes in the brain, share many phenotypical and functional characteristics with peripheral macrophages, suggesting that they may participate in an innate immune response against microorganisms invading the central nervous system (CNS). In this study, we demonstrate that the microglial cells constitutively exhibit antibacterial activity in vitro against Streptococcus pneumoniae. By using a Pneumococcal surface protein C (PspC)-deleted strain and its wild-type counterpart, we found that the extent of such an activity is significantly influenced by the presence of a PspC molecule on the bacterial surface. The PspC– mutant FP20 is indeed more susceptible than the PspC+ strain HB565 to microglial killing. Interestingly, this phenomenon is observed when using a medium supplemented with heat-inactivated foetal bovine serum (FBS). Electron microscopy studies indicate that the microglial cells interact more efficiently with PspC– than with PspC+ pneumococci. Moreover, upon infection with the PspC– mutant, microglial cells produce levels of TNF-, MIP-2, IL-10 and nitric oxide, significantly higher than those observed with PspC+ bacteria. These findings indicate that the lack of PspC significantly enhances the susceptibility of S. pneumoniae to both bactericidal activity and secretory response by the microglial cells, suggesting that this molecule may play an important role in the invasion of CNS by pneumococcus.     

p70S6 kinase mediates breast cancer cell survival in response to surgical wound fluid stimulation

In early breast cancer, local relapses represent a determinant and not simply an indicator of risk for distant relapse and death. Notably, 90% of local recurrences occur at or close to the same quadrant of the primary cancer. Relevance of PI3K/mTOR/p70S6K signaling in breast tumorigenesis is very well documented. However, the pathway/s involved in the process of breast cancer local relapse are not well understood. The ribosomal protein p70S6K has been implicated in breast cancer cell response to post‐surgical inflammation, supporting the hypothesis that it may be crucial also for breast cancer recurrence. Here, we show that p70S6K activity is required for the survival of breast cancer cells challenged in “hostile” microenvironments. We found that impairment of p70S6K activity in breast cancer cells strongly decreased their tumor take rate in nude mice. In line with this observation, if cells were challenged to grow in anchorage independence or in clonogenic assay, growth of colonies was strongly dependent on an intact p70S6K signaling. This in vitro finding was particularly evident when breast cancer cells were grown in the presence of wound fluids harvested following surgery from breast cancer patients, suggesting that the stimuli present in the post‐surgical setting at least partially relied on activity of p70S6K to stimulate breast cancer relapse. From a mechanistic point of view, our results indicated that p70S6K signaling was able to activate Gli1 and up‐regulate the anti‐apoptotic protein Bcl2, thereby activating a survival response in breast cancer cells challenged in hostile settings. Our work highlights a previously poorly recognized function of p70S6K in preserving breast cancer cell survival, which could eventually be responsible for local relapse and opens the way to the design of new and more specific therapies aiming to restrain the deleterious effects of wound response.     

Patau syndrome with long survival in a case of unusual mosaic trisomy 13

We report a 12-year-old patient with Patau syndrome, in whom two cell lines were present from birth, one with total trisomy 13 due to isochromosome (13q), and one with partial trisomy 13. A cytogenetic re-evaluation at 9 years of age brought to light in skin fibroblasts a third cell line, partially monosomic for chromosome 13. The derivatives (13) present in the three cell lines were characterized through fluorescence in situ hybridization (FISH) experiments with suitable probes; the results suggested a sequence of rearrangements which beginning from an isochromosome (13q) could have led to the other two derivatives. We report the clinical data at birth and at the age of 12; at this age pigmentary lesions with phylloid pattern were noted. Cytogenetic findings of the chromosomal analyses on different tissues, including skin fibroblasts from differently pigmented areas, are also reported.     

Does excessive daytime sleepiness contribute to explaining the association between obesity and ADHD symptoms?

