ERK1/2 pro-survival signalling is suppressed by pirtobrutinib in ibrutinib-resistant MYD88-mutated lymphoma cells.

Covalent Bruton's tyrosine kinase-inhibitors (cBTK-i) are highly active in MYD88-mutated (MYD88Mut) Waldenstrom's macroglobulinaemia and suppress nuclear factor kappa-light-chain-enhancer of activated B cells and extracellular signal-regulated kinases-1/2 (ERK1/2)-related signalling. BTKCys481 mutations are associated with cBTK-i acquired resistance and are accompanied by reactivation of ERK1/2 that promotes inflammatory cytokine secretion and paracrine-mediated resistance of BTK wild-type (BTKWT) tumour cells. Pirtobrutinib is a non-covalent BTK-inhibitor that binds at non-BTKCys481 sites. We show that pirtobrutinib blocked p-ERK1/2, ERK1/2-driven inflammatory cytokines, and overcame paracrine-mediated resistance in MYD88Mut lymphoma cells expressing mutated BTKCys481. Our results provide important mechanistic insights for the activity of pirtobrutinib in MYD88Mut lymphomas carrying BTKCys481 mutations.

Infectious disease associations in advanced stage, indolent lymphoma (follicular and nonfollicular): developing a lymphoma prevention strategy.

BACKGROUND: Eradication of Helicobacter pylori in gastric mucosa-associated lymphoid tumor can result in lymphoma remission. We prospectively identified/treated infections in nonbulky, advanced stage indolent lymphoma (follicular; nonfollicular lymphoma) eligible for observation. MATERIALS AND METHODS: Stool H. pylori, hepatitis C and Borrelia serologies, Borrelia and Chlamydia fixed tissue PCR, Chlamydia peripheral blood mononuclear cell PCR and hydrogen breath test for small bowel bacterial overgrowth (SBBO) were obtained. RESULTS: Fifty-six patients were enrolled. Positive infections: H. pylori (13); hepatitis C (3); SBBO (11). Negative: Borrelia (13); Chlamydophila psittaci (12, except one PCR). Lymphoma responses to antimicrobial therapy: H. pylori [one complete response (CR), 24+ months; one transient near CR]; hepatitis C [two CRs, 18+ and 30+ months; one partial response (PR) but hepatitis C virus persistent]; SBBO (one PR, 30+ months). Patients with associated infections, but without lymphoma CR, have required lymphoma treatment sooner than those without initial infections (treatment-free survival at 23.4 months median follow-up, 40.5% versus 74.7%, P = 0.01), indicating a different biology. CONCLUSION: Infections are common in advanced stage indolent lymphoma (37.5% in our series). Anecdotal lymphoma responses have been seen and three have been durable CRs (18 to 30+ months) with infection eradication alone. The identification and treatment of associated infections may be a first step towards developing a lymphoma prevention strategy.

Descriptive data on lifestyle, anthropometric status and mental health in italian elderly people.

OBJECTIVE: The objective of this paper is to provide descriptive information on anthropometric status, pathological conditions, cognitive impairment and lifestyle in apparently healthy elderly Italian people. DESIGN, SETTING AND SUBJECTS: In order to recruit the volunteers for the ZENITH study, 359 Italian participants (167 men and 192 women), aged between 70 and 85 years, free living in Rome, were selected. Volunteers underwent a full clinical examination, anthropometric measurements (height, weight), a lifestyle questionnaire and mental health assessment (cognitive impairment and depression). RESULTS: The prevalence of overweight and obesity was high (57% and 22% in men; 43% and 27% in women). Obesity was associated with low socio-economic profile in about 40% of participants. Although the sample was selected by family doctors and was apparently healthy, after medical screening the presence of several pathologies, particularly diabetes in 21% of participants was observed. There was a low prevalence of cognitive impairment in 4% of men and 7% of women and possible depression in 9% of men and 19% of women. The lifestyle questionnaire showed that most of their time was spent in light activities such as reading, watching TV or playing cards and significant differences between sex and BMI categories were observed (P=0.000). CONCLUSION: The results confirm the increasingly sedentary lifestyle of modern populations and demonstrate the need for sensitive and individualised strategies to design appropriate health promotion and disease prevention programs for older adults.

Effect of Red Orange Juice Consumption on Body Composition and Nutritional Status in Overweight/Obese Female: A Pilot Study.

