Peptides Regulating Proliferative Activity and Inflammatory Pathways in the Monocyte/Macrophage THP-1 Cell Line.

This study evaluates the effects of five different peptides, the Epitalon® tetrapeptide, the Vilon® dipeptide, the Thymogen® dipeptide, the Thymalin® peptide complex, and the Chonluten® tripeptide, as regulators of inflammatory and proliferative processes in the human monocytic THP-1, which is a human leukemia monocytic cell line capable of differentiating into macrophages by PMA in vitro. These peptides (Khavinson Peptides®), characterized by Prof. Khavinson from 1973 onwards, were initially isolated from animal tissues and found to be organ specific. We tested the capacity of the five peptides to influence cell cultures in vitro by incubating THP-1 cells with peptides at certain concentrations known for being effective on recipient cells in culture. We found that all five peptides can modulate key proliferative patterns, increasing tyrosine phosphorylation of mitogen-activated cytoplasmic kinases. In addition, the Chonluten tripeptide, derived from bronchial epithelial cells, inhibited in vitro tumor necrosis factor (TNF) production of monocytes exposed to pro-inflammatory bacterial lipopolysaccharide (LPS). The low TNF release by monocytes is linked to a documented mechanism of TNF tolerance, promoting attenuation of inflammatory action. Therefore, all peptides inhibited the expression of TNF and pro-inflammatory IL-6 cytokine stimulated by LPS on terminally differentiated THP-1 cells. Lastly, by incubating the THP1 cells, treated with the peptides, on a layer of activated endothelial cells (HUVECs activated by LPS), we observed a reduction in cell adhesion, a typical pro-inflammatory mechanism. Overall, the results suggest that the Khavinson Peptides® cooperate as natural inducers of TNF tolerance in monocyte, and act on macrophages as anti-inflammatory molecules during inflammatory and microbial-mediated activity.

Metabolic Alterations, Aggressive Hormone-Naïve Prostate Cancer and Cardiovascular Disease: A Complex Relationship.

Background: Epidemiological studies suggest a possible relationship between metabolic alterations, cardiovascular disease and aggressive prostate cancer, however, no clear consensus has been reached. Objective: The aim of the study was to analyze the recent literature and summarize our experience on the association between metabolic disorders, aggressive hormone-naïve prostate cancer and cardiovascular disease. Method: We identified relevant papers by searching in electronic databases such as Scopus, Life Science Journals, and Index Medicus/Medline. Moreover, we showed our experience on the reciprocal relationship between metabolic alterations and aggressive prostate cancer, without the influence of hormone therapy, as well the role of coronary and carotid vasculopathy in advanced prostate carcinoma. Results: Prostate cancer cells have an altered metabolic homeostatic control linked to an increased aggressivity and cancer mortality. The absence of discrimination of risk factors as obesity, systemic arterial hypertension, diabetes mellitus, dyslipidemia and inaccurate selection of vascular diseases as coronary and carotid damage at initial diagnosis of prostate cancer could explain the opposite results in the literature. Systemic inflammation and oxidative stress associated with metabolic alterations and cardiovascular disease can also contribute to prostate cancer progression and increased tumor aggressivity. Conclusions: Metabolic alterations and cardiovascular disease influence aggressive and metastatic prostate cancer. Therefore, a careful evaluation of obesity, diabetes mellitus, dyslipidemia, systemic arterial hypertension, together with a careful evaluation of cardiovascular status, in particular coronary and carotid vascular disease, should be carried out after an initial diagnosis of prostatic carcinoma.

Differential TBXA2 receptor transcript stability is dependent on the C924T polymorphism.

