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Despite recent advances in chronic heart failure (HF) therapy, the prognosis of HF patients remains poor, with high rates of HF rehospitalizations and death in the early months after discharge. This emphasizes the need for incorporating novel HF drugs, beyond the current approach (that of modulating the neurohumoral response). Recently, new antidiabetic oral medications (sodium-glucose cotransporter 2 inhibitors (SGLT2i)) have been shown to improve prognosis in diabetic patients with previous cardiovascular (CV) events or high CV risk profile. Data from DAPA-HF study showed that dapaglifozin is associated with a significant reduction in mortality and HF hospitalization as compared with placebo regardless of diabetes status. Recently, results from EMPEROR-Reduced HF trial were consistent with DAPA-HF trial findings, showing significant beneficial effect associated with empagliflozin use in a high-risk HF population with markedly reduced ejection fraction. Results from the HF with preserved ejection fraction trials using these same agents are eagerly awaited. This review summarizes the evidence for the use of gliflozins in HF treatment.

Methylglyoxal (MG), a potent glycotoxin that can be found in the diet, is one of the main precursors of Advanced glycation end products (AGEs). It is well known that modifications in lifestyle such as nutritional interventions can be of great value for preventing brain deterioration. This study aimed to evaluate in vivo how an oral MG treatment, that mimics a high MG dietary intake, could affect brain health. From our results, we demonstrated that MG administration affected working memory, and induced neuroinflammation and oxidative stress by modulating the Receptor for Advanced glycation end products (RAGE). The gene and protein expressions of RAGE were increased in the hippocampus of MG mice, an area where the activity of glyoxalase 1, one of the main enzymes involved in MG detoxification, was found reduced. Furthermore, at hippocampus level, MG mice showed increased expression of proinflammatory cytokines and increased activities of NADPH oxidase and catalase. MG administration also increased the gene and protein expressions of Presenilin-1, a subunit of the gamma-secretase protein complex linked to Alzheimer's disease. These findings suggest that high MG oral intake induces alteration directly in the brain and might establish an environment predisposing to AD-like pathological conditions.

In the middle of the coronavirus disease 19 (COVID-19) outbreak, the main efforts of the scientific community are rightly all focused on identifying efficient pharmacological treatments to cure the acute severe symptoms and developing a reliable vaccine. On the other hand, we cannot exclude that, in Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) positive subjects, the virus infection could have long-term consequences, leading to chronic medical conditions such as dementia and neurodegenerative disease. Considering the age of SARS-CoV-2 infected subjects, the neuroinvasive potential might lead/contribute to the development of neurodegenerative diseases. Here, we analyzed a possible link between SARS-CoV-2 infection and Alzheimer’s disease risk, hypothesizing possible mechanisms at the base of disease development. This reflection raises the need to start to experimentally investigating today the mechanistic link between Alzheimer’s disease (AD) and COVID-19 to be ready tomorrow.

BackgroundUnderstanding the earliest pathophysiological changes of Alzheimer’s disease (AD) may aid in the search for timely diagnostic biomarkers and effective disease-modifying therapies. The p53 protein is mostly known for its role in tumor suppression. However, emerging evidence supports that dysregulated p53 activity may contribute to various peripheral and brain alterations during the earliest stages of AD. This review describes the mechanisms through which p53 dysregulation may exacerbate AD pathology and how this could be used as a potential peripheral biomarker for early detection of the disease.Main bodyp53, known as the guardian of the genome, may underlie various compensation or defense mechanisms that prevent neurons from degeneration. These mechanisms include maintenance of redox homeostasis, regulation of inflammation, control of synaptic function, reduction of amyloid β peptides, and inhibition of neuronal cell cycle re-entry. Thereby, dysregulation of p53-dependent compensation mechanisms may contribute to neuronal dysfunction, thus leading to neurodegeneration. Interestingly, a conformational misfolded variant of p53, described in the literature as unfolded p53, which has lost its canonical structure and function, was observed in peripheral cells from mild cognitive impairment (MCI) and AD patients. In AD pathology, this peculiar conformational variant was caused by post-translational modifications rather than mutations as commonly observed in cancer. Although the presence of the conformational variant of p53 in the brain has yet to be formally demonstrated, the plethora of p53-dependent compensation mechanisms underscores that the guardian of the genome may not only be lost in the periphery during AD pathology.ConclusionThese findings revisit the role of p53 in the early development and exacerbation of AD pathology, both in the brain and periphery. The conformational variant of p53 represents a potential peripheral biomarker that could detect AD at its earliest stages.

Background: The biological mechanisms underlying early- and advanced-stage epithelial ovarian cancers (EOCs) are still poorly understood. This study explored kinase-driven metabolic signalling in early and advanced EOCs, and its role in tumour progression and response to carboplatin–paclitaxel treatment. Methods: Tumour epithelia were isolated from two independent sets of primary EOC (n=72 and 30 for the discovery and the validation sets, respectively) via laser capture microdissection. Reverse phase protein microarrays were used to broadly profile the kinase-driven metabolic signalling of EOC with particular emphasis on the LBK1–AMPK and AKT–mTOR axes. Signalling activation was compared between early and advanced lesions, and carboplatin–paclitaxel-sensitive and -resistant tumours. Results: Advanced EOCs were characterised by a heterogeneous kinase-driven metabolic signature and decreased phosphorylation of the AMPK–AKT–mTOR axis compared to early EOC (P

