Glycolytic competence in gastric adenocarcinomas negatively impacts survival outcomes of patients treated with salvage paclitaxel-ramucirumab.

INTRODUCTION: For energy production, cancer cells maintain a high rate of glycolysis instead of oxidative phosphorylation converting glucose into lactic acid. This metabolic shift is useful to survive in unfavorable microenvironments. We investigated whether a positive glycolytic profile (PGP) in gastric adenocarcinomas may be associated with unfavorable outcomes under an anticancer systemic therapy, including the anti-angiogenic ramucirumab. MATERIALS AND METHODS: Normal mucosa (NM) and primary tumor (PT) of 40 metastatic gastric adenocarcinomas patients who received second-line paclitaxel-ramucirumab (PR) were analyzed for mRNA expression of the following genes: HK-1, HK-2, PKM-2, LDH-A, and GLUT-1. Patients were categorized with PGP when at least a doubling of mRNA expression (PT vs. NM) in all glycolytic core enzymes (HK-1 or HK-2, PKM-2, LDH-A) was observed. PGP was also related to TP53 mutational status. RESULTS: Mean LDH-A, HK-2, PKM-2 mRNA expression levels were significantly higher in PT compared with NM. 18 patients were classified as PGP, which was associated with significantly worse progression-free and overall survival times. No significant association was observed between PGP and clinical-pathologic features, including TP53 positive mutational status, in 28 samples. CONCLUSIONS: Glycolytic proficiency may negatively affect survival outcomes of metastatic gastric cancer patients treated with PR systemic therapy. TP53 mutational status alone does not seem to explain such a metabolic shift.

Correlation between chronic treatment with proton pump inhibitors and bacterial overgrowth in the stomach: any possible beneficial role for selected lactobacilli?

BACKGROUND: The inhibition of physiological gastric acid secretion induced by proton pump inhibitors (PPIs), the most widely used drugs in the world, may cause a significant bacterial overgrowth in the gastrointestinal tract as a side effect. This study was undertaken firstly to correlate PPI intake with concentration of specific bacterial groups in the stomach as well as possible Helicobacter pylori infection, and secondly to assess the efficacy of the 4 lactobacilli L. rhamnosus LR06 (DSM 21981), L. pentosus LPS01 (DSM 21980), L. plantarum LP01 (LMG P-21021), and L. delbrueckii subsp. delbrueckii LDD01 (DSM 22106) in the restoration of a physiological gastric barrier. METHODS: Total bacteria, sulphite-reducing bacteria (SRB), total coliforms, and total lactobacilli were quantified in samples of gastric juice from 29 subjects taking PPIs for at least 3 months compared with 36 control subjects. The presence of H. pylori was also assessed.The subjects treated with PPIs with a concentration of total bacteria in the gastric juice higher than 10(5) cells/mL were selected for an intervention study with the 4 lactobacilli L. rhamnosus LR06, L. pentosus LPS01, L. plantarum LP01, and L. delbrueckii subsp. delbrueckii LDD01. After 15 days of supplementation, the same bacterial groups were quantified to compare these values with the baseline. RESULTS: No significant correlation was found between the presence of H. pylori and PPI intake. The baseline quantification of bacterial groups (log10 CFU/mL of gastric juice, PPI group vs. control) showed: total bacteria 8.35 versus 3.95 (P<0.001); total coliforms 4.98 versus 2.35 (P<0.001); SRB 5.71 versus 2.28 (P=0.065); and total lactobacilli 3.85 versus 2.20 (P=0.005). After 15 days of treatment with the 4 lactobacilli, the quantification of bacterial groups gave the following results: total bacteria 7.91 versus 8.35 at time zero (P=0.002); total coliforms 4.21 versus 4.98 at time zero (P<0.001); SRB 4.94 versus 5.71 at baseline (P=0.060); and total lactobacilli 7.20 versus 3.85 at baseline (P=0.040). CONCLUSIONS: A significant impairment of intragastric acidity is sufficient to induce a relevant bacterial overgrowth, with particular reference to SRB and total coliforms. This fact can contribute to an increase in the risk of infections and intestinal diseases. It could be crucial to restore the physiological "gastric barrier." The 2-week supplementation with the 4 lactobacilli tested proved to be effective in significantly reducing total bacteria and coliforms in the gastric milieu in subjects chronically treated with PPIs. It is therefore possible to hopothesise a beneficial role for such lactobacilli in clinical practice.