BACKGROUND: In order to better characterize the molecular mechanisms involved in processing mutated transcripts, we investigated the post-transcriptional role of the C924T polymorphism (rs4523) located in the 3' region of the TBXA2R gene. METHODS AND RESULTS: Experiments of dose response with Actinomycin D on MEG-01 human cell line showed a significant decrease on cell viability that was more evident on cells treated for 24h. In addition, we showed that treatments with 5-10µM, 15µM and 20µM of actinomycin D reduced cell viability by 44%, 72% and 75%, respectively, compared to the control group. Conversely, the samples treated with 1µM of actinomycin D did not show significant difference on cell viability as compared to the control group. Analysis of the steady state mRNA level of TBXA2R by qRT-PCR evidenced an increase in mRNA stability for the wild type (C) compared to the mutant (T) allele. Furthermore, the expression levels of TBXA2R on wild type (CC) and mutant type (TT) patients, based on C924T polymorphism, were analyzed. The wild type showed a higher expression of TBXA2 receptor also with two different degrees of glycosylation (55 and 64kDa), when compared to the mutant. These observations correlated with platelet aggregation, which was reduced in TT, independently of the platelet aggregation stimuli. CONCLUSIONS: The instability of the TBXA2R transcript and the lack of effect on platelet aggregation might suggest a protective role for the TBXA2R TT genotype against atherothrombosis and its complications in high-risk aspirin-treated patients.

Mast cell activation and autism.

Autism spectrum disorders (ASD) are neurodevelopmental disorders characterized by varying degrees of dysfunctional communication and social interactions, repetitive and stereotypic behaviors, as well as learning and sensory deficits. Despite the impressive rise in the prevalence of autism during the last two decades, there are few if any clues for its pathogenesis, early detection or treatment. Increasing evidence indicates high brain expression of pro-inflammatory cytokines and the presence of circulating antibodies against brain proteins. A number of papers, mostly based on parental reporting on their children's health problems, suggest that ASD children may present with "allergic-like" problems in the absence of elevated serum IgE and chronic urticaria. These findings suggest non-allergic mast cell activation, probably in response to environmental and stress triggers that could contribute to inflammation. In utero inflammation can lead to preterm labor and has itself been strongly associated with adverse neurodevelopmental outcomes. Premature babies have about four times higher risk of developing ASD and are also more vulnerable to infections, while delayed development of their gut-blood-brain barriers makes exposure to potential neurotoxins likely. Perinatal mast cell activation by infectious, stress-related, environmental or allergic triggers can lead to release of pro-inflammatory and neurotoxic molecules, thus contributing to brain inflammation and ASD pathogenesis, at least in a subgroup of ASD patients. This article is part of a Special Issue entitled: Mast cells in inflammation.

Anti-Obesity and Anti-Inflammatory Effects of Novel Carvacrol Derivatives on 3T3-L1 and WJ-MSCs Cells.

(1) Background: Obesity, a complex metabolic disease resulting from an imbalance between food consumption and energy expenditure, leads to an increase in adipocytes and chronic inflammatory conditions. The aim of this paper was to synthesize a small series of carvacrol derivatives (CD1-3) that are able to reduce both adipogenesis and the inflammatory status often associated with the progression of the obesity disease. (2) Methods: The synthesis of CD1-3 was performed using classical procedures in a solution phase. Biological studies were performed on three cell lines: 3T3-L1, WJ-MSCs, and THP-1. The anti-adipogenic properties of CD1-3 were evaluated using western blotting and densitometric analysis by assessing the expression of obesity-related proteins, such as ChREBP. The anti-inflammatory effect was estimated by measuring the reduction in TNF-α expression in CD1-3-treated THP-1 cells. (3) Results: CD1-3-obtained through a direct linkage between the carboxylic moiety of anti-inflammatory drugs (Ibuprofen, Flurbiprofen, and Naproxen) and the hydroxyl group of carvacrol-have an inhibitory effect on the accumulation of lipids in both 3T3-L1 and WJ-MSCs cell cultures and an anti-inflammatory effect by reducing TNF- α levels in THP-1 cells. (4) Conclusions: Considering the physicochemical properties, stability, and biological data, the CD3 derivative-obtained by a direct linkage between carvacrol and naproxen-resulted in the best candidate, displaying anti-obesity and anti-inflammatory effects in vitro.

Mitochondrial and metabolic alterations in cancer cells.