Mice emit ultrasonic vocalizations (USVs) in different social conditions: pups maternal separation, juveniles play, adults mating and social investigation. The USVs measurement has become an important instrument for behavioural phenotyping in neurodevelopmental disorders (NDDs). Recently, we have demonstrated that the deletion of the NFκB1 gene, which encodes the p50 NF-κB subunit, causes NDDs phenotype in mice. In this study, we investigated the ultrasonic communication and the effects of an early social enrichment in mice lacking the NF-κB p50 subunit (p50 KO). In particular, USVs of wild-type (WT), p50 KO and KO exposed to early social enrichment (KO enriched) were recorded using an ultrasound sensitive microphone and analysed by Avisoft software. USVs analysis showed that p50 KO pups emit more and longer vocalizations compared to WT pups. On the contrary, in adulthood, p50 KO mice emit less USVs than WT mice. We also found significant qualitative differences in p50 KO mice USVs compared to WT mice; the changes specifically involved two USVs categories. Early social enrichment had no effect on USVs number, duration and type in p50 KO mice. Together, these data revealed social communication alterations in a mouse model of NDDs; these deficits were not recovered by early social enrichment, strengthening the fact that genetic background prevails on environmental enrichment.

Abiraterone acetate (AbiAc) inhibits tumor growth when administered to immunodeficient mice engrafted with the in vitro cell model of human adrenocortical carcinoma (ACC). Here, we developed and validated a zebrafish model engrafted with cortisol-secreting ACC cells to study the effects of AbiAc on tumor growth. The experimental conditions for AbiAc absorption in AB zebrafish embryos including embryo number, AbiAc concentration, and absorption time curve by liquid chromatography–tandem mass spectrometry were set up. The AbiAc effect on steroid production in AB zebrafish embryos was measured as well. ACC cells (the NCI-H295R cell line, the primary cell ACC29, and the negative control cell SW13) were treated with drug-induced liver injury fluorescent dye, and ∼240 cells per 4 nL was injected in the subperidermal space of the yolk sac of AB zebrafish embryos (n = 80 ± 10). The cell area was measured with Noldus DanioScopeTM software. AbiAc absorption in AB zebrafish embryos was stage dependent. Abiraterone (Abi) concentration decreased, whereas its main metabolite, Δ4A, increased. Accordingly, we demonstrated that zebrafish expressed mRNA encoding the enzyme 3β-hydroxysteroid dehydrogenase, which converts Abi in Δ4A. Furthermore, ABiAc reduced cortisol production and increased progesterone in zebrafish embryos. Three days after cell injection, the cortisol-secreting ACC cell area in solvent-treated embryos was significantly higher than that in 1 µM AbiAC‒treated embryos, whereas no AbiAc effect was observed in SW13 cells, which lack the Abi target enzyme CYP17A1.Zebrafish embryos xenografted with ACC tumor cells could be a useful, fast, and reproducible experimental model to preclinically test the activity of new drugs in human ACC.

Aberrant immune activity during neurodevelopment could participate in the generation of neurological dysfunctions characteristic of several neurodevelopmental disorders (NDDs). Numerous epidemiological studies have shown a link between maternal infections and NDDs risk, animal models of maternal immune activation (MIA) have confirmed this association. Activation of maternal immune system during pregnancy induces behavioral and functional alterations in offspring but the biological mechanisms at the basis of these effects are still poorly understood. In this study, we investigated the effects of prenatal lipopolysaccharide (LPS) exposure in peripheral and central inflammation, cortical cytoarchitecture and behavior of offspring (LPS-mice). LPS-mice reported a significant increase in interleukin-1&beta, (IL-1&beta, ) serum level, glial fibrillary acidic protein (GFAP)- and ionized calcium-binding adapter molecule 1 (Iba1)-positive cells in the cortex. Furthermore, cytoarchitecture analysis in specific brain areas, showed aberrant alterations in minicolumns&rsquo, organization in LPS-mice adult brain. In addition, we demonstrated that LPS-mice presented behavioral alterations throughout life. In order to better understand biological mechanisms whereby LPS induced these alterations, dams were treated with meloxicam. We demonstrated for the first time that exposure to LPS throughout pregnancy induces structural permanent alterations in offspring brain. LPS-mice also present severe behavioral impairments. Preventive treatment with meloxicam reduced inflammation in offspring but did not rescue them from structural and behavioral alterations.

Despite recent advances in chronic heart failure treatment, prognosis of acute heart failure patients remains poor with a heart failure rehospitalization rate or death reaching approximately 25% during the first 6 months after discharge. In addition, about half of these patients have preserved ejection fraction for which there are no evidence-based therapies. Disappointing results from heart failure clinical trials over the past 20 years emphasize the need for developing novel approaches and pathways for testing new heart failure drugs and devices. Indeed, many trials are being conducted without matching the mechanism and action of the drug with the clinical event. The implementation of these novel approaches should be coupled with the training of a new generation of heart failure physicians and scientists in the art and science of clinical trials. Currently, drug development is led by opinion leaders and experts who, despite their huge personal experience, were never trained systematically on drug development. The aim of this article is to propose a training program of 'drug development in Heart Failure'. A physician attending this course would have to be trained with a major emphasis on heart failure pathophysiology to better match mechanisms of death and rehospitalization with mechanism of action of the drug. Applicants will have to prove their qualifications and special interest in heart failure drug development before enrollment. This article should serve as a roadmap on how to apply emerging general principles in an innovative drug-development-in-heart-failure-process as well as the introduction of a new educational and mentorship program focusing on younger generations of researchers.