Selenium and zinc internalized by Lactobacillus buchneri Lb26 (DSM 16341) and Bifidobacterium lactis Bb1 (DSM 17850): improved bioavailability using a new biological approach.

BACKGROUND: Minerals, often referred to as micronutrients, are one of the 5 fundamental groups of nutrients needed to sustain life. Micronutrient malnutrition affects >50% of the worldwide population. In particular, zinc (Zn) deficiency is considered an emerging public health problem in India and in other developing countries. Selenium (Se) is another trace mineral essential for humans and animals. Dietary Se exists primarily as selenomethionine and selenocysteine. In addition, Se may be present in its inorganic form (selenite) in some vegetables. To increase the daily intake of these minerals, numerous food supplements containing different inorganic and organic forms of Zn or Se are commercially available. At any rate, it is quite well known that inorganic salts have a very low bioavailability. Organic salts, commonly based on gluconate, orotate, citrate, or other molecules, are characterized by a higher systemic effect. The innovative opportunity of using certain species of probiotics enriched with the 2 minerals could represent an interesting alternative to these preparations. Diet integration with bacteria able to internalize Zn and Se may embody a new application of probiotics. METHODS: To overcome the difficulties of in vivo animal or human trials, in this work a cell culture model using Caco-2 cells in bicameral chambers (Transwell system) was developed and validated to quantify the bioavailability of some commercial forms of Se and Zn compared with the organic forms accumulated intracellularly by Lactobacillus buchneri Lb26 (DSM 16341) and Bifidobacterium lactis Bb1 (DSM 17850), respectively. RESULTS: The experimental data collected demonstrated a significantly higher bioavailability of Se and Zn internalized by L. buchneri Lb26 (DSM 16341) and B. lactis Bb1 (DSM 17850), respectively, compared with the inorganic and even organic forms tested. In particular, the Se accumulated at the intracellular level by L. buchneri Lb26 proved to be 5.9, 9.4, and 65 times more absorbable than sodium selenite, seleno-L-methionine, and seleno-L-cysteine, respectively. In contrast, Zn internalized by B. lactis Bb1 showed an absorption that was >16 times higher by Caco-2 cells compared with zinc gluconate and a 31.5 times higher absorption compared with zinc sulfate. Most notably, Se and Zn internalized by the 2 probiotics studied are the only forms able to reach the Transwell basolateral compartment at a concentration higher than the concentration found in the apical compartment, therefore suggesting a considerably higher in vivo ability to be absorbed into the bloodstream. Both organic and inorganic forms of Se and Zn were predominantly found in the apical compartment, thus demonstrating their poor ability to diffuse into the cell and become bioavailable in all subcellular areas. CONCLUSIONS: The opportunity of delivering minerals in a highly bioavailable form by means of a probiotic bacterium has not been deeply investigated to date. This is the first study reporting quantitative data on the bioavailability and percentage of absorption of minerals internalized by specific probiotics. The most noticeable aspect is the significantly higher absorption of both probiotic Se and Zn compared with their organic forms, with particular reference to seleno-L-methionine, seleno-L-cysteine, and zinc gluconate.

Genetic activation of the MET pathway and prognosis of patients with high-risk, radically resected gastric cancer.