Metabolic alterations have been observed in many cancer types. The deregulated metabolism has thus become an emerging hallmark of the disease, where the metabolism is frequently rewired to aerobic glycolysis. This has led to the concept of "metabolic reprogramming", which has therefore been extensively studied. Over the years, it has been characterized the enhancement of aerobic glycolysis, where key mutations in some of the enzymes of the TCA cycle, and the increased glucose uptake, are used by cancer cells to achieve a "metabolic phenotype" useful to gain a proliferation advantage. Many studies have highlighted in detail the signaling pathways and the molecular mechanisms responsible for the glycolytic switch. However, glycolysis is not the only metabolic process that cancer cells rely on. Oxidative Phosphorylation (OXPHOS), gluconeogenesis or the beta-oxidation of fatty acids (FAO) may be involved in the development and progression of several tumors. In some cases, these metabolisms are even more crucial than aerobic glycolysis for the tumor survival. This review will focus on the contribution of these alterations of metabolism to the development and survival of cancers. We will also analyze the molecular mechanisms by which the balance between these metabolic processes may be regulated, as well as some of the therapeutical approaches that can derive from their study.

Dissecting the Functional Role of the TRIM8 Protein on Cancer Pathogenesis.

TRIM/RBCC are a large family of proteins that include more than 80 proteins, most of which act as E3 ligases and catalyze the direct transfer of Ubiquitin, SUMO and ISG15 on specific protein substrates. They are involved in oncogenesis processes and in cellular immunity. On this topic, we focus on TRIM8 and its multiple roles in tumor pathologies. TRIM8 inhibits breast cancer proliferation through the regulation of estrogen signaling. TRIM8 downregulation in glioma is involved in cell proliferation, and it is related to patients' survival. Several studies suggested that TRIM8 regulates the p53 suppressor signaling pathway: it is involved in the NF-kB pathway (Nuclear Factor kappa light- chain-enhancer of activated B cells) and in STAT3 (Signal Transducer and Activator of Transcription 3) of the JAK-STAT pathway. In this review, we summarize how the association between these different pathways reflects a dual role of TRIM8 in cancer as an oncogene or a tumor suppressor gene.

The kidney, COVID-19, and the chemokine network: an intriguing trio.

On December 30th 2019, some patients with pneumonia of unknown etiology were reported in the Program for Monitoring Emerging Diseases (ProMED), a program run by the International Society for Infectious Diseases (ISID), hypothesized to be related to subjects who had had contact with the seafood market in Wuhan, China. Chinese authorities instituted an emergency agency aimed at identifying the source of infection and potential biological pathogens. It was subsequently named by the World Committee on Virus Classification as 2019-nCoV (2019-novel coronavirus) or severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). A number of studies have demonstrated that 2019-nCoV and the SARS-CoV shared the same cell entry receptor named angiotensin-converting enzyme 2 (ACE2). This is expressed in human tissues, not only in the respiratory epithelia, but also in the small intestines, heart, liver, and kidneys. Here, we examine the most recent findings on the effects of SARS-CoV-2 infection on kidney diseases, mainly acute kidney injury, and the potential role of the chemokine network.

The Epithelial-to-Mesenchymal Transition as a Possible Therapeutic Target in Fibrotic Disorders.

Fibrosis is a chronic and progressive disorder characterized by excessive deposition of extracellular matrix, which leads to scarring and loss of function of the affected organ or tissue. Indeed, the fibrotic process affects a variety of organs and tissues, with specific molecular background. However, two common hallmarks are shared: the crucial role of the transforming growth factor-beta (TGF-ß) and the involvement of the inflammation process, that is essential for initiating the fibrotic degeneration. TGF-ß in particular but also other cytokines regulate the most common molecular mechanism at the basis of fibrosis, the Epithelial-to-Mesenchymal Transition (EMT). EMT has been extensively studied, but not yet fully explored as a possible therapeutic target for fibrosis. A deeper understanding of the crosstalk between fibrosis and EMT may represent an opportunity for the development of a broadly effective anti-fibrotic therapy. Here we report the evidences of the relationship between EMT and multi-organ fibrosis, and the possible therapeutic approaches that may be developed by exploiting this relationship.