Context: Bone loss and nonvertebral fractures have been reported in patients with differentiated thyroid carcinoma (DTC) undergoing thyroid-stimulating hormone (TSH) suppressive therapy. Radiological vertebral fractures (VFs) are an early and clinically crucial marker of bone fragility. Objective and Design: A cross-sectional study to evaluate the prevalence and determinants of radiological VFs in women receiving L-thyroxine (L-T4) therapy for DTC. Patients and Interventions: A total of 179 consecutive women (median age, 59 years; n = 178 postmenopausal) who had undergone thyroidectomy for DTC and were currently receiving L-T4 were evaluated for radiological VFs and bone mineral density (BMD). There were three TSH target levels [, 0.5 mU/L, group 1 (n = 83); 0.5 to 1.0 mU/L, group 2 (n = 50);>1.0 mU/L, group 3 (n = 46)]. Results: VFs were found in 51 patients (28.5%), with significantly (P < 0.001) higher prevalence in group 1 (44.6%) as compared with group 2 (24.0%) and group 3 (4.3%). VF prevalence was not significantly different among patients in group 1 with normal BMD, osteopenia, or osteoporosis, whereas in groups 2 and 3, VFs were more frequent in patients with osteoporosis than in those with either osteopenia or normal BMD. In the whole population, VFs were significantly and independently associated with TSH level

Background: The administration of abiraterone acetate (abiraterone) leads to an adrenocorticotropic hormone (ACTH)-driven increase in mineralocorticoid hormones, requiring glucocorticoid supplementation that may stimulate the growth of prostate cancer (PCa). Amiloride is a drug that selectively reduces the aldosterone-sensitive Na+/K+ exchange and could be effective in the management of mineralocorticoid excess syndrome (MCES). Methods: The efficacy of amiloride + hydrochlorothiazide (HCT) in the clinical management of abiraterone-induced MCES was assessed in 5 consecutive patients with castration-resistant PCa (CRPC). Then, using the in vitro experimental model of PCa cell lines, the possible effects of drugs usually used in the clinical management of CRPC patients on PCa cell viability were investigated. Results: Amiloride/HCT led to a complete disappearance of all clinical and biochemical signs of abiraterone-induced MCES in the 5 treated patients. The in vitro study showed that abiraterone treatment significantly decreased cell viability of both androgen receptor (AR)-expressing VCaP (vertebral-cancer of the prostate) and LNCaP (lymph node carcinoma of the prostate) cells, with no effect on AR-negative PC-3 cells. Prednisolone, spironolactone, and eplerenone increased LNCaP cell viability, while amiloride reduced it. The non-steroid aldosterone antagonist PF-03882845 did not modify PCa cell viability. Conclusions: The combination of amiloride/HCT was effective in the management of abiraterone-induced MCES. Amiloride did not negatively interfere with the abiraterone inhibition of PCa cell viability in vitro.

Mutations in WDR45 gene, coding for a beta-propeller protein, have been found in patients affected by Neurodegeneration with Brain Iron Accumulation, NBIA5 (also known as BPAN). BPAN is a movement disorder with Non Transferrin Bound Iron (NTBI) accumulation in the basal ganglia as common hallmark between NBIA classes (Hayflick et al., 2013). WDR45 has been predicted to have a role in autophagy, while the impairment of iron metabolism in the different NBIA subclasses has not currently been clarified. We found the up-regulation of the ferrous iron transporter (-)IRE/Divalent Metal Transporter1 and down-regulation of Transferrin receptor in the fibroblasts of two BPAN affected patients with splicing mutations 235+1G>A (BPAN1) and 517_519ΔVal 173 (BPAN2). The BPAN patients showed a concomitant increase of intracellular ferrous iron after starvation. An altered pattern of iron transporters with iron overload is highlighted in BPAN human fibroblasts, supporting for a role of DMT1 in NBIA. We here present a novel element, about iron accumulation, to the existing knowledge in field of NBIA. Attention is focused to a starvation-dependent iron overload, possibly accounting for iron accumulation in the basal ganglia. Further investigation could clarify iron regulation in BPAN.

Background: Many studies suggest oxidative stress as an early feature of Alzheimer’s Disease (AD). However, evidence of established oxidative stress in AD peripheral cells is still inconclusive, possibly due to both, differences in the type of samples and the heterogeneity of oxidative markers used in different studies. Objective: The aim of this study was to evaluate blood-based redox alterations in Alzheimer’s Disease in order to identify a peculiar disease profile. Method: To that purpose, we measured the activity of Superoxide Dismutase, Catalase and Glutathione Peroxidase both in the extracellular and the intracellular blood compartments of AD, MCI and control subjects. The amount of an open isoform of p53 protein (unfolded p53), resulting from oxidative modifications was also determined. Results: Decreased SOD, increased GPx activity and higher p53 open isoform were found in both AD and MCI plasma compared to controls. In blood peripheral mononuclear cells, SOD activity was also decreased in both AD and MCI, and unfolded p53 increased exquisitely in younger AD males compared to controls. Conclusion: Overall, these data highlight the importance of considering both extracellular and intracellular compartments, in the determination of antioxidant enzyme activities as well as specific oxidation end-products, in order to identify peculiar blood-based redox alterations in AD pathology.

