Storage and uptake of D-serine into astrocytic synaptic-like vesicles specify gliotransmission.

Glial cells are increasingly recognized as active players that profoundly influence neuronal synaptic transmission by specialized signaling pathways. In particular, astrocytes have been shown recently to release small molecules, such as the amino acids l-glutamate and d-serine as "gliotransmitters," which directly control the efficacy of adjacent synapses. However, it is still controversial whether gliotransmitters are released from a cytosolic pool or by Ca(2+)-dependent exocytosis from secretory vesicles, i.e., by a mechanism similar to the release of synaptic vesicles in synapses. Here we report that rat cortical astrocytes contain storage vesicles that display morphological and biochemical features similar to neuronal synaptic vesicles. These vesicles share some, but not all, membrane proteins with synaptic vesicles, including the SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) synaptobrevin 2, and contain both l-glutamate and d-serine. Furthermore, they show uptake of l-glutamate and d-serine that is driven by a proton electrochemical gradient. d-Serine uptake is associated with vesicle acidification and is dependent on chloride. Whereas l-serine is not transported, serine racemase, the synthesizing enzyme for d-serine, is anchored to the membrane of the vesicles, allowing local generation of d-serine. Finally, we reveal a previously unexpected mutual vesicular synergy between d-serine and l-glutamate filling in glia vesicles. We conclude that astrocytes contain vesicles capable of storing and releasing d-serine, l-glutamate, and most likely other neuromodulators in an activity-dependent manner.

Advances in the pharmacological treatment of gastro-oesophageal cancer.

Despite a sharp decline in the incidence of gastric cancer during the second half of the 20th century, this malignancy remains the second leading cause of cancer mortality in the world. The incidence and mortality rate of gastric cancer increase with age; at present, the median ages at diagnosis are 67 years for men and 72 years for women in the US. This article reviews and discusses current medical treatment options for both the general population and elderly gastric cancer patients. Management of localized gastric cancer has changed significantly over recent years. Adjuvant chemoradiation is not generally recommended outside the US. After decades of trials of adjuvant chemotherapy with inconclusive results, a significant survival benefit for perioperative combination chemotherapy - as compared with surgery alone - in patients with resectable or locally advanced gastro-oesophageal cancer was recently demonstrated in the UK MAGIC trial. A further large, randomized trial from Japan demonstrated a significant survival benefit for adjuvant chemotherapy with S-1 after D2 resection for gastric cancer. However, both trials are applicable only to the population in which the trials were conducted. Specific data on elderly patients are missing. For patients with metastatic disease, oral fluoropyrimidines, such as capecitabine, have been developed. In Asian patients, treatment with the oral fluoropyrimidine S-1 is safe and effective. Docetaxel, oxaliplatin and irinotecan have demonstrated activity against gastric cancer in appropriately designed, randomized, phase III trials and have increased the available treatment options significantly. In addition, according to preliminary data, trastuzumab in combination with chemotherapy has significantly improved activity when compared to chemotherapy alone in patients with human epidermal receptor (HER)-2-positive gastric and gastro-oesophageal cancers. Thus, therapeutic decisions in patients with advanced gastric cancer may be adapted to the molecular subtype and co-morbidities of the individual patient. Data from retrospective analyses suggest that oxaliplatin seems to be better tolerated than cisplatin in elderly patients.

Mitochondrial fatty acid oxidation in obesity

Significance: Current lifestyles with high-energy diets and little exercise are triggering an alarming growth in obesity. Excess of adiposity is leading to severe increases in associated pathologies, such as insulin resistance, type 2 diabetes, atherosclerosis, cancer, arthritis, asthma, and hypertension. This, together with the lack of efficient obesity drugs, is the driving force behind much research. Recent Advances: Traditional anti-obesity strategies focused on reducing food intake and increasing physical activity. However, recent results suggest that enhancing cellular energy expenditure may be an attractive alternative therapy. Critical Issues: This review evaluates recent discoveries regarding mitochondrial fatty acid oxidation (FAO) and its potential as a therapy for obesity. We focus on the still controversial beneficial effects of increased FAO in liver and muscle, recent studies on how to potentiate adipose tissue energy expenditure, and the different hypotheses involving FAO and the reactive oxygen species production in the hypothalamic control of food intake. Future Directions: The present review aims to provide an overview of novel anti-obesity strategies that target mitochondrial FAO and that will definitively be of high interest in the future research to fight against obesity-related disorders.