The main objective of this research was to determine whether a commercial orange juice rich in anthocyanins could have an effect on body weight and on clinical parameters related to obesity including antioxidant status, lipid profile, and metabolic and inflammatory biomarkers. 11 women with an average BMI of 34.4 ± 4.8 kg/m2 were enrolled in a pilot study. Over a period of 12 weeks they received 500 mL daily dose into two doses (250 mL) of commercial red orange juice (COJ). The biochemical parameters were measured at baseline and at the end of the study (12 weeks). One month later upon free diet, a follow-up was performed measuring the same variables. The daily consumption of 500 mL of COJ had no significant effects on body weight, while there was a decrease in total cholesterol and LDL cholesterol. The grade of obesity implies different changes in inflammation biomarkers. In obese women, our data do not seem to support evidence that commercial red orange juice consumption acts as functional food preventing obesity and metabolic disorders such as insulin resistance and/or inflammatory status.

3-Aminobenzamide inhibition of protein kinase C at a cellular level.

3-Aminobenzamide, a known inhibitor of poly-(ADP-ribose)-polymerase has been found in the cell line U-937 to inhibit protein kinase C at the same concentration as poly-(ADP-ribose)-polymerase. 3-Aminobenzamide was not able, however, to inhibit the isolated enzyme. An indirect mechanism of protein kinase C inhibition is proposed.

Prevention of necrosis and activation of apoptosis in oxidatively injured human myeloid leukemia U937 cells.

A 3 h exposure to 1 mM H2O2 followed by 6 h post-challenge growth in peroxide-free medium induces necrosis in U937 cells. Addition of the poly(ADP-ribose)polymerase inhibitor 3-aminobenzamide during recovery prevents necrosis and triggers apoptosis, as shown by the appearance of apoptotic bodies, extensive blebbing and formation of multimeric DNA fragments as well as 50 kb double stranded DNA fragments. Thus, the same initial damage can be a triggering event for both apoptotic and necrotic cell death. Furthermore, necrosis does not appear to be a passive response to overwhelming damage.

Products of the phospholipase A(2) pathway mediate the dihydrorhodamine fluorescence response evoked by endogenous and exogenous peroxynitrite in PC12 cells.

A short-term exposure of PC12 cells to tert-butylhydroperoxide promotes a rapid oxidation of dihydrorhodamine sensitive to nitric oxide synthase inhibitors and peroxynitrite scavengers. This response was not directly caused by peroxynitrite, but rather appeared to be mediated by peroxynitrite-dependent activation of phospholipase A(2). The following lines of evidence support this inference: (i) the peroxynitrite-dependent dihydrorhodamine fluorescence response was blunted by low concentrations of two structurally unrelated phospholipase A(2) inhibitors; (ii) under similar conditions, the phospholipase A(2) inhibitors prevented release of arachidonic acid; (iii) low levels of arachidonic acid restored the dihydrorhodamine fluorescence response in nitric oxide synthase- as well as phospholipase A(2)-inhibited cells; (iv) the dihydrorhodamine fluorescence response induced by authentic peroxynitrite was also blunted by phospholipase A(2) inhibitors and restored upon addition of reagent arachidonic acid. We conclude that endogenous, or exogenous, peroxynitrite does not directly oxidize dihydrorhodamine in intact cells. Rather, peroxynitrite appears to act as a signalling molecule promoting release of arachidonic acid, which in turn leads to formation of species causing the dihydrorhodamine fluorescence response.

Peroxynitrite-mediated release of arachidonic acid from PC12 cells.

A short term exposure of PC12 cells to a concentration of tert-butylhydroperoxide (tB-OOH) causing peroxynitrite-dependent DNA damage and cytotoxiticity promoted a release of arachidonic acid (AA) that was sensitive to phospholipase A(2) (PLA(2)) inhibitors and insensitive to phospholipase C or diacylglycerol lipase inhibitors. The extent of AA release was also mitigated by nitric oxide synthase (NOS) inhibitors and peroxynitrite scavengers. Low levels (10 microM) of authentic peroxynitrite restored the release of AA mediated by tB-OOH in NOS-inhibited cells whereas concentrations of peroxynitrite of 20 microM, or higher, effectively stimulated a PLA(2) inhibitor-sensitive release of AA also in the absence of additional treatments. These results are consistent with the possibility that endogenous as well as exogenous peroxynitrite promotes activation of PLA(2).

Apoptosis and necrosis following exposure of U937 cells to increasing concentrations of hydrogen peroxide: the effect of the poly(ADP-ribose)polymerase inhibitor 3-aminobenzamide.