Patients with adrenocortical carcinoma (ACC) frequently suffer from cortisol excess, which portends a negative prognosis. Rapid control of cortisol hypersecretion and tumor growth are the main goals of ACC therapy. Abiraterone acetate (AA) is a potent inhibitor of 17alpha-hydroxylase/17,20-lyase, a key enzyme of adrenal steroidogenesis.This study sought to investigate the therapeutic use of AA in preclinical models of ACC.AA antisecretive and antiproliferative effects were investigated in vitro using NCI-H295R and SW13 ACC cell lines and human primary ACC cell cultures, as well as in vivo using immunodeficient mice.Steroid secretion, cell viability, and proliferation were analyzed in untreated and AA-treated ACC cells. The ability of AA to affect the Wnt/beta-catenin pathway in NCI-H295R cells was also analyzed. Progesterone receptor (PgR) gene was silenced by the RNA interference approach. The antitumor efficacy of AA was confirmed in vivo in NCI-H295R cells xenografted in immunodeficient mice.AA reduced the secretion of both cortisol and androgens, increased production of progesterone, and induced a concentration-dependent decrease of cell viability in the NCI-H295R cells and primary secreting ACC cultures. AA also reduced beta-catenin nuclear accumulation in NCI-H295R cells. AA administration to NCI-H295R-bearing mice enhanced progesterone levels and inhibited tumor growth. The cytotoxic effect of AA was prevented by either blocking PgR or by gene silencing.AA is able to inhibit hormone secretion and growth of ACC both in vitro and in vivo. It also reduces beta-catenin nuclear accumulation. The cytotoxic effect of AA seems to require PgR.

It is well recognized that mitochondrial dysfunction contributes to neurodegeneration occurring in Alzheimer’s disease (AD). However, evidences of mitochondrial defects in AD peripheral cells are still inconclusive. Here, some mitochondrial-encoded and nuclear-encoded proteins, involved in maintaining the correct mitochondria machine, were investigated in terms of protein expression and enzymatic activity in peripheral blood mononuclear cells (PBMCs) isolated from AD and Mild Cognitive Impairment (MCI) patients and healthy subjects. In addition mitochondrial DNA copy number was measured by real time PCR. We found some differences and some similarities between AD and MCI patients when compared with healthy subjects. For example, cytochrome C and cytochrome B were decreased in AD, while MCI showed only a statistical reduction of cytochrome C. On the other hand, both AD and MCI blood cells exhibited highly nitrated MnSOD, index of a prooxidant environment inside the mitochondria. TFAM, a regulator of mitochondrial genome replication and transcription, was decreased in both AD and MCI patients’ blood cells. Moreover also the mitochondrial DNA amount was reduced in PBMCs from both patient groups. In conclusion these data confirmed peripheral mitochondria impairment in AD and demonstrated that TFAM and mtDNA amount reduction could be two features of early events occurring in AD pathogenesis.

Alterations in genes that regulate neurodevelopment can lead to cortical malformations, resulting in malfunction during postnatal life. The NF-κB pathway has a key role during neurodevelopment by regulating the maintenance of the neural progenitor cell pool and inhibiting neuronal differentiation. In this study, we evaluated whether mice lacking the NF-κB p50 subunit (KO) present alterations in cortical structure and associated behavioral impairment. We found that, compared with wild type (WT), KO mice at postnatal day 2 present an increase in radial glial cells, an increase in Reelin protein expression levels, in addition to an increase of specific layer thickness. Moreover, adult KO mice display abnormal columnar organization in the somatosensory cortex, a specific decrease in somatostatin- and parvalbumin-expressing interneurons, altered neurite orientation, and a decrease in Synapsin I protein levels. Concerning behavior, KO mice, in addition to an increase in locomotor and exploratory activity, display impairment in social behaviors, with a reduction in social interaction. Finally, we found that risperidone treatment decreased hyperactivity of KO mice, but had no effect on defective social interaction. Altogether, these data add complexity to a growing body of data, suggesting a link between dysregulation of the NF-κB pathway and neurodevelopmental disorders pathogenesis.

Objective: Adrenocortical carcinoma (ACC) is a rare disease often associated with hormonal hypersecretion. Hypercortisolism is the most common endocrine syndrome associated with ACC. Abiraterone acetate is a selective and irreversible inhibitor of cytochrome P450 17A1 that is currently used in the treatment of metastatic castration-resistant prostate cancer. The drug induces a profound suppression of both androgen and cortisol secretion and it is a plausible candidate for medical treatment of Cushing's syndrome. We evaluated the hormonal effects of abiraterone administration in a patient with Cushing's syndrome induced by heavily pre-treated metastatic ACC.Methods: A 50-year-old woman with ACC, suffering from severe and unmanageable Cushing's syndrome despite mitotane therapy, received oral abiraterone at 500 mg/day.Results: After 9 days of treatment, the patient experienced a dramatic improvement in autonomy and deambulation, reduction of edema, better control of blood pressure and glycemia, serum potassium normalization, restoration of the sleep-wake rhythm, and disappearance of mental confusion episodes. Hormonal laboratory tests showed a marked reduction in circulating androgen levels and urinary cortisol excretion (urinary free cortisol levels lowered from 1,863 to 520 μg/24 h). Abiraterone therapy was interrupted due to subsequent deterioration of the patient's condition caused by the progression of ACC.Conclusions: Abiraterone can be an effective medical treatment in the management of Cushing's syndrome induced by ACC. This preliminary observation warrants confirmation in a prospective clinical trial.Abbreviations: ACC = adrenocortical carcinoma

Presently, there is no scientific evidence supporting a definite role for follow-up after gastrectomy for cancer, and clinical practices are quite different around the globe. The aim of this consensus conference was to present an ideal prototype of follow-up after gastrectomy for cancer, based on shared experiences and taking into account the need to rationalize the diagnostic course without losing the possibility of detecting local recurrence at a potentially curable stage. On June 19–22, 2013 in Verona (Italy), during the 10th International Gastric Cancer Congress (IGCC) of the International Gastric Cancer Association, a consensus meeting was held, concluding a 6-month, Web-based, consensus conference entitled “Rationale of oncological follow-up after gastrectomy for cancer.” Forty-eight experts, with a geographical distribution reflecting different health cultures worldwide, participated in the consensus conference, and 39 attended the consensus meeting. Six statements were finally approved, displayed in a plenary session and signed by the vast majority of the 10th IGCC participants. These statements are attached as an annex to the Charter Scaligero on Gastric Cancer. After gastrectomy for cancer, oncological follow-up should be offered to patients; it should be tailored to the stage of the disease, mainly based on cross-sectional imaging, and should be discontinued after 5 years.