In pancreatic carcinoma, dual EGFR/HER2 targeting with cetuximab/trastuzumab is more effective than treatment with trastuzumab/erlotinib or lapatinib alone: implication of receptors' down-regulation and dimers' disruption.

We previously demonstrated the synergistic therapeutic effect of the cetuximab (anti-epidermal growth factor receptor [EGFR] monoclonal antibody, mAb)-trastuzumab (anti-HER2 mAb) combination (2mAbs therapy) in HER2(low) human pancreatic carcinoma xenografts. Here, we compared the 2mAbs therapy, the erlotinib (EGFR tyrosine kinase inhibitor [TKI])-trastuzumab combination and lapatinib alone (dual HER2/EGFR TKI) and explored their possible mechanisms of action. The effects on tumor growth and animal survival of the three therapies were assessed in nude mice xenografted with the human pancreatic carcinoma cell lines Capan-1 and BxPC-3. After therapy, EGFR and HER2 expression and AKT phosphorylation in tumor cells were analyzed by Western blot analysis. EGFR/HER2 heterodimerization was quantified in BxPC-3 cells by time-resolved FRET. In K-ras-mutated Capan-1 xenografts, the 2mAbs therapy gave significantly higher inhibition of tumor growth than the erlotinib/trastuzumab combination, whereas in BxPC-3 (wild-type K-ras) xenografts, the erlotinib/trastuzumab combination showed similar growth inhibition but fewer tumor-free mice. Lapatinib showed no antitumor effect in both types of xenografts. The efficacy of the 2mAbs therapy was partly Fc-independent because F(ab')(2) fragments of the two mAbs significantly inhibited BxPC-3 growth, although with a time-limited therapeutic effect. The 2mAbs therapy was associated with a reduction of EGFR and HER2 expression and AKT phosphorylation. BxPC-3 cells preincubated with the two mAbs showed 50% less EGFR/HER2 heterodimers than controls. In pancreatic carcinoma xenografts, the 2mAbs therapy is more effective than treatments involving dual EGFR/HER2 TKIs. The mechanism of action may involve decreased AKT phosphorylation and/or disruption of EGFR/HER2 heterodimerization.

Definition of key variables for the induction of optimal NY-ESO-1-specific T cells in HLA transgene mice.

An attractive treatment of cancer consists in inducing tumor-eradicating CD8(+) CTL specific for tumor-associated Ags, such as NY-ESO-1 (ESO), a strongly immunogenic cancer germ line gene-encoded tumor-associated Ag, widely expressed on diverse tumors. To establish optimal priming of ESO-specific CTL and to define critical vaccine variables and mechanisms, we used HLA-A2/DR1 H-2(-/-) transgenic mice and sequential immunization with immunodominant DR1- and A2-restricted ESO peptides. Immunization of mice first with the DR1-restricted ESO(123-137) peptide and subsequently with mature dendritic cells (DCs) presenting this and the A2-restriced ESO(157-165) epitope generated abundant, circulating, high-avidity primary and memory CD8(+) T cells that efficiently killed A2/ESO(157-165)(+) tumor cells. This prime boost regimen was superior to other vaccine regimes and required strong Th1 cell responses, copresentation of MHC class I and MHC class II peptides by the same DC, and resulted in upregulation of sphingosine 1-phosphate receptor 1, and thus egress of freshly primed CD8(+) T cells from the draining lymph nodes into circulation. This well-defined system allowed detailed mechanistic analysis, which revealed that 1) the Th1 cytokines IFN-gamma and IL-2 played key roles in CTL priming, namely by upregulating on naive CD8(+) T cells the chemokine receptor CCR5; 2) the inflammatory chemokines CCL4 (MIP-1beta) and CCL3 (MIP-1alpha) chemoattracted primed CD4(+) T cells to mature DCs and activated, naive CD8(+) T cells to DC-CD4 conjugates, respectively; and 3) blockade of these chemokines or their common receptor CCR5 ablated priming of CD8(+) T cells and upregulation of sphingosine 1-phosphate receptor 1. These findings provide new opportunities for improving T cell cancer vaccines.

Role of Inflammatory Monocytes in Vaccine-Induced Reduction of Helicobacter felis Infection.