A 3-hr exposure of U937 cells to hydrogen peroxide (H2O2) followed by a 6-hr posttreatment incubation in fresh culture medium promotes apoptosis or necrosis, depending on the oxidant concentration. Addition of 3-aminobenzamide (3AB) during the recovery phase prevented necrosis and caused apoptosis. 3AB did not, however, affect the apoptotic response of cells treated with apogenic concentrations of H2O2. Cells exposed for 3 hr to 1.5 mM H2O2, while showing some signs of suffering, maintained a normal nuclear organization and good organelle morphology. At the biochemical level, the oxidant promoted the formation of Mb-sized DNA fragments and rapidly depleted both the adenine nucleotide and non-protein sulphydryl pools, which did not recover during posttreatment incubation in the absence or presence of 3AB. These results allow a novel interpretation of the concentration-dependent switch from apoptosis to necrosis. We propose that H2O2 activates the apoptotic response at the early times of peroxide exposure and that this process can be completed, or inhibited, during the posttreatment incubation phase. Inhibition of apoptosis leads to necrosis and can be prevented by 3AB via a mechanism independent of inhibition of poly(ADP-ribose)polymerase. As a corollary, the necrotic response promoted by high concentrations of H2O2 in U937 cells appears to be the result of specific inhibition of the late steps of apoptosis.

Isolation and preliminary characterization of a Chinese hamster ovary cell line with high-degree resistance to hydrogen peroxide.

We have isolated and conducted preliminary characterization of a cell line derived from the Chinese hamster ovary cell line AA8, which we have designated AG8 and which is highly resistant to the cytotoxic effects of H2O2 (approximately 17-fold when the H2O2 treatment was at 37 degrees; approximately 11-fold when the H2O2 treatment was at 4 degrees). AG8 cells were moderately (but significantly; P < 0.05) cross-resistant to CdCl2 (approximately 4-fold), NaAsO2 (approximately 2.3-fold), t-butyl hydroperoxide (approximately 2.9-fold), cumene hydroperoxide (approximately 3-fold), menadione (approximately 1.7-fold) and HgCl2 (approximately 1.5-fold), but were not significantly cross-resistant to hyperthermia (43 degrees), 254 nm UV light, 137Cs gamma-rays, and 42-MeV (p-->Be+) fast neutrons. As regards their biochemical status, AG8 and AA8 cells contain similar non-protein sulfhydryl levels per milligram of protein. Catalase activity (assessed by both spectrophotometry and polarography) was significantly higher in AG8 than in AA8 cells irrespective of whether enzyme activity was expressed per 10(6) cells (approximately 3.6-fold increase) or per milligram of protein (approximately 1.6-fold increase). AG8 cells also exhibited significantly greater glutathione reductase activity than wild-type cells when the data were expressed per 10(6) cells (approximately 2.9-fold) or per milligram of protein (approximately 1.3-fold). Glutathione peroxidase activity was immeasurably low in both cell lines. The susceptibility of the two cell lines to H2O2-mediated generation of DNA single-strand breaks (as measured by alkaline elution) indicated a slightly (approximately 1.5-fold) decreased yield in the resistant AG8 cell line. The two cell lines repaired these breaks with similar kinetics. In contrast, no measurable induction of DNA double-strand breaks (as measured by pulsed-field gel electrophoresis) was apparent in either cell line after survival-curve range concentrations of H2O2. On the basis of these data, it appears that the AG8 phenotype involves two previously identified resistance mechanisms, namely an adaptive component that may or may not involve increased antioxidant capacity, and a second component that does involve increased antioxidant (primarily catalase) capacity.

The effect of hydrogen peroxide/L-histidine-induced DNA single- vs. double-strand breaks on poly(ADP-ribose)polymerase.

L-Histidine markedly increases the ability of hydrogen peroxide to induce DNA cleavage and this effect is associated with a 3-aminobenzamide-inhibitable decline in NAD+ levels, an event which very likely reflects an enhanced stimulation of the enzyme poly(ADP-ribose)polymerase. 3-Aminobenzamide slowed down the removal of alkaline elution-detected strand breaks induced by either H2O2 alone (producing only DNA single-strand breaks) or associated with L-histidine (resulting in the formation of both single-strand breaks and DNA double-strand breaks), and the extent of inhibition was similar under the two experimental conditions. 3-Aminobenzamide did not affect the rate of rejoining of DNA double-strand breaks generated by the cocktail H2O2/L-histidine. The above results suggest that these double-strand breaks have hardly any effect on the induction of poly(ADP-ribose)polymerase activity, a conclusion that is consistent with the observation that the activity of this enzyme appears to be basically identical under conditions that abolish the formation of DNA double-strand breaks, in the absence of measurable variations in the level of induction of DNA single-strand breaks (e.g. in the presence of an excess of L-glutamine, a competitive inhibitor of L-histidine uptake). Finally, 3-aminobenzamide did not affect the toxicity of the oxidant, both in the absence and presence of L-histidine.