Preclinical data indicate a direct anti-tumor effect of zoledronic acid (ZA) outside the skeleton, but its molecular mechanism is still not completely clarified. The aim of this study was to investigate the anti-cancer effects of ZA in human breast cancer cell lines, suggesting that they may in part be mediated via the miR-21/PTEN/Akt signaling pathway. The effect of ZA on cell viability was measured by MTT assay, and cell death induction was analyzed using either a double AO/EtBr staining and M30 ELISA assay. A Proteome Profiler Human Apoptosis Array was executed to evaluate the molecular basis of ZA-induced apoptosis. Cell cycle analysis was executed by flow cytometry. The effect of ZA on miR-21 expression was quantified by qRT-PCR, and the amount of PTEN protein and its targets were analyzed by Western blot. ZA inhibited cell growth in a concentration- and time-dependent manner, through the activation of cell death pathways and arrest of cell cycle progression. ZA downregulated the expression of miR-21, resulting in dephosphorilation of Akt and Bad and in a significant increase of p21 and p27 proteins expression. These results were observed also in MDA-MB-231 cells, commonly used as an experimental model of bone metastasis of breast cancer. This study revealed, for the first time, an involvement of the miR-21/PTEN/Akt signaling pathway in the mechanism of ZA anti-cancer actions in breast cancer cells. We would like to underline that this pathway is present both in the hormone responsive BC cell line (MCF-7) as well as in a triple negative cell line (MDA-MB-231). Taken together these results reinforce the use of ZA in clinical practice, suggesting the role of miR-21 as a possible mediator of its therapeutic efficacy.

Oxidative stress has been implicated in the pathogenesis of Alzheimer's disease (AD). Both AD and arguably its earlier form, mild cognitive impairment (MCI), have elevated membrane oxidative damage in brain. The tumor suppressor and transcription factor p53 plays a pivotal function in neuronal apoptosis triggered by oxidative stress. Apoptosis contributes to neuronal death in many neurological disorders, including AD. In this study, we investigated p53 expression in a specific region of the cerebral cortex, namely the inferior parietal lobule (IPL), in MCI and AD brain, to test the hypothesis that alterations of this pro-apoptotic protein may be involved in neuronal death in the progression of AD. By immunoprecipitation assay, we also investigated whether 4-hydroxy-2-transnonenal (HNE), an aldehydic product of lipid peroxidation, was bound in excess to p53 in IPL from subjects with MCI and AD compared to control. Overall, the data provide evidence that p53 is involved in the neuronal death in both MCI and AD, suggesting that the observed alterations are early events in the progression of AD. In addition, HNE may be a novel non-protein mediator of oxidative stress-induced neuronal apoptosis.

Oxidative stress, a hallmark of Alzheimer disease (AD), has been shown to induce lipid peroxidation and apoptosis disrupting cellular homeostasis. Normally, the aminophospholipid phosphatidylserine (PtdSer) is asymmetrically distributed on the cytosolic leaflet of the lipid bilayer. Under oxidative stress conditions, asymmetry is altered, characterized by the appearance of PtdSer on the outer leaflet, to initiate the first stages of an apoptotic process. PtdSer asymmetry is actively maintained by the ATP-dependent translocase flippase, whose function is inhibited if covalently bound by lipid peroxidation products, 4-hydroxynonenal (HNE) and acrolein, within the membrane bilayer in which they are produced. Additionally, pro-apoptotic proteins Bax and caspase-3 have been implemented in the oxidative modification of PtdSer resulting in subsequent asymmetric collapse, while anti-apoptotic protein Bcl-2 has been found to prevent this process. The current investigation focused on detection of PtdSer on the outer leaflet of the bilayer in synaptosomes from brain of subjects with AD and amnestic mild cognitive impairment (MCI), as well as expression levels of apoptosis-related proteins Bcl-2, Bax, and caspase-3. Fluorescence and Western blot analysis suggest PtdSer exposure on the outer leaflet is significantly increased in brain from subjects with MCI and AD contributing to early apoptotic elevation of pro- and anti-apoptotic proteins and finally neuronal loss. MCI is considered a possible transition point between normal cognitive aging and probable AD. Brain from subjects with MCI is reported to have increased levels of tissue oxidation; therefore, the results of this study could mark the progression of patients with MCI into AD. This study contributes to a model of apoptosis-specific oxidation of phospholipids consistent with the notion that PtdSer exposure is required for apoptotic-cell death.

We found that pergolide, a dopamine D1/D2 receptor agonist used in the clinical therapy of Parkinson's disease, protects SH-SY5Y neuroblastoma cells from cell death induced by a brief pulse (15 min) of 1 mM H(2)O(2). Neuroprotection was found when pergolide was added to the culture medium either simultaneously with (EC(50)=60 nM) or 2 h before (EC(50)=40 nM) H(2)O(2) treatment. These effects were not blocked by different dopamine receptor antagonists. Our data suggest that pergolide, independently of dopamine receptor stimulation, may interfere with the early phases of the oxidative stress-induced neurotoxic process.