Despite the proven ability of immunization to reduce Helicobacter infection in mouse models, the precise mechanism of protection has remained elusive. In this study, we evaluated the role of inflammatory monocytes in the vaccine-induced reduction of Helicobacter felis infection. We first showed by using flow cytometric analysis that Ly6C(low) major histocompatibility complex class II-positive chemokine receptor type 2 (CCR2)-positive CD64(+) inflammatory monocytes accumulate in the stomach mucosa during the vaccine-induced reduction of H. felis infection. To determine whether inflammatory monocytes played a role in the protection, these cells were depleted with anti-CCR2 depleting antibodies. Indeed, depletion of inflammatory monocytes was associated with an impaired vaccine-induced reduction of H. felis infection on day 5 postinfection. To determine whether inflammatory monocytes had a direct or indirect role, we studied their antimicrobial activities. We observed that inflammatory monocytes produced tumor necrosis factor alpha and inducible nitric oxide synthase (iNOS), two major antimicrobial factors. Lastly, by using a Helicobacter in vitro killing assay, we showed that mouse inflammatory monocytes and activated human monocytes killed H. pylori in an iNOS-dependent manner. Collectively, these data show that inflammatory monocytes play a direct role in the immunization-induced reduction of H. felis infection from the gastric mucosa.

Differential effects of polyphenols and alcohol of red wine on the expression of adhesion molecules and inflammatory cytokines related to atherosclerosis: a randomized clinical trial

Background: Few clinical studies have focused on the alcoholindependent cardiovascular effects of the phenolic compounds of red wine (RW). Objective: We aimed to evaluate the effects of ethanol and phenolic compounds of RW on the expression of inflammatory biomarkers related to atherosclerosis in subjects at high risk of cardiovascular disease. Design: Sixty-seven high-risk, male volunteers were included in a randomized, crossover consumption trial. After a washout period, all subjects received RW (30 g alcohol/d), the equivalent amount of dealcoholized red wine (DRW), or gin (30 g alcohol/d) for 4 wk. Before and after each intervention period, 7 cellular and 18 serum inflammatory biomarkers were evaluated. Results: Alcohol increased IL-10 and decreased macrophage-derived chemokine concentrations, whereas the phenolic compounds of RW decreased serum concentrations of intercellular adhesion molecule- 1, E-selectin, and IL-6 and inhibited the expression of lymphocyte function-associated antigen 1 in T lymphocytes and macrophage-1 receptor, Sialil-Lewis X, and C-C chemokine receptor type 2 expression in monocytes. Both ethanol and phenolic compounds of RW downregulated serum concentrations of CD40 antigen, CD40 ligand, IL-16, monocyte chemotactic protein-1, and vascular cell adhesion molecule-1. Conclusion: The results suggest that the phenolic content of RW may modulate leukocyte adhesion molecules, whereas both ethanol and polyphenols of RW may modulate soluble inflammatory mediators in high-risk patients. The trial was registered in the International Standard Randomized Controlled Trial Number Register at http://www. isrctn.org/ as ISRCTN88720134

Differential effects of polyphenols and alcohol of red wine on the expression of adhesion molecules and inflammatory cytokines related to atherosclerosis: a randomized clinical trial

Background: Few clinical studies have focused on the alcoholindependent cardiovascular effects of the phenolic compounds of red wine (RW). Objective: We aimed to evaluate the effects of ethanol and phenolic compounds of RW on the expression of inflammatory biomarkers related to atherosclerosis in subjects at high risk of cardiovascular disease. Design: Sixty-seven high-risk, male volunteers were included in a randomized, crossover consumption trial. After a washout period, all subjects received RW (30 g alcohol/d), the equivalent amount of dealcoholized red wine (DRW), or gin (30 g alcohol/d) for 4 wk. Before and after each intervention period, 7 cellular and 18 serum inflammatory biomarkers were evaluated. Results: Alcohol increased IL-10 and decreased macrophage-derived chemokine concentrations, whereas the phenolic compounds of RW decreased serum concentrations of intercellular adhesion molecule- 1, E-selectin, and IL-6 and inhibited the expression of lymphocyte function-associated antigen 1 in T lymphocytes and macrophage-1 receptor, Sialil-Lewis X, and C-C chemokine receptor type 2 expression in monocytes. Both ethanol and phenolic compounds of RW downregulated serum concentrations of CD40 antigen, CD40 ligand, IL-16, monocyte chemotactic protein-1, and vascular cell adhesion molecule-1. Conclusion: The results suggest that the phenolic content of RW may modulate leukocyte adhesion molecules, whereas both ethanol and polyphenols of RW may modulate soluble inflammatory mediators in high-risk patients. The trial was registered in the International Standard Randomized Controlled Trial Number Register at http://www. isrctn.org/ as ISRCTN88720134

