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Triple negative breast cancers (TNBCs) are very aggressive and have a poor prognosis due to lack of efficacious therapies. The only effective treatment is chemotherapy that however is frequently hindered by the occurrence of drug resistance. We approached this problem in vitro and in vivo on a triple negative and a hormone sensitive breast cancer cell lines: 4T1 and TS/A. A main defense mechanism of tumors is the extrusion of intracellular protons derived from the metabolic shift to glycolysis, and necessary to maintain an intracellular pH compatible with life. The resulting acidic extracellular milieu bursts the malignant behavior of tumors and impairs chemotherapy. Therefore, we investigated the efficacy of combined therapies that associate cisplatin (Cis) with proton exchanger inhibitors, such as esomeprazole (ESO) and 5‐(N‐ethyl‐N‐isopropyl)amiloride (EIPA). Our results demonstrate that in the 4T1 triple negative model the combined therapy Cis plus EIPA is significantly more effective than the other treatments. Instead, in the TS/A tumor the best therapeutic result is obtained with ESO alone. Remarkably, in both 4T1 and TS/A tumors these treatments correlate with increase of CD8+ T lymphocytes and dendritic cells, and a dramatic reduction of M2 macrophages and other suppressor myeloid cells (MDSC) in the tumor infiltrates., We show that association of cisplatin and sodium/hydrogen exchanger one inhibitors decreases tumor growth, improves the tumor microenvironment state, and increases survival more efficiently than cisplatin alone in triple negative breast cancer‐bearing mice, supporting the exploitation of this combo therapy in chemoresistant TNBCs. At variance, esomeprazole alone is more efficacious than cisplatin or various combo therapies in hormone sensitive breast cancer in mice. Thus, proton transport inhibitors should be considered as valuable nontoxic anticancer drugs.

Objectives The present study aims to verify the relationship between glucose consumption and uptake of 18F-2-deoxy-glucose (FDG) in the skeletal muscle (SM) of experimental models of streptozotocin-induced diabetes mellitus (STZ-DM). Methods The study included 36 Balb/c mice. Two weeks after intraperitoneal administration of saline (control group, n = 18) or 150 mg streptozotocin (STZ-DM group, n = 18), the two cohorts were submitted to an oral glucose tolerance test and were further subdivided into three groups (n = 6 each): untreated and treated with metformin (MTF) at low or high doses (10 or 750 mg/kg daily, respectively). Two weeks thereafter, all mice were submitted to dynamic micro–positron emission tomography (PET) imaging after prolonged fasting. After sacrifice, enzymatic pathways and response to oxidative stress were evaluated in harvested SM. Results On PET imaging, the FDG uptake rate in hindlimb SM was significantly lower in nondiabetic mice as compared with STZ-DM–untreated mice. MTF had no significant effect on SM FDG uptake in untreated mice; however, its high dose induced a significant decrease in STZ-DM animals. Upon conventional analysis, the SM standard uptake value was higher in STZ-DM mice, while MTF was virtually ineffective in either control or STZ-DM models. This metabolic reprogramming was not explained by any change in cytosolic glucose metabolism. By contrast, it closely agreed with the catalytic function of hexose-6P-dehydrogenase (H6PD; i.e., the trigger of a specific pentose phosphate pathway selectively located within the endoplasmic reticulum). In agreement with this role, the H6PD enzymatic response to both STZ-DM and MTF matched the activation of the NADPH-dependent antioxidant responses to the increased generation of reactive oxygen species caused by chronic hyperglycemia. Ex vivo analysis of tracer kinetics confirmed that the enhanced SM avidity for FDG occurred despite a significant reduction in glucose consumption, while it was associated with increased radioactivity transfer to the endoplasmic reticulum. Conclusions These data challenge the current dogma linking FDG uptake to the glycolytic rate. They instead introduce a new model considering a strict link between the uptake of this glucose analog, H6PD reticular activity, and oxidative damage in diabetes, at least under fasting condition., Highlights • The accepted link between glucose consumption and uptake of hexose analogs such as FDG and 2DG is looser than expected. • FDG selectively tracks the activity of a pentose-phosphate shunt triggered by H6PD. • FDG uptake in the skeletal muscles tracks the hyperglycemia-related oxidative damage. • FDG uptake might represent an index of NADPH renewal indicating the ER-PPP response to oxidative stress.

Background. We recently reported that enhanced [18F]-fluorodeoxyglucose (FDG) uptake in skeletal muscles predicts disease aggressiveness in patients with amyotrophic lateral sclerosis (ALS). The present experimental study aimed to assess whether this predictive potential reflects the link between FDG uptake and redox stress that has been previously reported in different tissues and disease models. Methods. The study included 15 SOD1G93A mice (as experimental ALS model) and 15 wildtype mice (around 120-days-old). Mice were submitted to micro-PET imaging. Enzymatic pathways and response to oxidative stress were evaluated in harvested quadriceps and hearts by biochemical, immunohistochemical and immunofluorescence analysis. Colocalization between the endoplasmic reticulum (ER) and the fluorescent FDG analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) was performed in fresh skeletal muscle sections. Finally, mitochondrial ultrastructure and bioenergetics were evaluated in harvested quadriceps and hearts.Results. FDG retention was significantly higher in hindlimb skeletal muscles of symptomatic SOD1G93A mice with respect to control ones. This difference was not explained by any acceleration in glucose degradation through glycolysis or cytosolic pentose phosphate pathway (PPP). Similarly, it was independent of inflammatory infiltration. Rather, the high FDG retention in SOD1G93A skeletal muscle was associated with an accelerated generation of reactive oxygen species. This redox stress selectively involved the ER and the local PPP triggered by hexose-6P-dehydrogenase. ER involvement was confirmed by the colocalization of the 2-NBDG with a vital ER tracker. The oxidative damage in transgenic skeletal muscle was associated with a severe impairment in the crosstalk between ER and mitochondria combined with alterations in mitochondrial ultrastructure and fusion/fission balance. The expected respiratory damage was confirmed by a deceleration in ATP synthesis and oxygen consumption rate. These same abnormalities were represented to a markedly lower degree in the myocardium, as a sample of non-voluntary striated muscle.Conclusion. Skeletal muscle of SOD1G93A mice reproduces the increased FDG uptake observed in ALS patients. This finding reflects the selective activation of the ER-PPP in response to significant redox stress associated with alterations of mitochondrial ultrastructure, networking, and connection with the ER itself. This scenario is less severe in cardiomyocytes suggesting a relevant role for either communication with synaptic plaque or contraction dynamics.