175 Background: Abiraterone acetate (AA) deeply inhibits androgen synthesis but leads to an ACTH driven increase in mineralocorticoid hormones requiring glucocorticoid supplementation that may impair its antineoplastic efficacy. New strategies for the management of the AA induced mineral corticoid excess syndrome (MCES) are warranted. Methods: We analyzed in vitro the interaction in terms of proliferative activity of AA plus/minus prednisone with the steroid aldosterone receptor antagonists: eplerenone, spironolactone, a non-steroidal aldosterone receptor antagonist (PF-03882845) and the epithelial sodium channel antagonist amiloride. LNCaP were grown in a medium with charcoal-treated serum and concentration-response curves for each studied drug were performed. Besides, the activity of amiloride plus hydrochlorothiazide was assessed in the clinical management of AA induced MCES in 5 consecutive patients with castrate resistant prostate cancer. The recovery of AA induced MCES symptoms and signs was the primary end point. Results: Prednisone, spironolactone and eplerenone induced an increase in the LNCAP proliferation rate and antagonized the AA-induced reduction of the cell proliferation in a concentration-dependent manner, while PF-03882845 did not. Amiloride at high concentrations induced cell death. When combined with AA +/- prednisone, amiloride at low concentration did not interfere with AA anti-proliferative activity however an additive inhibitory effect was observed at higher concentrations. The association of amiloride with hydrochlorothiazide led to a complete disappearance of all clinical and biochemical signs of abiraterone induced MCES in the 5 treated patients. Conclusions: Amiloride and PF-03882845 do not negatively interfere with the AA inhibition of proliferative activity of prostate cancer cells in vitro. The association of amiloride plus hydrochlorothiazide is efficacious in the management AA induced MCES.

Reverse Transcriptase-Polymerase Chain Reaction technique was used to determine the levels of the mRNAs encoding different receptors belonging to the D2 family in various brain areas of 6-, 12-, 18-, and 24-month-old rats. We found a progressive, age-dependent reduction in the mRNA levels of D2 short and D2 long receptors in corpus striatum, substantia nigra, and frontal cortex. D2 short and D2 long receptor mRNA levels were unchanged in hippocampus and olfactory tubercle, while they increased in pituitary. D3 receptor mRNA levels were reduced in olfactory tubercle and unchanged in striatum. D4 receptor mRNA levels were unmodified in pituitary gland. These data suggest that the molecular mechanisms responsible for regulating the pattern of expression of the various dopamine receptors are differentially regulated by aging.

Pizzi M, Arrighi V, Galli P, Memo M, Spano PF. Lack of vasoactive intestinal peptide-releasing property in prolactin cells from ovariectomized rats: contribution of post-transductional impairments. Eur J Endocrinol 1994;130:361–5. ISSN 0804–4643 We have demonstrated recently that in menopausal women and in ovariectomized rats the deficiency of circulating oestrogens impairs vasoactive intestinal peptide (VIP) efficacy in stimulating prolactin (PRL) release. The present study was designed to investigate whether the lack of VIP-induced PRL release after ovariectomy is a consequence of a defect at the receptor-transductional or post-transductional level. For this purpose we evaluated the VIP receptor function, by measuring VIP-stimulated cyclic adenosine monophosphate (AMP) formation, and the efficacy of the cyclic AMP-dependent PRL release in pituitary cells from control and ovariectomized animals. We observed that VIP induced a significantly higher stimulation of adenylate cyclase in pituitary homogenates from ovariectomized rats than in those from control animals. This effect appeared to be linked to an increased efficiency of the Gs coupling protein, because superimposable results were obtained by using the non-hydrolysable guanosine triphosphate (GTP) analogue, 5-guanylylimidodiphosphate. On the contrary, the cyclic AMP analogue, 8-Br-cAMP, that potently stimulated PRL release from control pituitary cells was completely ineffective in cells from ovariectomized rats. The present data indicate that in PRL-secreting cells from ovariectomized rats a defect in the post-transductional mechanism that couples the VIP receptor to PRL secretion, rather than a reduction of receptor function, possibly accounts for the lack of VIP efficacy. Marina Pizzi, Dept. Biomed. Sci. & Biotech., School of Medicine, Via Valsabbina 19, 25123 Brescia, Italy

Mild cognitive impairment (MCI) is a syndrome defined as cognitive decline, but not sufficient to meet the criteria for any specific dementia. Although subjects with MCI may have an increased risk to develop AD, this clinical state encompasses several subtypes of cognitive dysfunction of different etiologies, none of which necessarily progresses to AD. The current inability of clinical criteria to accurately identify this at-risk group for AD development is fuelling the interest in biomarkers able to supplement clinical approaches. We recently described a blood-based cytofluorimetric method for conformationally altered p53 protein detection that allows the discrimination of AD patients from control subjects and patients affected by other dementias. The same protein also predicted progression to AD in preclinical patients with MCI two years before clinical diagnosis of AD was made. Herein, we describe these findings and discuss the potential of the test in diagnosing AD.