Mitochondrial fatty acid oxidation in obesity

Significance: Current lifestyles with high-energy diets and little exercise are triggering an alarming growth in obesity. Excess of adiposity is leading to severe increases in associated pathologies, such as insulin resistance, type 2 diabetes, atherosclerosis, cancer, arthritis, asthma, and hypertension. This, together with the lack of efficient obesity drugs, is the driving force behind much research. Recent Advances: Traditional anti-obesity strategies focused on reducing food intake and increasing physical activity. However, recent results suggest that enhancing cellular energy expenditure may be an attractive alternative therapy. Critical Issues: This review evaluates recent discoveries regarding mitochondrial fatty acid oxidation (FAO) and its potential as a therapy for obesity. We focus on the still controversial beneficial effects of increased FAO in liver and muscle, recent studies on how to potentiate adipose tissue energy expenditure, and the different hypotheses involving FAO and the reactive oxygen species production in the hypothalamic control of food intake. Future Directions: The present review aims to provide an overview of novel anti-obesity strategies that target mitochondrial FAO and that will definitively be of high interest in the future research to fight against obesity-related disorders.

Comparison of nuclear grade and immunohistochemical features in situ and invasive components of ductal carcinoma of breast

PURPOSE:To compare the prognostic and predictive features between in situ and invasive components of ductal breast carcinomas. METHODS:We selected 146 consecutive breast samples with ductal carcinoma in situ (DCIS) associated with adjacent invasive breast carcinoma (IBC). We evaluated nuclear grade and immunohistochemical expression of estrogen receptor (ER), progesterone receptor (PR), human epidermal growth factor receptor 2 (HER2), cytokeratin 5/6 (CK5/6), and epidermal growth factor receptor (EGFR) in both components, in situ and invasive, and the Ki-67 percentage of cells in the invasive part. The DCIS and IBC were classified in molecular surrogate types determined by the immunohistochemical profile as luminal (RE/PR-positive/ HER2-negative), triple-positive (RE/RP/HER2-positive), HER2-enriched (ER/PR-negative/HER2-positive), and triple-negative (RE/RP/HER2-negative). Discrimination between luminal A and luminal B was not performed due to statistical purposes. Correlations between the categories in the two groups were made using the Spearman correlation method. RESULTS:There was a significant correlation between nuclear grade (p<0.0001), expression of RE/RP (p<0.0001), overexpression of HER2 (p<0.0001), expression of EGFR (p<0.0001), and molecular profile (p<0.0001) between components in situ and IBC. CK 5/6 showed different distribution in DCIS and IBC, presenting a significant association with the triple-negative phenotype in IBC, but a negative association among DCIS. CONCLUSIONS: Our results suggest that classical prognostic and predictive features of IBC are already determined in the preinvasive stage of the disease. However the role of CK5/6 in invasive carcinoma may be different from the precursor lesions.

Morphological and immunohistochemical characterization of angiogenic and apoptotic factors and the expression of thyroid receptors in the ovary of tilapia Oreochromis niloticus in captivity

Morphological and immunohistochemical characterization of angiogenic and apoptotic factors and the expression of thyroid receptors in the ovary of tilapia Oreochromis niloticus in captivity were studied. The morphological evaluation of the ovaries was performed by histological paraffin embedded and stained with HE. The immunohistochemical expressions of CDC47, VEGF, Flk-1, angiopoietin, Tie-2 and thyroid receptor (TRα) were performed by the technique of streptavidein-biotin-peroxidase. Apoptosis was assessed using the TUNEL kit. The relative expression of thyroid hormone receptors (TRα and TRβ) was assessed by RT-PCR real time. The nuclear expression of CDC47 increased with the stage of maturation of the oocyte and was observed in the follicle cells. Apoptotic bodies were observed in the follicular cells of atretic follicles and postovulatory follicles from the ovaries of 150g and 350g fish. Expression of VEGF and its receptor Flk-1 was also observed in the follicular cells, and the expression of both increased with the maturity of the oocyte, with a higher intensity observed in the full-grown follicle. The expression of angiopoietin and of its receptor (Tie 2) was discrete and moderate respectively. TRα expression was independent of follicular development. However, the 350 g tilapia exhibited higher expression of TRβ compared with the 50 g tilapia. We conclude that the proliferative activity and the expression of VEGF and its receptor increase with follicular maturation and that the TRs expression increases with ovarian maturity in tilapia (Oreochromis niloticus).