PURPOSE: To investigate whether prognosis of patients with high-risk gastric cancer may depend on MET copy number gain (CNG) or an activating truncation within a deoxyadenosine tract element (DATE) in the promoter region of the MET ligand HGF. PATIENTS AND METHODS: A single-institution cohort of 230 patients with stage II/III gastric cancer was studied. Formalin-fixed paraffin-embedded tumor specimens were used for DNA extraction. Quantitative polymerase chain reaction (qPCR) for MET CNG and sequencing for HGF DATE truncation (< 25 deoxyadenosines instead of 30) were used. Results were analyzed for association with disease-free survival (DFS) and overall survival (OS). To assess the reliability of the qPCR measurement, a random sample of cases was reanalyzed using an alternative assay (fluorescent in situ hybridization [FISH]) with calculation of the intracorrelation coefficient (ICC). RESULTS: In 216 assessable patients, MET CNG five or more copies and homozygous HGF-truncated DATE occurred in 21 patients (10%) and 30 patients (13%), respectively. Patients with MET CNG five or more copies (MET-positive) showed significantly worse prognosis with multivariate hazard ratio (HR) of 3.02 (95% CI, 1.71 to 5.33; P < .001) for DFS and multivariate HR of 2.91 (95% CI, 1.65 to 5.11; P < .001) for OS. The agreement between qPCR and FISH was high, with ICC = 0.9% (95% CI, 0.81% to 0.95%; the closer the ICC is to 1, the greater is the agreement). HGF-truncated DATE did not show relevant prognostic effect. CONCLUSION: In this study, qPCR revealed approximately 10% of white patients with gastric cancer harboring MET CNG of five or more copies. This marker was significantly associated with unfavorable prognosis. This information is relevant to the current clinical development of anti-MET compounds.

Peculiar response to methylphenidate in adolescent compared to adult rats: a phMRI study.

RATIONALE: Adolescent rodents differ markedly from adults in several neuro-behavioural parameters. Moreover, 'paradoxical' responses to psychostimulants have been reported at this age. OBJECTIVES: Thus, we investigated the responses of adolescent (post-natal day, PND, 34 to 43) and adult (PND >60) Sprague-Dawley male rats to the psychostimulant drug methylphenidate (MPH). We used pharmacological magnetic resonance imaging (phMRI) performed at 4.7 T under isoflurane anaesthesia. Following anatomical MRI, axial gradient echo images were collected continuously. After baseline recording (32 min), animals received MPH (0 or 4 mg/kg i.p.) and were recorded for further 32 min. RESULTS: Region-specific changes in the blood-oxygenation level dependent (BOLD) signal were evident as a function of age. As expected, among adults MPH induced an increase of BOLD signal in nucleus accumbens (NAcc) and prefrontal cortex (PFC), with no effects in the hippocampus (Hip). Notably, among adolescents, MPH induced a marked and generalised decrease of BOLD signal, which occurred earlier in NAcc and PFC whilst being delayed in the Hip. Any bias in BOLD responses was excluded by the measurement of physiological parameters. CONCLUSIONS: The present findings highlight the utility of phMRI in animal models. The peculiar negative BOLD effect found in adolescent rats may be suggestive of a reduced cerebro-vascular feedback and/or an increased MPH-induced neuronal activation. Data are relevant for a better understanding of brain/behavioural regulation during adolescent development. Moreover, a greater understanding of the differences between adult and adolescent drug responses will aid in the development of a more appropriate age-specific treatment strategy.

In vitro sensitivity of probiotics to human pancreatic juice.