This study aims to verify in experimental models of hyperglycemia induced by streptozotocin (STZ-DM) to what degree the high competition between unlabeled glucose and metformin (MET) treatment might affect the accuracy of cancer FDG imaging. The study included 36 "control" and 36 "STZ-DM" Balb/c mice, undergoing intraperitoneal injection of saline or streptozotocin, respectively. Two-weeks later, mice were subcutaneously implanted with breast (4 T1) or colon (CT26) cancer cells and subdivided in three subgroups for treatment with water or with MET at 10 or 750 mg/Kg/day. Two weeks after, mice were submitted to micro-PET imaging. Enzymatic pathways and response to oxidative stress were evaluated in harvested tumors. Finally, competition by glucose, 2-deoxyglucose (2DG) and the fluorescent analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) on FDG uptake was studied in 4 T1 and CT26 cultured cells. STZ-DM slightly decreased cancer volume and FDG uptake rate (MRF). More importantly, it also abolished MET capability to decelerate lesion growth and MRF. This metabolic reprogramming closely agreed with the activity of hexose-6-phosphate dehydrogenase within the endoplasmic reticulum. Finally, co-incubation with 2DG virtually abolished FDG and 2-NBDG uptake within the endoplasmic reticulum in cultured cells. These data challenge the current dogma linking FDG uptake to glycolytic flux and introduce a new model to explain the relation between glucose analogue uptake and hexoses reticular metabolism. This selective fate of FDG contributes to the preserved sensitivity of PET imaging in oncology even in chronic moderate hyperglycemic conditions.

The relevant role of pentose phosphate pathway (PPP) in cancer metabolic reprogramming has been usually outlined by studying glucose-6-phosphate dehydrogenase (G6PD). However, recent evidence suggests an unexpected role for a less characterized PPP, triggered by hexose-6-phosphate dehydrogenase (H6PD) within the endoplasmic reticulum (ER). Studying H6PD biological role in breast and lung cancer, here we show that gene silencing of this reticular enzyme decreases cell content of PPP intermediates and d-ribose, to a similar extent as G6PD silencing. Decrease in overall NADPH content and increase in cell oxidative status are also comparable. Finally, either gene silencing impairs at a similar degree cell proliferating activity. This unexpected response occurs despite the absence of any cross-interference between the expression of both G6PD and H6PD. Thus, overall cancer PPP reflects the contribution of two different pathways located in the cytosol and ER, respectively. Disregarding the reticular pathway might hamper our comprehension of PPP role in cancer cell biology.

The favourable kinetics of 18F-fluoro-2-deoxyglucose (FDG) permits to depict cancer glucose consumption by a single evaluation of late tracer uptake. This standard procedure relies on the slow radioactivity loss, usually attributed to the limited tumour expression of G6P-phosphatase (G6Pase). However, this classical interpretation intrinsically represents an approximation since, as in all tissues, cancer G6Pase activity is remarkable and is confined to the endoplasmic reticulum (ER), whose lumen must be reached by phosphorylated FDG to explain its hydrolysis and radioactivity release. The present study tested the impact of G6Pase sequestration on the mathematical description of FDG trafficking and handling in cultured cancer cells. Our data show that accounting for tracer access to the ER configures this compartment as the preferential site of FDG accumulation. This is confirmed by the reticular localization of fluorescent FDG analogues. Remarkably enough, reticular accumulation rate of FDG is dependent upon extracellular glucose availability, thus configuring the same ER as a significant determinant of cancer glucose metabolism.

The favourable kinetics of 18F-fluoro-2-deoxyglucose (FDG) permits to depict cancer glucose consumption by a single evaluation of late tracer uptake. This standard procedure relies on the slow radioactivity loss, usually attributed to the limited tumour expression of G6P-phosphatase (G6Pase). However, this classical interpretation intrinsically represents an approximation since, as in all tissues, cancer G6Pase activity is remarkable and is confined to the endoplasmic reticulum (ER), whose lumen must be reached by phosphorylated FDG to explain its hydrolysis and radioactivity release. The present study tested the impact of G6Pase sequestration on the mathematical description of FDG trafficking and handling in cultured cancer cells. Our data show that accounting for tracer access to the ER configures this compartment as the preferential site of FDG accumulation. This is confirmed by the reticular localization of fluorescent FDG analogues. Remarkably enough, reticular accumulation rate of FDG is dependent upon extracellular glucose availability, thus configuring the same ER as a significant determinant of cancer glucose metabolism.

PURPOSE: The endoplasmic reticulum (ER) contains hexose-6P-dehydrogenase (H6PD). This enzyme competes with glucose-6P-phosphatase for processing a variety of phosphorylated hexoses including 2DG-6P. The present study aimed to verify whether this ER glucose-processing machinery contributes to brain FDG uptake. METHODS: Effect of the H6PD inhibitor metformin on brain 18F-FDG accumulation was studied, in vivo, by microPET imaging. These data were complemented with the in vitro estimation of the lumped constant (LC). Finally, reticular accumulation of the fluorescent 2DG analogue 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2NBDG) and its response to metformin was studied by confocal microscopy in cultured neurons and astrocytes. RESULTS: Metformin halved brain 18F-FDG accumulation without altering whole body tracer clearance. Ex vivo, this same response faced the doubling of both glucose consumption and lactate release. The consequent fall in LC was not explained by any change in expression or activity of its theoretical determinants (GLUTs, hexokinases, glucose-6P-phosphatase), while it agreed with the drug-induced inhibition of H6PD function. In vitro, 2NBDG accumulation selectively involved the ER lumen and correlated with H6PD activity being higher in neurons than in astrocytes, despite a lower glucose consumption. CONCLUSIONS: The activity of the reticular enzyme H6PD profoundly contributes to brain 18F-FDG uptake. These data challenge the current dogma linking 2DG/FDG uptake to the glycolytic rate and introduce a new model to explain the link between 18-FDG uptake and neuronal activity.