Blockade of NK-1 receptors in the lateral commissural nucleus tractus solitarii of awake rats had no effect on the cardiovascular responses to chemoreflex activation

The neurotransmission of the chemoreflex in the nucleus tractus solitarii (NTS), particularly of the sympatho-excitatory component, is not completely understood. There is evidence that substance P may play a role in the neurotransmission of the chemoreflex in the NTS. Microinjection of substance P (50 pmol/50 nl, N = 12, and 5 nmol/50 nl, N = 8) into the commissural NTS of unanesthetized rats produced a significant increase in mean arterial pressure (101 ± 1 vs 108 ± 2 and 107 ± 3 vs 115 ± 4 mmHg, respectively) and no significant changes in heart rate (328 ± 11 vs 347 ± 15 and 332 ± 7 vs 349 ± 13 bpm, respectively) 2 min after microinjection. Previous treatment with WIN, an NK-1 receptor antagonist (2.5 nmol/50 nl), microinjected into the NTS of a specific group of rats, blocked the pressor (11 ± 5 vs 1 ± 2 mmHg) and tachycardic (31 ± 6 vs 4 ± 3 bpm) responses to substance P (50 pmol/50 nl, N = 5) observed 10 min after microinjection. Bilateral microinjection of WIN into the lateral commissural NTS (N = 8) had no significant effect on the pressor (50 ± 4 vs 42 ± 6 mmHg) or bradycardic (-230 ± 16 vs -220 ± 36 bpm) responses to chemoreflex activation with potassium cyanide (iv). These data indicate that the activation of NK-1 receptors by substance P in the NTS produces an increase in baseline mean arterial pressure and heart rate. However, the data obtained with WIN suggest that substance P and NK-1 receptors do not play a major role in the neurotransmission of the chemoreflex in the lateral commissural NTS.

Pharmacological differences between memory consolidation of habituation to an open field and inhibitory avoidance learning

Rats implanted bilaterally with cannulae in the CA1 region of the dorsal hippocampus or the entorhinal cortex were submitted to either a one-trial inhibitory avoidance task, or to 5 min of habituation to an open field. Immediately after training, they received intrahippocampal or intraentorhinal 0.5-µl infusions of saline, of a vehicle (2% dimethylsulfoxide in saline), of the glutamatergic N-methyl-D-aspartate (NMDA) receptor antagonist 2-amino-5-phosphono pentanoic acid (AP5), of the protein kinase A inhibitor Rp-cAMPs (0.5 µg/side), of the calcium-calmodulin protein kinase II inhibitor KN-62, of the dopaminergic D1 antagonist SCH23390, or of the mitogen-activated protein kinase kinase inhibitor PD098059. Animals were tested in each task 24 h after training. Intrahippocampal KN-62 was amnestic for habituation; none of the other treatments had any effect on the retention of this task. In contrast, all of them strongly affected memory of the avoidance task. Intrahippocampal Rp-cAMPs, KN-62 and AP5, and intraentorhinal Rp-cAMPs, KN-62, PD098059 and SCH23390 caused retrograde amnesia. In view of the known actions of the treatments used, the present findings point to important biochemical differences in memory consolidation processes of the two tasks.

Treatment of hemorrhagic shock with hypertonic saline solution modulates the inflammatory response to live bacteria in lungs

Shock and resuscitation render patients more susceptible to acute lung injury due to an exacerbated immune response to subsequent inflammatory stimuli. To study the role of innate immunity in this situation, we investigated acute lung injury in an experimental model of ischemia-reperfusion (I-R) followed by an early challenge with live bacteria. Conscious rats (N = 8 in each group) were submitted to controlled hemorrhage and resuscitated with isotonic saline (SS, 0.9% NaCl) or hypertonic saline (HS, 7.5% NaCl) solution, followed by intratracheal or intraperitoneal inoculation of Escherichia coli. After infection, toll-like receptor (TLR) 2 and 4 mRNA expression was monitored by RT-PCR in infected tissues. Plasma levels of tumor necrosis factor α and interleukins 6 and 10 were determined by ELISA. All animals showed similar hemodynamic variables, with mean arterial pressure decreasing to nearly 40 mmHg after bleeding. HS or SS used as resuscitation fluid yielded equal hemodynamic results. Intratracheal E. coli inoculation per se induced a marked neutrophil infiltration in septa and inside the alveoli, while intraperitoneal inoculation-associated neutrophils and edema were restricted to the interseptal space. Previous I-R enhanced lung neutrophil infiltration upon bacterial challenge when SS was used as reperfusion fluid, whereas neutrophil influx was unchanged in HS-treated animals. No difference in TLR expression or cytokine secretion was detected between groups receiving HS or SS. We conclude that HS is effective in reducing the early inflammatory response to infection after I-R, and that this phenomenon is achieved by modulation of factors other than expression of innate immunity components.