BACKGROUND: The resistance of gut flora with probiotic activity to pancreatic juice is usually tested with artificial pancreatic fluid. Previous studies evaluated the sensitivity of diverse probiotics to human gastric and biliary secretion; none tested the resistance of probiotics to human pancreatic juice. As most bacteria sensitive to artificial pancreatic fluid in vitro have a high rate of isolation from feces, the resistance to human pancreatic juice could be higher. AIM: The aim of this study was to compare the sensitivity of different strains of probiotics to artificial and human pancreatic juice. MATERIALS AND METHODS: The viability of 8 strains of Lactobacillus and 4 strains of Bifidobacterium was tested with standard artificial and human pancreatic juice withdrawn from 16 patients during endoscopic retrograde cholangiopancreatography procedure. The mortality rate (%) of various bacteria was measured after 5, 30, and 60 minutes contact time. The results were normalized for mortality rate induced by hypotonic condition and time exposure to 37 degrees C. RESULTS: When incubated with artificial and human pancreatic juice, the mortality rate of all strains of Lactobacillus at 5, 30, and 60 minutes was 10.1 versus 7.6, 20.5 versus 19.7, and 28.6 versus 29.8, respectively; whereas the mortality rate of all strains of Bifidobacterium was 8.0 versus 9.2, 33.3 versus 28.9, and 42.2 versus 44.4, respectively. CONCLUSIONS: All the tested strains were sensitive to artificial and human pancreatic juice depending on time contact. Bifidobacterium strains seem to be more sensitive than Lactobacillus strains in particular at higher time contact. There is no significant difference between sensitivity to simulated and human pancreatic juice. For this reason, probiotics activity may be tested with artificial pancreatic fluid using a standardized, easier, and less costly procedure.

Eicosapentaenoic acid stimulates the expression of myelin proteins in rat brain.

We have previously demonstrated that, in C6 glioma cells, eicosapentaenoic acid (EPA) stimulates the expression of proteolipid protein (PLP) via cAMP-mediated pathways. In this study, we investigated whether n-3 polyunsaturated fatty acids can affect myelinogenesis in vivo. A single dose of either EPA or docosahexaenoic acid (DHA) was injected intracerebroventricularly into 2-day-old rats, which were then killed after 3 days post-injection (p.i.). Total RNA was isolated from the medulla, cerebellum, and cortex, and the expression of myelin-specific mRNAs was analyzed by real-time PCR. The levels of PLP, myelin basic protein, and myelin oligodendrocyte protein mRNAs increased in nearly all brain regions of DHA- and EPA-treated animals, but the effect was more pronounced in EPA-treated rats. The enhancement in PLP transcript levels was followed by an increase in PLP translation in EPA-treated rats. A further indicator of accelerated myelination was the increase in 2'-3'-cyclic nucleotide 3'-phosphodiesterase (CNPase) protein levels. In EPA-treated rats, the increased expression of myelin genes coincided with a decrease of cAMP-response element-binding protein (CREB)-DNA binding in the cerebellum and cortex (1 hr p.i.). After 16 hr, this effect was still present in the same cerebral regions even though the decrease in EPA-treated rats was less pronounced than in controls. The down-regulation of CREB activity was due to a decrease in the levels of CREB phosphorylation. In conclusion, our data suggest that EPA stimulates the expression of specific myelin proteins through decreased CREB phosphorylation. These results corroborate the clinical studies of the n-3 PUFA beneficial effects on several demyelinating diseases.

Cholinesterase inhibition and alterations of hepatic metabolism by oral acute and repeated chlorpyrifos administration to mice.

Chlorpyrifos (CPF) is a broad spectrum organophosphorus insecticide bioactivated in vivo to chlorpyrifos-oxon (CPFO), a very potent anticholinesterase. A great majority of available animal studies on CPF and CPFO toxicity are performed in rats. The use of mice in developmental neurobehavioural studies and the availability of transgenic mice warrant a better characterization of CPF-induced toxicity in this species. CD1 mice were exposed to a broad range of acute (12.5-100.0mg/kg) and subacute (1.56-25mg/kg/day from 5 to 30 days) CPF oral doses. Functional and biochemical parameters such as brain and serum cholinesterase (ChE) and liver xenobiotic metabolizing system, including the biotransformation of CPF itself, have been studied and the no observed effect levels (NOELs) identified. Mice seem to be more susceptible than rats at least to acute CPF treatment (oral LD(50) 4.5-fold lower). The species-related differences were not so evident after repeated exposures. In mice a good correlation was observed between brain ChE inhibition and classical cholinergic signs of toxicity. After CPF-repeated treatment, mice seemed to develop some tolerance to CPF-induced effects, which could not be attributed to an alteration of P450-mediated CPF hepatic metabolism. CPF-induced effects on hepatic microsomal carboxylesterase (CE) activity and reduced glutathione (GSH) levels observed at an early stage of treatment and then recovered after 30 days, suggest that the detoxifying mechanisms are actively involved in the protection of CPF-induced effects and possibly in the induction of tolerance in long term exposure. The mouse could be considered a suitable experimental model for future studies on the toxic action of organophosphorus pesticides focused on mechanisms, long term and age-related effects.