Oxidative stress and its interference on myocardial metabolism play a major role in Doxorubicin (DXR) cardiotoxic cascade.Mice models of neuroblastoma (NB) were treated with 5 mg DXR/kg, either free (Free-DXR) or encapsulated in untargeted (SL[DXR]) or in NB-targeting Stealth Liposomes (pep-SL[DXR] and TP-pep-SL[DXR]). Control mice received saline. FDG-PET was performed at baseline (PET1) and 7 days after therapy (PET2). At PET2 Troponin-I and NT-proBNP were assessed. Explanted hearts underwent biochemical, histological, and immunohistochemical analyses. Finally, FDG uptake and glucose consumption were simultaneously measured in cultured H9c2 in the presence/absence of Free-DXR (1 μM).Free-DXR significantly enhanced the myocardial oxidative stress. Myocardial-SUV remained relatively stable in controls and mice treated with liposomal formulations, while it significantly increased at PET2 with respect to baseline in Free-DXR. At this timepoint, myocardial-SUV was directly correlated with both myocardial redox stress and hexose-6-phosphate-dehydrogenase (H6PD) enzymatic activity, which selectively sustain cellular anti-oxidant mechanisms. Intriguingly, in vitro, Free-DXR selectively increased FDG extraction fraction without altering the corresponding value for glucose.The direct correlation between cardiac FDG uptake and oxidative stress indexes supports the potential role of FDG-PET as an early biomarker of DXR oxidative damage.

Background The close connection between neuronal activity and glucose consumption accounts for the clinical value of 18F-fluoro-2-deoxyglucose (FDG) imaging in neurodegenerative disorders. Nevertheless, brain metabolic response to starvation (STS) might hamper the diagnostic accuracy of FDG PET/CT when the cognitive impairment results in a severe food deprivation. Methods Thirty six-week-old BALB/c female mice were divided into two groups: “control” group (n = 15) were kept under standard conditions and exposed to fasting for 6 h before the study; the remaining “STS” mice were submitted to 48 h STS (absence of food and free access to water) before imaging. In each group, nine mice were submitted to dynamic micro-PET imaging to estimate brain and skeletal muscle glucose consumption (C- and SM-MRGlu*) by Patlak approach, while six mice were sacrificed for ex vivo determination of the lumped constant, defined as the ratio between CMRGlu* and glucose consumption measured by glucose removal from the incubation medium (n = 3) or biochemical analyses (n = 3), respectively. Results CMRGlu* was lower in starved than in control mice (46.1 ± 23.3 vs 119.5 ± 40.2 nmol × min−1 × g−1, respectively, p

BACKGROUND: The close connection between neuronal activity and glucose consumption accounts for the clinical value of 18F-fluoro-2-deoxyglucose (FDG) imaging in neurodegenerative disorders. Nevertheless, brain metabolic response to starvation (STS) might hamper the diagnostic accuracy of FDG PET/CT when the cognitive impairment results in a severe food deprivation. METHODS: Thirty six-week-old BALB/c female mice were divided into two groups: "control" group (n = 15) were kept under standard conditions and exposed to fasting for 6 h before the study; the remaining "STS" mice were submitted to 48 h STS (absence of food and free access to water) before imaging. In each group, nine mice were submitted to dynamic micro-PET imaging to estimate brain and skeletal muscle glucose consumption (C- and SM-MRGlu*) by Patlak approach, while six mice were sacrificed for ex vivo determination of the lumped constant, defined as the ratio between CMRGlu* and glucose consumption measured by glucose removal from the incubation medium (n = 3) or biochemical analyses (n = 3), respectively. RESULTS: CMRGlu* was lower in starved than in control mice (46.1 ± 23.3 vs 119.5 ± 40.2 nmol × min-1 × g-1, respectively, p < 0.001). Ex vivo evaluation documented a remarkable stability of lumped constant as documented by the stability of GLUT expression, G6Pase activity, and kinetic features of hexokinase-catalyzed phosphorylation. However, brain SUV in STS mice was even (though not significantly) higher with respect to control mice. Conversely, a marked decrease in both SM-MRGlu* and SM-SUV was documented in STS mice with respect to controls. CONCLUSIONS: STS markedly decreases brain glucose consumption without altering measured FDG SUV in mouse experimental models. This apparent paradox does not reflect any change in lumped constant. Rather, it might be explained by the metabolic response of the whole body: the decrease in FDG sequestration by the skeletal muscle is as profound as to prolong tracer persistence in the bloodstream and thus its availability for brain uptake.

Unlike most cytokines, IL-1β lacks a secretory signal sequence raising the question of how is this cytokine processed and delivered outside the producing cells. After the seminal observation that IL-1β is actively secreted by human monocytes through a route alternative to the classic endoplas mic reticulum–Golgi, several different pathways have been proposed for IL-1β secretion in different cell types and culture conditions, some of which are unique to macrophage cell lines. Here we describe the most credited of these pathways. In particular, we will focus on IL-1β secretion from primary human blood monocytes. In fact, although data from macrophages or macrophage cell lines are predominant, secretion of IL-1β by monocytes is the most clinically relevant.