The Impact of Metabolic Syndrome and Its Components on Female Sexual Dysfunction: A Narrative Mini-Review.

BACKGROUND: The impact of metabolic syndrome on female sexual dysfunction received modest consideration in clinical practice. The aim of the research was to analyze the international literature to determine the relationship between the metabolic syndrome, its components and female sexual disorders. METHODS: We identified relevant full-length papers by electronic databases as Index Medicus/Medline, Scopus, Life Science Journals, from 2005 to the present. Studies were searched using the following as search query: metabolic syndrome, female sexual dysfunction, obesity, systemic arterial hypertension, diabetes mellitus, dyslipidemia. RESULTS: Women with metabolic syndrome showed higher prevalence of sexual inactivity and low sexual desire, orgasm and satisfaction respect to women without metabolic syndrome. Particularly metabolic components as diabetes mellitus, dy-slipidemia, systemic arterial hypertension were strongly associated with lower sexual desire, activity and Female Sexual Function Index total score. In contrast, other studies showed no relationship. CONCLUSION: Our study showed that in the clinical evaluation of women with metabolic syndrome routine inquiring about female sexual dysfunction should be recommended to ameliorate sexual function and quality of life. However more prospective and longitudinal studies on the sexual effects of metabolic syndrome should also be suggested to know the factors related to women's sexuality better.

A Method to Study the C924T Polymorphism of the Thromboxane A2 Receptor Gene.

The thromboxane A2 receptor (TBXA2R) gene is a member of the G-protein coupled superfamily with seven-transmembrane regions. It is involved in atherogenesis progression, ischemia, and myocardial infarction. Here we present a methodology of patient genotyping to investigate the post-transcriptional role of the C924T polymorphism (rs4523) situated at the 3' region of the TBXA2 receptor gene. This method relies on DNA extraction from whole blood, polymerase chain reaction (PCR) amplification of the TBXA2 gene portion containing the C924T mutation, and identification of wild type and/or mutant genotypes using a restriction digest analysis, specifically a restriction fragment length polymorphism (RFLP) on agarose gel. In addition, the results were confirmed by sequencing the TBXA2R gene. This method features several potential advantages, such as high efficiency and the rapid identification of the C924T polymorphism by PCR and restriction enzyme analysis. This approach allows a predictive study for plaque formation and atherosclerosis progression by analyzing patient genotypes for the TBXA2R C924T polymorphism. Application of this method has the potential to identify subjects who are more susceptible to atherothrombotic processes, in particular subjects in a high-risk, aspirin-treated group.

Angiotensin receptor blockers improve insulin signaling and prevent microvascular rarefaction in the skeletal muscle of spontaneously hypertensive rats.

OBJECTIVE: Spontaneously hypertensive rats are an example of an animal model of genetic hypertension with insulin resistance. The aim of this study was to investigate insulin signaling in the heart and in the skeletal muscle of spontaneously hypertensive rats, as well as to evaluate the effects of renin-angiotensin system blockade. DESIGN AND METHODS: We investigated eight untreated spontaneously hypertensive rats of 12 weeks of age and eight age-matched normotensive Wistar-Kyoto controls. In addition, eight spontaneously hypertensive rats were treated for 8 weeks with the angiotensin receptor blocker olmesartan, and eight spontaneously hypertensive rats with the angiotensin-converting enzyme inhibitor enalapril. The heart and a skeletal muscle (quadriceps femoris) were promptly dissected and frozen. Insulin signaling was evaluated by Western blot analysis of involved proteins; in addition, microvessel density was indirectly evaluated by immunohistochemistry. RESULTS: Blood pressure values were normalized by both olmesartan and enalapril. In the heart, no statistically significant difference in the expression of proteins involved in insulin signaling was observed between untreated spontaneously hypertensive rats and Wistar-Kyoto controls. On the contrary, in the skeletal muscle of untreated spontaneously hypertensive rats, we noted a significant reduction of insulin receptors, of insulin-receptor substrate-1, and of phosphorylated-mammalian target of rapamycin. The treatment with olmesartan normalized insulin signaling, including expression of glucose transporter-4, whereas the treatment with enalapril was ineffective for the insulin receptor and less effective than olmesartan on the insulin-receptor substrate-1, phosphorylated-mammalian target of rapamycin and glucose transporter-4. There was a significant reduction in microvessel density in the skeletal muscle of spontaneously hypertensive rats compared with Wistar-Kyoto controls, and this was completely prevented by both olmesartan and enalapril. CONCLUSION: These results suggest that changes in insulin signaling occur in the skeletal muscle but not in the heart of untreated spontaneously hypertensive rats. In the skeletal muscle, insulin signaling was restored by olmesartan, whereas enalapril was less effective. Effective antihypertensive treatment with olmesartan or enalapril was associated with prevention of microvascular rarefaction.