We studied the adaptive changes of the locomotor effects of lisuride, a selective agonist for dopamine (DA) D 2 receptors, and the functional state of D 1 and D 2 receptors after repeated administration of lisuride at a dose supposed to act preferentially on DA autoreceptors. Rats were treated daily with saline or lisuride, at a dose that causes a significant reduction in locomotor activity when given to naive rats (25 μg/kg i.p.), for 33 days and the effect of different challenging doses of the drug on locomotor activity was measured at different times during and after the treatment. The functional state of D 1 and D 2 DA receptors was evaluated by measuring SKF 82526-stimulated and LY 171555-inhibited adenylate cyclase (AC) activity in the caudatus/putamen, nucleus accumbens and substantia nigra and naive and chronically treated rats. There was a progressive decline in the ability of lisuride to decrease locomotor activity in rats given daily injections of lisuride, and there was a marked reduction in the threshold dose of lisuride for causing hypermotility. The functional state of DA receptors, positively or negatively linked to AC activity, was not modified by the treatment. The most suitable explanation of the reported adaptive behavioral changes is a down-regulation of DA autoreceptors after chronic treatment with presynaptic doses of lisuride.

Functional modifications of both D-1 and D-2 dopamine (DA) receptor subtypes following repeated administration of lisuride, a DA agonist that acts selectively on D-2 DA receptors were studied. The functional state of D-1 and D-2 DA receptors was evaluated from measurements of SKF 82526-stimulated and bromocriptine-inhibited adenylate cyclase activity in different brain regions of rats treated daily for 26 days with saline or lisuride (100 μg/kg i.p.). Persistent stimulation by lisuride of DA receptors in striatum, nucleus accumbens, substantia nigra, frontal cortex, hippocampus and pituitary gland induced a down-regulation of D-2 receptors without changing the functional activity of D-1 receptors.

The role of angiogenesis in tumour progression is a major subject in modern oncology and a correlation between angiogenesis and poor outcome has been demonstrated for human neuroblastomas. However, the role of angiogenesis in the maturation phase of neuroblastic tumours has never been considered. Human carcinoembryonic antigen-related cell adhesion molecule 1 (CEACAM1), a potent pro-angiogenic factor and mediator of vascular endothelial growth factor (VEGF)-induced angiogenesis, plays a crucial role during the activation phase of angiogenesis and it has been shown to be expressed in the microvessels of the developing central nervous system as well as in newly formed immature blood vessels in many different tumours and under physiological conditions. The present study has investigated the role of CEACAM1/VEGF-mediated angiogenesis across the whole spectrum of neuroblastic tumours, from undifferentiated to fully differentiated mature ganglioneuromas. CEACAM1 is peculiarly expressed in the microvessels of areas of active tumour maturation among differentiating neuroblastic/ganglion cells, whereas it is completely absent in the vessels of poorly differentiated/undifferentiated as well as in entirely mature Schwannian-rich areas. Interestingly, VEGF expression has been found in differentiating neuroblastic/ganglion cells adjacent to CEACAM1-positive microvessels. In keeping with these observations, VEGF expression was found in human neuroblastoma SH-SY5Y cells during differentiation after retinoic acid treatment. Moreover, conditioned medium from SH-SY5Y cells collected at different stages of differentiation induced progressive in vitro up-regulation of CEACAM1 expression in human umbilical vein endothelial cells (HUVECs) that was abrogated by the specific VEGF receptor-2/KDR inhibitor SU5416. Taken together, these data point to a role for CEACAM1/VEGF cross-talk during the maturation phase of neuroblastic tumours. This may mimic physiological events leading to maturation of the vasculature in the developing normal central nervous system. On the other hand, in poorly differentiated/undifferentiated lesions, VEGF-sustained angiogenesis does not reproduce physiological steps, but rather is associated with tumour aggressiveness and may involve other molecular pathways.

We originally suggested that inhibition of tumor necrosis factor-related apoptosis inducing ligand (TRAIL) death pathway could be taken into consideration as a potential therapeutic strategy for Alzheimer's disease (AD). However, because the critical role of TRAIL in immune surveillance, the neutralization of TRAIL protein by an antibody to prevent its binding to death receptors is definitely a risky approach. Here, we demonstrated that the blockade of the TRAIL death receptor DR5 with a specific antibody completely prevented amyloid beta peptide (A beta) neurotoxicity in both neuronal cell line and primary cortical neurons. DR5 was demonstrated to be a key factor in TRAIL death pathway. In fact, whereas TRAIL expression was enhanced dose-dependently by concentrations of beta amyloid ranging from 10 nM to 1 microM, only the highest toxic dose of A beta (25 microM) induced the increased expression of DR5 and neuronal cell death. In addition, the increased expression of DR5 receptor after beta amyloid treatment was sustained by p53 transcriptional activity, as demonstrated by the data showing that the p53 inhibitor Pifithrin alpha prevented both beta amyloid-induced DR5 induction and cell death. These data suggest a sequential activation of p53 and DR5 upon beta amyloid exposure. Further insight into the key role of DR5 in AD was suggested by data showing a significant increase of DR5 receptor in cortical slices of AD brain. Thus, these findings may give intracellular TRAIL pathway a role in AD pathophysiology, making DR5 receptor a possible candidate as a pharmacological target.

Here we demonstrate that pramipexole, an antiparkinsonian dopamine receptor agonist drug, exerts neuroprotective effects against beta-amyloid neurotoxicity. Using a specific protocol to test individually oligomers, fibrils, or unaggregated amyloid beta-peptide, we found pramipexole able to protect cells against oligomers and fibrils. Unaggregated amyloid beta-peptide was found unable to cause cell death. Fibrils and oligomers were also found to produce elevated amount of free radicals, and this effect was prevented by pramipexole. We propose pramipexole may become in the future a coadjuvant in the treatment of neuropathologies, besides Parkinson's disease, where amyloid beta-peptide-mediated oxidative injury exerts a relevant role.