CTLA-4 blockade with monoclonal antibodies can lead to cancer regression in patients with metastatic melanoma (MM). CTLA-4 gene polymorphisms may influence the response to anti-CTLA-4 antibodies although few data are available regarding this issue. We analyzed six CTLA-4 single nucleotide polymorphisms (−1661A>G, −1577G>A, −658C>T, −319C>T, +49A>G, and CT60G>A) in 14 Italian MM patients and 45 healthy subjects. We found a significant association between the −1577G/A and CT60G/A genotypes and improved overall survival (Pc < 0.006, Bonferroni corrected), further confirmed by the diplotype analysis (−1577&CT60 GG-AA diplotype, p < 0.001). A positive trend toward an association between these genotypes and response to therapy was also observed.

Estrogen synthesis suppression induced by aromatase inhibitors in breast cancer (BC) patients may be affected by single nucleotide polymorphisms (SNPs) of the gene encoding aromatase enzyme, CYP19A1. We assessed the association between plasma estrone sulfate (ES), letrozole treatment, and four SNPs of CYP19A1 gene (rs10046 C>T, rs4646 G>T, rs749292 C>T, rs727479 T>G) which seem to be related to circulating estrogen levels. Patients were enrolled into a prospective, Italian multi-center clinical trial (Gruppo Italiano Mammella, GIM-5) testing the association of CYP19A1 SNPs with the efficacy of letrozole adjuvant therapy, in postmenopausal early BC patients. SNPs were identified from peripheral blood cell DNA. Plasma ES concentrations were evaluated by Radio Immuno Assay. Blood samples were obtained immediately before letrozole therapy (N = 204), at 6-weeks (N = 178), 6 (N = 152) and 12-months (N = 136) during treatment. Medians (IQR) of ES were 160 pg/mL (85–274) at baseline, 35 pg/mL (12–64) at 6-weeks, 29 pg/mL (17–48) at 6 months and 25 pg/mL (8–46) after 12 months treatment. No statistically significant association was evident between polymorphisms and ES circulating levels during letrozole therapy. Letrozole suppression of the aromatase enzyme function is not affected by polymorphisms of CYP19A1 gene in postmenopausal BC patients.

Conflicting observations have been reported about the role of CTLA-4 gene polymorphisms in the clinical outcome of allogeneic hematopoietic stem cell transplantation (HSCT). We have investigated three polymorphisms of the CTLA-4 gene (−318C>T, +49A>G, CT60G>A) in 133 donor/recipient pairs who underwent HLA-matched sibling donor HSCT for hematological malignancies. We found no association of the clinical outcome of the HSCT with either recipient or donor −318C>T and CT60G>A polymorphisms. At variance, we found a significant association of donor +49A>G G/G genotype with longer overall survival (OS; log-rank test, P = 0.04), and the number of +49A>G G-alleles in the recipient with longer OS (P = 0.027), longer disease-free survival (P = 0.036) and reduced relapse rate (P = 0.042). However, only recipient +49A>G polymorphism was retained as independent prognostic factor in a multivariate analysis, suggesting that the expression of CTLA-4 on the cells of recipient may be relevant for the clinical outcome of HSCT.

Pyrrolidinedithiocarbamate (PDTC) is a compound displaying antioxidant, pro-oxidant and metal chelator properties in different cell types. It has been described that PDTC may exert either anti-apoptotic or apoptotic activity. Moreover it is known that this agent regulates the activity of redox-sensitive transcription factors, such as AP-1 and NF-kappaB. Using cerebellar granule cells (CGCs), a well-described model of neuronal primary cultures, we investigated the effects of different concentrations of this compound on cell viability and the intracellular mechanisms involved. PDTC used at concentrations, as low as 1 microM, exerts cytotoxic effects on CGC through the activation of the apoptotic machinery with a maximal efficacy for concentration of 10 microM. The PDTC-dependent apoptosis is correlated to a biphasic and long-lasting increase of AP-1 binding to the DNA, apparently without affecting the NF-kappaB whose activity was reduced only at much higher concentrations (100 microM). PDTC treatment enhanced ERK phosphorylation (maximal effect 1h) and p38 phosphorylation (maximal effect 7h) that was accompanied by an increase of both mRNA and protein of c-Jun. In conclusion the results presented show that PDTC exerts apoptotic effects on CGC, that are correlated to the activation of stress-pathways, involving mainly AP-1 and MAPKs.

IL-1β acts in concert with anti-inflammatory cytokines, in particular, IL-1R antagonist (IL-1Ra), to ensure the correct development and outcome of the inflammation: imbalance in the IL-1β/IL-1Ra ratio is implicated in many human diseases and may lead to dramatic consequences. In this article, we show that single TLR engagement induces IL-1β and, with a little delay, IL-1Ra. Differently, costimulation of TLR2, TLR4, and TLR7/8 enhances IL-1β secretion but severely inhibits IL-1Ra production. The IL-1β/IL-1Ra unbalance after activation of multiple TLRs depends on the insurgence of oxidative stress, because of enhanced production of reactive oxygen species and failure of the antioxidant systems. Increased reactive oxygen species levels increase ATP externalization by monocytes, resulting in enhanced inflammasome activation and IL-1β secretion. Oxidative stress then induces cell responses to stress, including inhibition of protein synthesis, which, in turn, is responsible for the impaired production of IL-1Ra. IL-1Ra secretion is restored by exogenous antioxidants that oppose oxidative stress. Similar effects are evident also on other cytokines: TNF-α is induced, whereas IL-6 is inhibited by costimulation. Our findings provide a molecular basis to the imbalance between proinflammatory and regulatory cytokine circuits that occur in various pathologic conditions, and suggest new strategies for controlling inflammation.