Intracellular mechanisms of metabolism regulation: the role of signaling via the mammalian target of rapamycin pathway and other routes.

The development of diabetes mellitus is the consequence of defects in the action of insulin in skeletal muscles and adipose tissue other than pancreas and liver defects. Insulin action defects are mostly the results of defects of the insulin intracellular signaling transduction pathway. This review summarizes the main mechanisms involved in insulin signaling and possible intracellular defects that lead to insulin resistance. We also report preliminary experimental data that suggest the existence of intracellular alternatives to insulin metabolic pathways stimulated by nutrients such as amino acids (AAs). Indeed, we found that oral supplements with AAs stimulated both glucose transporter-4 and protein synthesis through independent insulin signals in rat hearts. Evidence suggests that the mammalian target of rapamycin and/or other molecules could be involved in this insulin-independent metabolic pathway. This hypothesis suggests the presence of an ancestral metabolic pathway in eukaryotic cells that is not active when insulin intracellular signaling is efficient but can be activated by alternative stimuli, such as AAs, when insulin signaling is impaired. Our observations provide molecular evidence that supports the use of anabolic nutrients such as AAs, together with standard therapies, to overcome insulin resistance syndrome.

Psychobiome Feeding Mind: Polyphenolics in Depression and Anxiety.

Recently gut bacterial populations seem to be involved in many functions and in the pathogenesis of several medical conditions. Traditionally the intestinal microbiome has been recognized to play an important role in metabolizing food compounds in simpler chemical structures for the absorption of different nutrients, and in maintenance control of gastrointestinal pathogens species. Bacterial populations are implicated in a complicated network of interactions within the immune system, epithelial cells local endocrine system, that affects the peripheral and the central nervous system, via blood circulation. Microbiome influencing the mind via immune, endocrine and metabolic signalling, is able to exert some clinical effects in different mental diseases. It releases endocrine substances through several pathways involved in the modulation of neuroinflammation and production of several neurotrasmitter precursors. It has recently been named psychobiome. It is known that phenolic compounds are able to influence microbiome proliferation and to exert several roles, especially regarding neuroinflammation in depressive and anxious behaviour. The clinical effects are reported in the literature. The aim of this study is to highlight the interaction between polyphenols and microbiota- gut-brain axis.

Carvacrol reduces adipogenic differentiation by modulating autophagy and ChREBP expression.