Recent studies suggest a significant association between obesity and attention-deficit/hyperactivity disorder (ADHD). The factors underlying this newly described comorbidity are still unclear and unexplored. In the present article, we propose that excessive daytime sleepiness (EDS) contributes to explaining the association between ADHD and obesity. The background for this hypothesis comes from studies on the association between ADHD and EDS, as well as from investigations on EDS in obese individuals. Available studies suggest that ADHD behaviours are significantly associated with EDS. Moreover, increasing evidence indicates that obesity is significantly associated with EDS independently of sleep-disordered breathing (SDB) or any other sleep disorders. Given the relationship between EDS and ADHD behaviors, we hypothesize that the higher than expected rates of EDS in obese individuals contribute to explaining the association between obesity and ADHD behaviors. We further speculate on the role of the brain derived neurotrophic factor (BDNF) and other molecules such as the proinflammatory cytokines IL-6 and TNF-alpha. Our hypothesis generates potentially relevant clinical and therapeutic implications. From a clinical standpoint, it may suggest to systematically look for ADHD symptoms (including hyperactivity and impulsivity) in obese patients described as sleepy. With regard to the therapeutic implications, we suggest that wake-promoting agents with anorexigenic effect, such as mazindol, might be particularly indicated for the treatment of ADHD symptoms in obese patients, since they might address both ADHD symptoms and weight reduction. In conclusion, considering the burden that ADHD adds to obesity, we believe that further studies on the comorbidity between obesity and ADHD are necessary. Research on the role of EDS might allow advancements in this field, suggesting a more effective management and, ultimately, a better quality of life of patients with both obesity and ADHD.     

Attention-deficit/hyperactivity disorder, Tourette's syndrome, and restless legs syndrome: the iron hypothesis

Preliminary but increasing evidence suggests that attention-deficit/hyperactivity disorder (ADHD), Tourette's syndrome (TS), and restless legs syndrome (RLS) may be comorbid. In the present article, we hypothesize that ADHD, TS, and RLS may be part of a spectrum, and that iron deficiency contributes to the pathophysiology underlying this spectrum. Iron deficiency might lead to ADHD, RLS and TS symptoms via its impact on the metabolism of dopamine and other catecholamines, which have been involved into the pathophysiology of ADHD, TS, and RLS. We speculate that the catecholaminergic systems are differently impacted in each of the three disorders, contributing to a different specific phenotypic expression of iron deficiency. MRI studies assessing brain iron levels in ADHD, TS, and childhood RLS, as well as genetic studies on the specific molecular pathways involved in iron deficiency, are greatly needed to confirm the iron hypothesis underlying ADHD, TS, and RLS. This body of research may set the basis for controlled trials assessing the effectiveness and tolerability, as well as the most appropriate dose, duration and type (oral vs. intravenous) of iron supplementation. In conclusion, the iron hypothesis may help us progress in the understanding of pathophysiological links between ADHD, RLS, and TS, suggesting that iron supplementation might be effective for all these three impairing conditions.     

Cyclophosphamide inhibits the generation and function of CD8(+) regulatory T cells

CD8(+) regulatory T cells (Treg) and CD4(+)CD25(+) Treg infiltrate human cancers, thus favoring tumor immune escape. Therefore, in the setting of antitumor therapeutic protocols, it is important to associate antitumor treatment with agents that are able to inhibit Treg function. Cyclophosphamide (CY) has been demonstrated to be effective in counteracting CD4(+)CD25(+) Treg activity. Hence, we tested its inhibitory efficacy on human CD8(+) Treg. Because CY is a prodrug, 4-hydroperoxycyclophosphamide (4-HC), a derivative of CY that in aqueous solution is converted to 4-hydroxycyclophosphamide, an active metabolite of CY, was used. 4-HC significantly inhibited CD8(+) Treg generation and function but only at the higher tested concentration (0.5 μg/mL), that is, in the therapeutic range of the drug. The lower 4-HC concentration tested (0.1 μg/mL) was almost ineffective. 4-HC inhibitory effects were related to apoptosis/necrosis induction. When CD8(+)CD28(+) non-Treg were analyzed for comparative purposes, significantly lower cytotoxic rates among these cells were observed than among CD8(+) Treg, which were differentiated because they did not express the CD28 molecule. These data demonstrate that CD8(+) Treg are inhibited through cytotoxic phenomena by CY, thus supporting the use of this drug at adequate concentrations and schedules of administration as a Treg inhibitor in combinatorial chemo- or immunotherapeutic anticancer protocols.