CTLA-4 expression/function can be affected by single nucleotide polymorphisms (SNPs) of CTLA-4 gene, which have been widely associated with susceptibility or progression to autoimmune diseases and cancer development. In this study, we analyzed six CTLA-4 SNPs (-1661A>G, -1577G>A, -658C>T, -319C>T, +49A>G, CT60G>A) in 197 DNA samples from 43 B-lymphoblastoid cell lines (B-LCLs), 40 systemic sclerosis (SSc) patients, 14 pre-analyzed melanoma patients and 100 Italian healthy subjects. Genotyping of -1661A>G, -1577G>A, -658C>T and CT60G>A was performed by newly developed multiplex pyrosequencing (PSQ) assays, whereas -319C>T and +49A>G by T-ARMS PCR and direct sequencing. Genotype/allele frequency were analyzed using χ(2) or Fisher exact test. Our study provides the first multiplex PSQ method that allows simultaneous genotyping of two CTLA-4 SNP pairs (i.e. -1661A>G/-658C>T and -1577G>A/CT60G>A) by two multiplex PSQ reactions. Herein, we show the CTLA-4 genotype distribution in the B-LCLs providing the first and best characterized cell line panel typed for functionally relevant CTLA-4 SNPs. We also report the significant association of the -1661A/G genotype, -1661 & -319 AC-GT diplotype and -319 & CT60 TG haplotype with susceptibility to SSc without Hashimoto's thyroiditis occurrence. Furthermore, we confirmed previous genotyping data referred to melanoma patients and provided new genotyping data for Italian healthy subjects.

Stromal cell-derived factor-1 (SDF-1), the ligand of the CXCR4 receptor, is a chemokine involved in chemotaxis and brain development that also acts as co-receptor for HIV-1 infection. We previously demonstrated that CXCR4 and SDF-1alpha are expressed in cultured type-I cortical rat astrocytes, cortical neurones and cerebellar granule cells. Here, we investigated the possible functions of CXCR4 expressed in rat type-I cortical astrocytes and demonstrated that SDF-1alpha stimulated the proliferation of these cells in vitro. The proliferative activity induced by SDF-1alpha in astrocytes was reduced by PD98059, indicating the involvement of extracellular signal-regulated kinases (ERK1/2) in the astrocyte proliferation induced by CXCR4 stimulation. This observation was further confirmed showing that SDF-1alpha treatment selectively activated ERK1/2, but not p38 or stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK). Moreover, both astrocyte proliferation and ERK1/2 phosphorylation, induced by SDF-1alpha, were inhibited by pertussis toxin (PTX) and wortmannin treatment indicating the involvement of a PTX sensitive G-protein and of phosphatidyl inositol-3 kinase in the signalling of SDF-1alpha. In addition, Pyk2 activation represent an upstream components for the CXCR4 signalling to ERK1/2 in astrocytes. To our knowledge, this is the first report demonstrating a proliferative effect for SDF-1alpha in primary cultures of rat type-I astrocytes, and showing that the activation of ERK1/2 is responsible for this effect. These data suggest that CXCR4/SDF-1 should play an important role in physiological and pathological glial proliferation, such as brain development, reactive gliosis and brain tumour formation.

Background: Obesity is an independent adverse prognostic factor in early breast cancer patients, but it is still controversial whether obesity may affect adjuvant endocrine therapy efficacy. The aim of our study (ancillary to the two clinical trials Gruppo Italiano Mammella (GIM)4 and GIM5) was to investigate whether the circulating oestrogen levels during treatment with the aromatase inhibitor letrozole are related to body mass index (BMI) in postmenopausal women with breast cancer. Methods: Plasma concentration of oestrone sulphate (ES) was evaluated by radioimmunoassay in 370 patients. Plasma samples were obtained after at least 6 weeks of letrozole therapy (steady-state time). Patients were divided into four groups according to BMI. Differences among the geometric means (by ANOVA and ANCOVA) and correlation (by Spearman's rho) between the ES levels and BMI were assessed. Results: Picomolar geometric mean values (95% confidence interval, n=patients) of circulating ES during letrozole were 58.6 (51.0–67.2, n=150) when BMI was

Recombinant antibodies are efficiently secreted by cells of the nervous system. Thus, their local expression in the CNS of transgenic mice could be used to perturb the function of the corresponding antigen. As a first application of this approach, we have generated transgenic mice that express antibodies against the neuropeptide substance P, under the transcriptional control of the promoter of the neuronal gene vgf . The transgenic antibodies are expressed in a tissue-specific and developmentally regulated manner and are effective in competing with the endogenous substance P, as demonstrated by a marked Inhibition of neurogenic inflammation and by motor deficits. This phenotypic knockout approach may provide a complementary alternative to gene knockout by homologous recombination.

Incidence of sepsis is increasing, representing a tremendous burden for health-care systems. Death in acute sepsis is attributed to hyperinflammatory responses, but the underlying mechanisms are still unclear. We report here that proton pump inhibitors (PPIs), which block gastric acid secretion, selectively inhibited tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) secretion by Toll-like receptor (TLR)-activated human monocytes in vitro, in the absence of toxic effects. Remarkably, the oversecretion of IL-1β that represents a hallmark of monocytes from patients affected by cryopyrin-associated periodic syndrome is also blocked. Based on these propaedeutic experiments, we tested the effects of high doses of PPIs in vivo in the mouse model of endotoxic shock. Our data show that a single administration of PPI protected mice from death (60% survival versus 5% of untreated mice) and decreased TNF-α and IL-1β systemic production. PPIs were efficacious even when administered after lipopolysaccharide (LPS) injection. PPI-treated mice that survived developed a long-term cross-tolerance, becoming resistant to LPS- and zymosan-induced sepsis. In vitro, their macrophages displayed impaired TNF-α and IL-1β to different TLR ligands. PPIs also prevented sodium thioglycollate-induced peritoneal inflammation, indicating their efficacy also in a non-infectious setting independent of TLR stimulation. Lack of toxicity and therapeutic effectiveness make PPIs promising new drugs against sepsis and other severe inflammatory conditions.