Developmental neurotoxicity of organophosphorous pesticides: fetal and neonatal exposure to chlorpyrifos alters sex-specific behaviors at adulthood in mice.

Developmental exposure to the organophosphorous insecticide chlorpyrifos (CPF) induces long-term effects on brain and behavior in laboratory rodents. We evaluated in adult mice the behavioral effects of either fetal and/or neonatal CPF exposure at doses not inhibiting fetal and neonatal brain cholinesterase. CPF (3 or 6 mg/kg) was given by oral treatment to pregnant females on gestational days 15-18 and offspring were treated sc (1 or 3 mg/kg) on postnatal days (PNDs) 11-14. Serum and brain acetylcholinesterase (AChE) activity was evaluated at birth and 24 h from termination of postnatal treatments. On PND 70, male mice were assessed for spontaneous motor activity in an open-field test and in a socioagonistic encounter with an unfamiliar conspecific. Virgin females underwent a maternal induction test following presentation of foster pups. Both sexes were subjected to a plus-maze test to evaluate exploration and anxiety levels. Gestational and postnatal CPF exposure (higher doses) affected motor activity in the open field and enhanced synergically agonistic behavior. Postnatal CPF exposure increased maternal responsiveness toward pups in females. Mice of both sexes exposed to postnatal CPF showed reduced anxiety response in the plus-maze, an effect greater in females. Altogether, developmental exposure to CPF at doses that do not cause brain AChE inhibition induces long-term alterations in sex-specific behavior patterns of the mouse species. Late neonatal exposure on PNDs 11-14 was the most effective in causing behavioral changes. These findings support the hypothesis that developmental CPF may represent a risk factor for increased vulnerability to neurodevelopmental disorders in humans.

[Accuracy of the cytopathological telediagnosis of palpable limphoadenopathies. A study design simulating the diagnostic support from remote access to health workers in the developing countries]. / Accuratezza della telediagnosi nella citopatologia delle linfoadenopatie superficiali palpabili. Un modello di simulazione di assistenza telematica ad aree remote di paesi in via di sviluppo.

It has been estimated the accuracy of the cytologic diagnosis, carried out by a cytopathologist, on digital images (DID) of fine needle aspiration lymphonode biopsies (FNAB), in order to validate the telematic support to health workers practising in specialist-lacking remote areas of the developing countries. A blind-cross check simulation design was drawn-up. A non-pathologist practictioner selected the microscopic fields of 39 cases of FNAB, took the pictures with a digital camera, stored them and submitted to a cytopathologist for DID. The DID were compared against the original diagnosis in direct view (DVC). The histologic diagnosis on the surgical biopsies were the golden standard to refer to. The results demonstrate that: 1) DVC and DID have similar and satisfactory sensitivity rates (88.2 e 85.3%) and positive predictive values (96.8 e 90.1%); 2) DID have lower specificity and efficiency rates against DVC (40.0 vs 80.0 and 79.5 vs 79.2%); 3) the negative predictive values are low both in DVC and DID (50.0 e 28.6%), but the data ought to be confirmed in a well-balanced ratio malignancy-benignancy; d) the accuracy of DID could be increased by improving the selection process of the microscopic fields; e) the remote access-assisted cytologic diagnosis of FNAB can be a procedure to carry out easily and cheaply in areas where the histopathology facilities are unavailable. The accuracy is the highest in metastasis, in high-grade non-Hodgkin lymphoma and in lymphocite depletion and mixed cell sub-groups of Hodgkin lymphoma.