OBJECTIVE: Obesity is the result of white adipose tissue accumulation where excess of food energy is stored to form triglycerides. De novo lipogenesis (DNL) is the continuous process of new fat production and is driven by the transcription factor ChREBP. During adipogenesis, white adipocytes change their morphology and the entire cell volume is occupied by one large lipid droplet. Recent studies have implicated an essential role of autophagy in adipogenic differentiation, cytoplasmic remodelling and mitochondria reorganization. The phenolic monoterpenoid carvacrol (2-methyl-5-[1-methylethyl]phenol), produced by numerous aromatic plants, has been shown to reduce lipid accumulation in murine 3T3-L1 cells during adipogenic differentiation by modulating genes associated with adipogenesis and inflammation. Therefore, the aim of this study was to evaluate whether carvacrol could affect autophagy and ChREBP expression during adipogenic differentiation. METHODS: The study was carried on by using the murine 3T3-L1 and the human WJ-MSCs (Wharton's jelly-derived mesenchymal stem cells) cell lines. Cells undergoing adipogenic differentiation were untreated or treated with carvacrol. Adipogenic differentiation was assessed by analyzing cellular lipid accumulation with Oil-Red O staining and by ultrastructural examination with TEM. Autophagy was evaluated by western immunoblotting of autophagy markers LC3B and p62/SQSTM and by ultrastructural examination of autophagic bodies. Autophagic flux was evaluated by using autophagy inhibitor cloroquine (CQ). ChREBP expression levels was assessed by both western blotting and immunoelectron microscopy and ChREBP activity by analysis of adipogenic target genes expression. RESULTS: We found that carvacrol reduced adipogenic differentiation of about 40% and 30% in, respectively, 3T3-L1 and in WJ-MSCs cells. The effect of carvacrol on adipogenic differentiation correlated with both reduction of autophagy and reduction of ChREBP expression. CONCLUSION: The results support the notion that carvacrol, through its effect on autophagy (essential for adipocyte maturation) and on ChREBP activity, could be used as a valuable adjuvant to reduce adipogenic differentiation.

Autophagy processes are dependent on EGF receptor signaling.

Autophagy is a not well-understood conserved mechanism activated during nutritional deprivation in order to maintain cellular homeostasis. In the present study, we investigated the correlations between autophagy, apoptosis and the MAPK pathways in melanoma cell lines. We demonstrated that during starvation the EGF receptor mediated signaling activates many proteins involved in the MAPK pathway. Our data also suggest a previously unidentified link between the EGFR and Beclin-1 in melanoma cell line. We demonstrated that, following starvation, EGFR binds and tyrosine-phosphorylates Beclin-1, suggesting that it may play a key inhibitory role in the early stage of starvation, possibly through the Beclin-1 sequestration. Furthermore, EGFR releases Beclin-1 and allows initiating steps of the autophagic process. Interestingly enough, when the EGFR pathway was blocked by anti-EGF antibodies, immunoprecipitated Beclin-1 did not bind the phospho-EGFR. In addition, an extended binding of p-Bcl2 either with Beclin-1 or with Bax was observed with a decreased activation of the stress-induced JNK kinase, thus avoiding the transduction pathways that activate autophagy and apoptosis, respectively. For this reason, we advance the hypothesis that the activation of the EGFR is a necessary event that allows the ignition and progression of the autophagic process, at least in melanoma cells.

Analytical Management of Patients Undergoing Oral Anticoagulant Therapy Could Have a Strong Impact on Clinical Outcomes: A Follow-up Study.

INTRODUCTION: Oral anticoagulant therapy, such as vitamin K antagonists (VKAs), is prominent for the prevention of cerebral ischemic stroke or systemic embolism and all-cause mortality in patients with atrial fibrillation, venous thromboembolism, and mechanical or biological valve. VKA treatment requires monitoring of the international normalized ratio (INR) in order to maintain it in a therapeutic range, avoiding side effects, the main and most significant of which is bleeding. The aim of the present study was to evaluate the event rates of several clinical composite outcomes, such as bleeding, thromboembolic events, and all-cause death. METHODS: We compared three organizational models distinguished by a total (from 1 January to 31 December 2015 in which PT/INR analysis with the relative internal and external quality controls was performed by the surveillance center) or partial (from 15 January to 15 July 2016 and from 15 August to 15 November 2016, in which the surveillance center had the ability to view only the PT/INR results or all patients analyses, including blood count, creatinine, liver enzymes, etc., respectively) analytical patient management. The present longitudinal follow-up study included 1225 patients, recruited from 1 January 2015 to 15 November 2016 at a surveillance center for the prevention of cerebral ischemic stroke and systemic embolism in Chieti (Italy). RESULTS: The results show a significant rise of the incidence rate ratio in patients undergoing VKA treatment during the period 15 January to 15 July 2016 compared to the previous one regarding total bleeding, especially for minor bleeding and digestive bleeding; thromboembolic events; and all-death cause. CONCLUSIONS: These findings show that analytical and clinical data and information should be under the direct supervision and responsibility of the surveillance center. In fact, this approach seems to highlight the best results in terms of safety and therapeutic effectiveness.