Complement C3 ‘slow’ and ‘fast’ allotypes are associated with immune-mediated disorders and may affect the outcome of renal transplantation. We report a tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) that provides a rapid, reproducible and cost-effective method to genotype both complement C3 ‘slow’ and ‘fast’ alleles by a single tube reaction.

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is the major negative regulator of T-cell responses, although growing evidence supports its wider role as an immune attenuator that may also act in other cell lineages. Here, we have analyzed the expression of CTLA-4 in human monocytes and monocyte-derived dendritic cells (DCs), and the effect of its engagement on cytokine production and T-cell stimulatory activity by mature DCs. CTLA-4 was highly expressed on freshly isolated monocytes, then down-modulated upon differentiation toward immature DCs (iDCs) and it was markedly upregulated on mature DCs obtained with different stimulations (lipopolysaccharides [LPS], Poly:IC, cytokines). In line with the functional role of CTLA-4 in T cells, treatment of mDCs with an agonistic anti-CTLA-4 mAb significantly enhanced secretion of regulatory interleukin (IL)-10 but reduced secretion of IL-8/IL-12 pro-inflammatory cytokines, as well as autologous CD4+ T-cell proliferation in response to stimulation with recall antigen purified protein derivative (PPD) loaded-DCs. Neutralization of IL-10 with an anti-IL-10 antibody during the mDCs-CD4+ T-cell co-culture partially restored the ability of anti-CTLA-4-treated mDCs to stimulate T-cell proliferation in response to PPD. Taken together, our data provide the first evidence that CTLA-4 receptor is expressed by human monocyte-derived mDCs upon their full activation and that it exerts immune modulatory effects.

We present a strategy to study functional and/or developmental processes occurring in the nervous system, as well as in other systems, of mice. This strategy is based on the local expression of specific monoclonal antibodies (mAbs) by cells of the nervous system. As an application of this strategy, we report the cloning of the anti-substance P rat mAb NC1/34HL. Functional substance P-binding antibodies were reconstituted from the cloned variable domains by using vectors for expression in myeloma cells. With these and other vectors a general system for the cloning and expression of mAbs under a series of promoters (of the rat VGF8a gene, the neurofilament light-chain gene, and the methallothionein gene) has been created. The activity of these plasmids was confirmed by expressing the recombinant NC1/34HL mAb in GH3 pituitary cells, PC12 pheochromocytoma cells, and COS cells. DNA from the described constructs can be used to target the expression of the NC1/34HL mAb to the central nervous system of transgenic mice. This procedure will allow us to perturb substance P activity in a controlled way in order to dissect its multiple roles.

Tetraprimer amplification refractory mutation system PCR (T-ARMS-PCR) is a simple and inexpensive genotyping method for differentiating both alleles of a polymorphism/mutation (both single-nucleotide polymorphisms and small insertions/deletions) with a single-tube PCR (1). In T-ARMS-PCR, a pair of common (outer) primers produces a non–allele-specific control amplicon and in combination with 2 allele-specific (inner) primers (designed to anneal in the opposite orientation) produces 2 allele-specific amplicons. These allele-specific amplicons have different sizes because the polymorphism/mutation is asymmetrically located with respect to the common primers. Thus, the amplicons can be separated by standard gel electrophoresis. T-ARMS-PCR has also been designed in a multiplex fashion to genotype more than one polymorphism/mutation by a single-tube PCR (2). We describe a modified multiplex T-ARMS-PCR, the hexaprimer ARMS-PCR (H-ARMS-PCR), which is for when 2 polymorphisms are close in the sequence. H-ARMS-PCR uses only 6 primers and provides direct information about haplotype structure. The CTLA4 gene (cytotoxic T-lymphocyte–associated protein 4; also known as CD152) is a negative regulator of T-cell function (3). The CTLA4 polymorphisms −318 C>T (rs5742909) and +49 A>G (rs231775) are associated with susceptibility to autoimmune diseases and cancer (3)(4). To genotype these 2 polymorphisms, which are only 365 bp apart in the 5′ region of the CTLA4 gene, we designed an H-ARMS-PCR that combines a single pair of common primers and 2 pairs of allele-specific primers in the same tube (Fig. 1A⇓ ). Figure 1. Polymorphism genotyping by H-ARMS-PCR. (A), H-ARMS-PCR for CTLA4 −318 C>T (rs5742909) and …

A panel of homozygous cell lines, previously typed by primed lymphocyte test for their DPw specificity, have been studied by restriction fragment length polymorphism analysis, using a DPβ-specific probe. Highly stringent hybridization and washing conditions were used to prevent cross-hybridization with DR- and DQ-specific fragments. Three out of six enzymes employed allowed us to distinguish clustered or single DPw specificities, and by MspI digestion it was possible to detect different patterns within a single specificity such as DPw4. Some of the cell lines have been further studied with synthetic oligonucleotides derived from the polymorphic regions of the second exon of DPβ1 gene, and, in general, a correlation with the primed lymphocyte test-defined specificities and restriction fragment length polymorphism was found. These data suggest a more extended complexity of the DP region, in addition to that defined as the DPw1-DPw6 segregant series.

Summary Systemic sclerosis (SSc) is a complex and heterogeneous autoimmune disorder with a multi-factorial pathogenesis. Like other autoimmune disorders, the possible role of specific cytotoxic T lymphocyte antigen-4 (CTLA-4) gene polymorphisms in predisposing to SSc has been hypothesized, but it remains controversial. CTLA-4 promoter (−318C/T) and exon 1 (+49 A/G) polymorphisms have been analysed in 43 Italian females with SSc and in 93 unrelated matched healthy controls by a newly designed tetra-primer amplification refractory mutation system–polymerase chain reaction (T-ARMS–PCR) method. No significant association has been found with either polymorphisms. Nevertheless, SSc patients without concomitant Hashimoto's thyroiditis (HT) were carrying both the −318T allele (P = 0·031) and the +49 G allele (P = 0·076) more frequently than SSc patients with HT [defined by positivity for anti-thyroperoxidase (TPO) and anti-thyroglobulin (TGA) autoantibodies] than controls. Haplotype analysis confirms this association (P = 0·028), and suggests the predominant role of the −318T, whereas that of the +49 G, if any, seems weak. Thus, in Italian SSc patients the CTLA-4 −318C/T promoter polymorphism appears to be associated with the susceptibility to develop SSc without thyroid involvement. Larger studies are needed to confirm these findings and to clarify whether the −318C/T polymorphism is the functional responsible or whether it reflects the presence of another linked genetic element in the same chromosomal region.