In vitro inhibitory effect of aflatoxin B1 on acetylcholinesterase activity in mouse brain.

Growing concern on the problem of mycotoxins in the alimentary chain underlines the need to investigate the mechanisms explaining the cholinergic effects of aflatoxin B(1) (AFB(1)). We examined the effect of AFB(1), a mycotoxin produced by Aspergillus flavus, on mouse brain acetylcholinesterase (AChE) and specifically on its molecular isoforms (G(1) and G(4)) after in vitro exposure. AFB(1) (from 10(-9) to 10(-4)M), inhibited mouse brain AChE activity (IC(50) = 31.6 x 10(-6)M) and its G(1) and G(4) molecular isoforms in a dose-dependent manner. Michaelis-Menten parameters indicate that the K(m) value increased from 55.2 to 232.2% whereas V(max) decreased by 46.2-75.1%. The direct, the Lineweaver-Burk and the secondary plots indicated a non-competitive-mixed type antagonism, induced when the inhibitor binds to the free enzyme and to the enzyme-substrate complex. AFB(1)-inhibited AChE was partially reactivated by pyridine 2-aldoxime (2-PAM) (10(-4)M) but the AChE-inhibiting time courses of AFB(1) (10(-4)M) and diisopropylfluorophosphate (DFP) (2 x 10(-7)M) differed. Overall these data suggest that AFB(1) non-competitively inhibits mouse brain AChE by blocking access of the substrate to the active site or by inducing a defective conformational change in the enzyme through non-covalent binding interacting with the AChE peripheral binding site, or through both mechanisms.

QSAR in ecotoxicity: an overview of modern classification techniques.

This study deals with classification for toxicity prediction. Using a data set of 235 pesticides and 153 descriptors, we built several models using seven classification algorithms: nearest mean classifier, linear discriminant analysis, quadratic discriminant analysis, regularized discriminant analysis, soft independent modeling of class analogy, K nearest neighbors classification, classification, and regression tree. The performance of the models was then compared with the classifier, the end-points, the number of descriptor, and the diversity of the data set. Finally, we made a critical analysis of the models and descriptors.

Bone marrow extracellular large geometric crystals in IgG/lambda MGUS.

We describe the peculiar histopathology of the bone marrow in a case of IgG/lambda MGUS. Striking eosinophilic crystals with a rectangular, rhomboid or square shape lay in the interstitium, sometimes in optically empty spaces, but failed to elicit a foreign body giant cell reaction. Their histochemical properties, immunoreactivity for anti-lambda light chain antiserum, and ultrastructural features strongly supported their relationship with the paraprotein synthesized by the monoclonal plasma cells. The crystals were not observed on bone marrow aspirate smear, suggesting that they had formed during trephine biopsy processing or, alternatively, that they had been removed during the smear preparation. We feel that pathologists should be aware of the existence of this type of crystals, which differ from both the amyloid deposits and the proteinaceous material sometimes observed in plasma cell proliferations. Their presence in the bone marrow should alert the clinician to investigate the involvement of other organs with immunoglobulin deposits.

Synthesis and cholinesterase activity of phenylcarbamates related to Rivastigmine, a therapeutic agent for Alzheimer's disease.

In order to develop new cholinesterase agents effective against Alzheimer's disease (AD) we synthesized some phenylcarbamates structurally related to Rivastigmine and evaluated their in vitro and in vivo biological activity. Among the compounds which displayed the most significant in vitro activity, 1-[1-(3-dimethylcarbamoyloxyphenyl)ethyl]piperidine (31b), in addition to a simple and cheaper synthesis, showed lower toxicity and very similar therapeutic index in comparison with Rivastigmine.