Low levels of hydrogen peroxide and L-histidine induce DNA double-strand breakage and apoptosis.

The results presented in this study demonstrate that L-histidine triggers a lethal response in U937 cells exposed to nontoxic, albeit growth-inhibitory, levels of H2O2. Treatment for 1 h with the cocktail H2O2/L-histidine promotes the formation of a low level of DNA double-strand breaks that are rapidly rejoined, and this process is followed by secondary DNA fragmentation at about 7 h of post-treatment incubation, at which time cells are still viable. The appearance of oligonucleosomal DNA fragments associated with the detection of morphological changes typical of apoptosis strongly suggests that a portion of the cells was undergoing an apoptotic process. The relative level of cells with fragmented chromatin never exceeded 15-20% throughout the 20 h post-treatment incubation. Treatment with high concentrations of H2O2 in the presence of L-histidine was found to trigger necrotic cell death. The results presented in this paper provide further experimental evidence in support of the notion that DNA double-strand breaks mediate the lethal effects of the cocktail H2O2/L-histidine and suggest that this type of DNA lesion can promote both apoptotic and necrotic cell death, depending on the concentration of the oxidant.

The antioxidant butylated hydroxytoluene induces apoptosis in human U937 cells: the role of hydrogen peroxide and altered redox state.

Exposure of U937 cells to the antioxidant 2,6-di-tert-butyl-4-methylphenol (BHT), unlike exposure to other antioxidants such as N,N'-diphenyl-1,4-phenylenediamine, Trolox or alpha-tocopherol, promotes a time- and concentration-dependent induction of apoptosis. This response was prevented by the iron chelator o-phenanthroline and by the thiol reagent N-acetylcysteine but was increased remarkably in cells pre-exposed to the catalase inhibitor 3-amino-1,2,4-triazole or to L-buthionine-[S,R]-sulfoximine, a specific inhibitor of glutathione synthesis. Furthermore, the BHT-induced apoptotic response was markedly enhanced by cytochrome P450 inhibitors. Taken together, the experimental results presented in this study indicate that BHT efficiently induces apoptosis in U937 cells and that this response is not caused by products of cytochrome P450 metabolism. Instead, apoptosis appeared to be causally linked to an altered cellular redox state in which hydrogen peroxide plays a pivotal role.

Establishment and characterization of two cell lines derived from human glioblastoma multiforme.

We established and characterized two cell lines derived from glioblastoma multiforme. Both cell lines exhibited tumor cell morphology and growth kinetics and showed variable expression of glial fibrillary acidic protein (GFAP), S-100, fibronectin and vimentin. Cytofluorimetrical analysis of tumor samples showed a diploid DNA distribution, whereas permanent culture cells evolved to the hyperdiploid DNA content. Karyotype studies revealed cytogenetical abnormalities described in glial tumors including gain of chromosome 7, loss of chromosome 10 and presence of double minutes (DMs). Enhanced expression of Ha-ras and c-myc genes resulted from high p-21 and p-62 levels. The contemporary presence of TGF-alpha and EGF-Rc transcripts suggested an autocrine mechanism in the cell lines growth.

New horizons on the role of cannabinoid CB1 receptors in palatable food intake, obesity and related dysmetabolism.

Excessive consumption of high-energy, palatable food contributes to obesity, which results in the metabolic syndrome, heart disease, type-2 diabetes and death. Current knowledge on the function of the hypothalamus as the brain 'feeding centre' recognizes this region as the main regulator of body weight in the central nervous system. Because of their intrinsically fast and adaptive activities, feeding-controlling neural circuitries are endowed with synaptic plasticity modulated by neurotransmitters and hormones that act at different hierarchical levels of integration. In the hypothalamus, among the chemical mediators involved in this integration, endocannabinoids (eCBs) are ideal candidates for the fast (that is, non-genomic), stress-related fine-tuning of neuronal functions. In this article, we overview the role of the eCB system (ECS) in the control of energy intake, and particularly in the consumption of high-energy, palatable food, and discuss how such a role is affected in the brain by changes in the levels of feeding-regulated hormones, such as the adipose tissue-derived anorexigenic mediator leptin, as well as by high-fat diets. The understanding of the molecular mechanisms underlying the neuronal control of feeding behaviours by eCBs offers many potential opportunities for novel therapeutic approaches against obesity. Highlights of the latest advances in the development of strategies that minimize central ECS overactivity in 'western diet'-driven obesity are discussed.