Background: We describe a simple tetra-primer amplification refractory mutation system PCR (T-ARMS-PCR) for detecting MUTYH mutations, which are associated with colorectal adenomas and colorectal cancer. Methods: We designed specific T-ARMS-PCR assays for 6 mutations (Y165C, G382D, 1395_7delGGA, Y90X, 1103delC, and R231H) selected on the basis of the frequency of their occurrence. We also designed a set of 3 multiplex T-ARMS PCR assays, each for detection of 2 mutations. We tested DNA samples from patients with attenuated or classic adenomatous polyposis coli and no detectable APC germline mutations. Results: All mutations were easily detected with both the specific and multiplex T-ARMS-PCR assays. Results were confirmed by DNA HPLC analysis in all 54 patients, and each mutation was confirmed by direct DNA sequencing. Conclusions: T-ARMS-PCR does not require any special equipment, and it provides rapid, reproducible, and cost-effective detection of common MUTYH mutations. Multiplex T-ARMS-PCR allows the detection of 6 common MUTYH mutations with use of as few as 3 single tube PCR reactions. It could be useful to carry out large population-based epidemiologic studies.

Chemokines are a family of proteins that chemoattract and activate cells by interacting with specific receptors on the surface of their targets. They are grouped into four classes based on the position of key cysteine residues: C, CC, CXC, and CX3C. Stromal cell-derived factor 1 (SDF1), the ligand of the CXCR4 receptor, is a CXC chemokine involved in chemotaxis and brain development that also acts as coreceptor for HIV-1 infection. It has been proposed that CXCR4 is overexpressed and required for proliferation in human brain tumor cells. We previously demonstrated that CXCR4 and SDF1 are expressed in culture of cortical type I rat astrocytes, cortical neurons, and cerebellar granule cells. In this study, we analyzed the expression of CXCR4 and SDF1 in four human brain tumor tissues, showing that CXCR4 is expressed in all tumors analyzed, whereas SDF1 is expressed only in two tumor tissues. We also investigated the possible functions of CXCR4 expressed in rat type I cortical astrocytes, demonstrating that SDF1alpha stimulates the proliferation of these cells in vitro. Moreover, we studied by western blot the intracellular pathway involved in cell proliferation, demonstrating that SDF1alpha induces the ERK1/2 phosphorylation that is reduced by the PD98059 compound, an MEK inhibitor.

Many activities of neuronal cells, such as synaptic transmission, inflammation, neuroprotection, and neurotoxicity, are regulated by the activity of the transcription factor nuclear factor-kappaB (NF-kappaB). In resting cells, NF-kappaB activity is present both in the cytoplasm, as an inducible-inactive complex, and in the nucleus as a constitutive form. The activation of its inducible form is related to processing of IkappaB(s), which occurs through the proteasome. To understand whether NF-kappaB is involved in the survival of cerebellar granule cells (CGCs) maintained under conditions of mild depolarization (25 mM KCl), these cells were treated with different proteasome inhibitors. The results presented show that these pharmacological tools reduce CGC survival with changes in nuclear morphology and induction of apoptosis. Furthermore, we demonstrate that PSI-induced apoptosis is reverted by inhibitors of transcription and translation, as well as by specific caspase inhibitors. These issues are also associated with a redistribution of NF-kappaB, in that a reduced amount of nuclear NF-kappaB and an increased p65 cytoplasmic level have been observed. Finally, we propose that, at least in part, p65 metabolism could also be regulated by the ubiquitin-proteasome complex. Altogether, the results presented define an important role for NF-kappaB in maintainig CGC survival.

The present findings show that an atypical non-steroidal anti-inflammatory drug, such as acetaminophen, retains the ability to recover amyloid beta-peptides driven neuronal apoptosis through the impairment of oxidative stress. Moreover, this compound reduces the increased NF-kappaB binding activity, which occurs in these degenerative conditions. Therapeutic interventions aimed at reducing the inflammatory response in Alzheimer's disease (AD) recently suggested the application of non-steroidal anti-inflammatory drugs. Although the anti-inflammatory properties of acetaminophen are controversial, it emerged that in an amyloid-driven astrocytoma cell degeneration model acetaminophen proved to be effective. On these bases, we analyzed the role of acetaminophen against the toxicity exerted by different Abeta-peptides on rat primary hippocampal neurons and on a rat pheochromocytoma cell line. We found a consistent protection from amyloid beta-fragments 1-40 and 1-42-induced impairment of mitochondrial redox activity on both cell cultures, associated with a marked reduction of apoptotic nuclear fragmentation. An antioxidant component of the protective activity emerged from the analysis of the reduction of phospholipid peroxidation, and also from a significant reduction of cytoplasmic accumulation of peroxides in the pheochromocytoma cell line. Moreover, activation of NF-kappaB by amyloid-derived peptides was greatly impaired by acetaminophen pre-treatment in hippocampal cells. This evidence points out antioxidant and anti-transcriptional properties of acetaminophen besides the known capability to interfere with inflammation within the central nervous system, and suggests that it can be exploited as a possible therapeutic approach against AD.