Comparison of Serum Total Valproic Acid Levels and %CDT Values in Chronic Alcohol Addictive Patients in an Italian Clinic: A Retrospective Study.

BACKGROUND: Valproate is a broad-spectrum anticonvulsant that is effective in the treatment of tonic-clonic, myoclonic and absence seizures as well as in partial seizures as a second-line drug. It has been widely demonstrated in the literature that the effect of valproate on type-A γ-aminobutyric acid (GABA-A) receptors may reduce relapse to ethanol abuse. This retrospective study evaluated a 3-year period in which 42 patients from the Department of Alcoholism and Substance Abuse (DASA) were treated with valproate. OBJECTIVES: We compared different serum total valproic acid (VPA) concentrations, and the effectiveness of this drug in maintaining alcohol abstinence was evaluated by percentage of carbohydrate deficient transferrin (%CDT) values. METHOD: CDT is a biochemical marker used for identifying regular high alcohol consumption and monitoring abstinence in outpatients during treatment. Serum concentrations of valproate were divided into four groups: <10, 10-30, 31-50, and >50 µg/mL. RESULTS: This study shows that a mean serum total VPA concentration >30 µg/mL is more effective in maintaining alcohol abstinence than a lower one (p < 0.05). In this study, mean serum total VPA concentrations between 31 and 50 µg/mL showed the same effectiveness as higher ones (>50 µg/mL); in fact, there was no significant difference in mean %CDT values between these two groups (p > 0.05). After at least 12 months' treatment with valproate, mean platelet counts increased by 12 × 103/µL compared with baseline (254 ± 63 vs 242 × 103/µL, p > 0.05, respectively) in patients with mean serum total VPA levels <10 µg/mL; increased by 8 × 103/µL from baseline (253 ± 59 vs 245 × 103/µL, p > 0.05, respectively) in patients with levels between 10 and 30 µg/mL; decreased by 2 × 103/µL from baseline (265 ± 63 vs 267 × 103/µL, p > 0.05, respectively) in patients with levels between 31 and 50 µg/mL, and decreased by 48 × 103/µL from baseline (215 ± 56 vs 263 × 103/µL, p < 0.05, respectively) in patients with levels >50 µg/mL. CONCLUSION: A mean serum total concentration lower than the currently accepted therapeutic level (50-100 µg/mL) may have the same effectiveness in maintaining alcohol abstinence with a lower risk of presenting side effects.

P16 hypermethylation contributes to the characterization of gene inactivation profiles in primary gastric cancer.

The objective of this study was to investigate the contribution of p16 inactivation in gastric cancer and to compare it with p53. A cohort of 34 primary GCs were analyzed for p16 mutations and transcriptional silencing of the gene due to hypermethylation of the promoter. SSCP analysis and direct sequencing of exons 1 and 2 of the p16 gene were performed to detect any structural alterations. The methylation specific PCR (MSP) assay was applied to reveal hypermethylation of the 'CpG' island in the regulatory region using specific primer pairs for methylated and unmethylated nucleotides after a chemical reaction converting cytosines into uracile when unmethylated. SSCP and direct sequencing analysis did not detect any p16 mutations. The MSP assay showed 4 MSP(+) variants (11.8%). Three MSP(+) were stage III-IV disease and 1 MSP(+) was detected in an early stage disease (IB). All MSP(+) were diffuse type adenocarcinomas. The MSP(+) samples were different from previously reported samples harboring p53 mutations in the same cohort. These data increase the number of gastric cancers showing alterations of either p53 or p16 to 29.4% (10/34). Functional inactivation by hypermethylation of the p16 locus and p53 mutations could play a significant, complementary role in the pathogenesis of sporadic gastric cancer.