Antioxidant effect of zinc supplementation on both plasma and cellular red-ox status markers in a group of elderly Italian population.

OBJECTIVES: The aim of this work was to evaluate the effect of long term supplementation with two moderate dose of Zn on plasma and cellular red-ox status markers in elderly volunteers. DESIGN, SETTING AND SUBJECTS: In a double blind study 108 healthy volunteers, aged 70-85 years, were enrolled. They were randomly divided in 3 groups of treatment, receiving placebo, 15 mg/day and 30 mg/day of Zn for 6 months. Red-ox status markers were assessed at baseline and after 6 months evaluating carotenoids, vitamin A and E in plasma; glutathione (GSH), thiol groups (RSH), malondialdehyde (MDA), percentage of haemolysis and methemoglobin in erythrocytes. RESULTS: Zn supplementation had no significant effects on red-ox status markers except for vitamin A levels (from 1.94±0.44 to 2.18±0.48 µM in volunteers receiving 15 mg of Zn and from 1.95±0.46 to 2.26±0.56 µM in volunteers receiving 30 mg of Zn), which increased proportionally to zinc dose. CONCLUSIONS: It appears that, differently from unhealthy populations, long-term supplementation with two moderate doses of Zn in a healthy elderly population, with an adequate Zn nutritive status and macro and micronutrients intakes in the range of normality, is an inefficient way to increase antioxidant defences.

Qualitative screening in doping control by MALDI-TOF/TOF mass spectrometry: a proof-of-evidence.

The analysis of doping agents in biological fluids is of top significance in clinical and forensic toxicology. Herein we describe the study of a screening method for the detection of a mixture of drugs of potential abuse including cocaine and its metabolites. By using matrix-assisted laser desorption/ionization MALDI-TOF/TOF mass spectrometry. This screening procedure to detect the presence of different drugs, avoiding time consuming procedures could be useful in different fields of forensic analytical toxicology, including antidoping analysis.

tert-Butylhydroperoxide induces peroxynitrite-dependent mitochondrial permeability transition leading PC12 cells to necrosis.

A short-term exposure of PC12 cells to tert-butylhydroperoxide, followed by recovery in fresh culture medium, causes cell death and the extent of this response progressively increases during the 120 min of post-treatment incubation. Morphological and biochemical analyses of these cells revealed that the mode of cell death was necrosis. Cell killing induced by the hydroperoxide seems to be in part mediated by peroxynitrite because the lethal response was markedly and similarly reduced by the nitric oxide synthase inhibitor N omega-nitro-L-arginine methylester and by scavengers of nitric oxide or peroxynitrite. This peroxynitrite-dependent mechanism of cytotoxicity was blunted by antioxidants and inhibitors of mitochondrial permeability transition and the onset of cell death was preceded by mitochondrial depolarization and loss of cellular ATP. We conclude that tert-butylhydroperoxide promotes peroxynitrite-dependent PC12 cell necrosis causally linked to peroxidation of membrane lipids and mitochondrial permeability transition.

Resistance of female Fischer-344 rats to the hepatonecrogenic and hepatocarcinogenic actions of a choline-devoid diet.

A series of experiments was performed to investigate the response of female Fischer-344 rats to a choline-devoid (CD) diet. In contrast to findings previously made in male Fischer-344 rats, hepatocellular carcinomas did not develop in females chronically fed the diet. Their liver actually showed only minimal histopathological changes. For these reasons, the steatogenic and hepatonecrogenic actions of a CD diet were compared in male and female Fischer-344 rats. Young adult females were found to be largely resistant to both. The results were taken as further evidence of the primary and essential role played by an enhanced cell turnover in the pathogenesis of the carcinomas induced in male Fischer-344 rats by this non-chemical, nutritional model of hepatocarcinogenesis. A high incidence of preneoplastic and benign lesions developed in breast, pancreas or stomach of females chronically fed the CD diet. However, a similar spectrum and high incidence of lesions was observed in control rats fed a choline-supplemented diet. These findings were attributed to an enhanced occurrence of spontaneous lesions, most likely due to the high fat content (15%) of the two diets used.