The nuclear factor (NF)-κB family of transcription factors plays important roles in the regulation of many activities of neuronal cells, such as synaptic transmission, inflammation, neuroprotection, and neurotoxicity. In resting cells, NF-κB activity is present both in the cytoplasm, as an inducible-inactive complex, and in the nucleus, as a constitutive form. Regulation of its inducible activity relies on processing of IκB(s), which occurs through the proteasome. Here we show that in cerebellar granule cells (CGC) the induction of apoptosis, by potassium withdrawal (5 mM KCl), decreases the amount of nuclear NF-κB. To understand whether NF-κB was required for CGC survival, these cells, maintained under depolarizing conditions (25 mM KCl and serum), were treated with proteasome inhibitors. The results show that these treatments reduce the nuclear amount of NF-κB and increase p65 cytoplasmic levels, a process partially regulated via IκBα degradation. These events are also associated with an impairment in CGC survival, with changes in nuclear morphology, induction of DNA laddering, and oligonucleosome formation, consistent with apoptosis. According to the K+ deprivation model, PSI-induced apoptosis is reversed by inhibitors of transcription and translation as well as by specific caspase inhibitors. Together our results show an important role for NF-κB in maintaining CGC survival. Indeed, under conditions of mild depolarization (K25) necessary for CGC survival, NF-κB is distributed between cytosol and nucleus, whereas, under apoptotic conditions (K5), it is depleted from the nucleus, such as after proteasome inhibitor treatment. Therefore, NF-κB nuclear deprivation is involved in the induction of CGC apoptosis. J Neurosci. Res. 66:1064–1073, 2001. © 2001 Wiley-Liss, Inc.

Chemokines are a family of proteins that chemoattract and activate cells by interacting with specific receptors on the surface of their targets. The chemokine stromal cell-derived factor 1, (SDF1), binds to the seven-transmembrane G protein-coupled CXCR4 receptor and acts to modulate cell migration, differentiation, and proliferation. CXCR4 and SDF1 are reported to be expressed in various tissues including brain. Here we show that SDF1 and CXCR4 are expressed in cultured cortical type I rat astrocytes, cortical neurons, and cerebellar granule cells. In cortical astrocytes, prolonged treatment with lipopolysaccharide induced an increase of SDF1 expression and a down-regulation of CXCR4, whereas treatment with phorbol esters did not affect SDF1 expression and down-modulated CXCR4 receptor expression. We also demonstrated the ability of human SDF1alpha (hSDF1alpha) to increase the intracellular calcium level in cultured astrocytes and cortical neurons, whereas in the same conditions, cerebellar granule cells did not modify their intracellular calcium concentration. Furthermore, in cortical astrocytes, the simultaneous treatment of hSDF1alpha with the HIV-1 capside glycoprotein gp120 inhibits the cyclic AMP formation induced by forskolin treatment.

In this chapter we shall describe how ectopic antibody expression can be used to interfere with the function of molecules that are located in the extracellular environment of cells of the nervous system.

515 Background: BMI is associated with an increased risk of BC in postmenopausal women. Moreover, BC overweight women have poorer prognosis compared to women with normal weight. Data from ATAC (the anastrozole, tamoxifen alone or in combination study) trial reported that obesity reduce the efficacy of the aromatase inhibitor (AI) anastrozole in early BC. One possible explanation is that higher estrogens levels resulting from high BMI may lead to lower efficacy of the AI treatment. No clear data are available about the effects of BMI on estrogen circulating levels during AI treatment, and if the increase of AI dosage may result in more complete estrogen suppression in overweight women. Methods: We evaluated the correlation between BMI and plasma concentration of ES in postmenopausal women with early BC during adjuvant treatment with L (2.5 mg/die). Patients were participating in two prospective Italian clinical trials (GIM4 and GIM5) evaluating L treatments after adjuvant tamoxifen. Plasma samples were obt...

Background Trastuzumab is a humanized monoclonal antibody (mAb) currently used for the treatment of breast cancer (BC) patients with HER-2 overexpressing tumor subtype. Previous data reported the involvement of FcγRIIIA/IIA gene polymorphisms and/or antibody-dependent cellular cytotoxicity (ADCC) in the therapeutic efficacy of trastuzumab, although results on these issues are still controversial. This study was aimed to evaluate in vitro the functional relationships among FcγRIIIA/IIA polymorphisms, ADCC intensity and HER-2 expression on tumor target cells and to correlate them with response to trastuzumab. Patients and methods Twenty-five patients with HER-2 overexpressing BC, receiving trastuzumab in a neoadjuvant (NEO) or metastatic (MTS) setting, were genotyped for the FcγRIIIA 158V>F and FcγRIIA 131H>R polymorphisms by a newly developed pyrosequencing assay and by multiplex Tetra-primer-ARMS PCR, respectively. Trastuzumab-mediated ADCC of patients’ peripheral blood mononuclear cells (PBMCs) was evaluated prior to therapy and measured by 51Chromium release using as targets three human BC cell lines showing different levels of reactivity with trastuzumab. Results We found that the FcγRIIIA 158F and/or the FcγRIIA 131R variants, commonly reported as unfavorable in BC, may actually behave as ADCC favorable genotypes, in both the NEO (P ranging from 0.009 to 0.039 and from 0.007 to 0.047, respectively) and MTS (P ranging from 0.009 to 0.032 and P = 0.034, respectively) patients. The ADCC intensity was affected by different levels of trastuzumab reactivity with BC target cells. In this context, the MCF-7 cell line, showing the lowest reactivity with trastuzumab, resulted the most suitable cell line for evaluating ADCC and response to trastuzumab. Indeed, we found a statistically significant correlation between an increased frequency of patients showing ADCC of MCF-7 and complete response to trastuzumab in the NEO setting (P = 0.006). Conclusions Although this study was performed in a limited number of patients, it would indicate a correlation of FcγR gene polymorphisms to the ADCC extent in combination with the HER-2 expression levels on tumor target cells in BC patients. However, to confirm our findings further experimental evidences obtained from a larger cohort of BC patients are mandatory. Electronic supplementary material The online version of this article (doi:10.1186/s12967-015-0680-0) contains supplementary material, which is available to authorized users.