A common arginine to proline polymorphism is harboured at codon 72 of the human p53 gene. In this investigation, we found that fibroblasts and lymphocytes isolated from arginine allele homozygote centenarians and sexagenarians (Arg+) undergo an oxidative-stress-induced apoptosis at a higher extent than cells obtained from proline allele carriers (Pro+). At variance, the difference in apoptosis susceptibility between Arg+ and Pro+ is not significant when cells from 30-year-old people are studied. Further, we found that Arg+ and Pro+ cells from centenarians differ in the constitutive levels of p53 protein and p53/MDM2 complex, as well as in the levels of oxidative stress-induced p53/Bcl-xL complex and mitochondria-localised p53. Consistently, all these differences are less evident in cells from 30-year-old people. Finally, we investigated the in vivo functional relevance of the p53 codon 72 genotype in a group of old patients (66-99 years of age) affected by acute myocardial ischaemia, a clinical condition in which in vivo cell death occurs. We found that Arg+ patients show increased levels of Troponin I and CK-MB, two serum markers that correlate with the extent of the ischaemic damage in comparison to Pro+ patients. In conclusion, these data suggest that p53 codon 72 polymorphism contributes to a genetically determined variability in apoptotic susceptibility among old people, which has a potentially relevant role in the context of an age-related pathologic condition, such as myocardial ischaemia.

Thyrotropin-releasing hormone (TRH) and some of its stable analogues have recently been shown to improve functional recovery after neurologic dysfunctions, such as brain trauma and epilepsy, in both animals and humans. The exact mechanism by which TRH produces its neuroprotective effects is still uncertain. The present study provides the first evidence that TRH exerts a neuroprotective effect against N-methyl-D-aspartate (NMDA)-mediated excitotoxicity in rat hippocampal slices. TRH concentration dependently reduced NMDA toxicity by a mechanism that was highly sensitive to the protein kinase C blocker, bisindolilmaleimide. Delayed application of TRH, during NMDA exposure, still produced neuroprotection.

treatment group was significantlybetter than the appropriate controlgroup. Thus, prolonged treatmentwith propentofylline, unlike theAChEIs tested to date, produces animprovement in cognition that issustained for some weeks after drugwashout must be complete. How-ever, propentofylline, which hadbeen approved in Japan, was thisAutumn delisted and that rathermore damningly, the EuropeanCPMP has voted not to recom-mended the approval of propento-fylline in AlzheimerOs disease.

Human NT2-N neurons derived from retinoic acid treatment of the NTera 2 cell line were used to determine the consequences of ionotropic glutamate receptor (iGluR) hyperstimulation and possible modulatory role(s) exerted by metabotropic glutamate receptor (mGluR) activation. We found that NT2-N neurons express the NR1 subunit of N-methyl-D-aspartate (NMDA) iGluRs and mRNA encoding the 1a isoform of mGluRs. A 15 min pulse with 100 microM NMDA induced an increase in the levels of tau proteins in NT2-N cells. This effect was prevented by incubating NT2-N neurons in the presence of the mGluR agonist (1 S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid (1S,3R-ACPD). This phenomenon was related, in terms of doses and time, with the observed 1S,3R-ACPD-mediated protection against NMDA-induced NT2-N cell death. Our findings suggest that iGluRs and mGluRs might participate in the control of human neuron viability by differentially affecting the expression of tau proteins.

The idea that inflammatory processes contribute to the pathology of neurodegenerative diseases and in particular of Alzheimer’s disease (AD), has been supported by epidemiological and clinical studies. Multiple retrospective epidemiological analyses indicate that patients receiving anti-inflammatory drugs or suffering from conditions in which such drugs are routinely used, have a decreased risk of developing AD (see McGeer and McGeer, 1995, as review). In a preliminary double-blind clinical trial, the non steroidal anti-inflammatory drug (NSAID) indomethacin has proven to reduce progression of cognitive decline in AD patients (Rogers et al., 1993). Obviously, more comprehensive clinical trials need to be carried out, but the future of anti-inflammatory therapy in AD holds great promise.

By combining biochemical, molecular and immunohistochemical approaches, we have investigated the presence of metabotropic glutamate receptors (mGluRs) belonging to the subtype 5 in rat and human spinal cords and the developmental changes in their expression. A polyclonal antibody raised against the carboxy-terminal portion of mGluR5 was used to study the distribution of the receptor in rat foetal (Et15), neonatal (P8) and adult spinal cords and dorsal root ganglia (DRG). mGluR5 appeared to be predominantly expressed in regions containing the primary sensory afferents. Immunoblotting with anti-mGluR5 antibody revealed lower receptor protein levels in rat adult spinal cord when compared with P8 rat spinal cord. Reverse transcriptase-polymerase chain reaction showed both mGluR5a and mGluR5b mRNAs expression in rat spinal cord. The mGluR5a variant was found more abundant in young animals than in adults. The pattern of mGluR5 immunostaining was also studied in foetal (6-8, 10, 12 and 22 weeks of gestation) and adult human spinal cord. At all stages of human development, a strong mGluR5 immunoreactivity was observed in the dorsal roots and in the dorsal and dorsolateral funiculi with maximum levels of staining at week 12 of gestation. Foetal DRG neurons were heterogeneously labeled. mGluR5 was also diffusely detectable in the mantle layer. In adult human spinal cords, immunoreactivity was confined to laminae I and II of the dorsal horns. These results demonstrate for the first time the presence of mGluR5 in human spinal cord. The distribution of this receptor suggests a role in the development of somatosensory pathways and in the control of nociceptive neurotransmission.

In the present study, the monoclonal antibody Alz-50 has been used to determine and compare the immunohistochemical localization of phosphorylated tau proteins in the developing and normal adult spinal cord. At all stages of fetal life Alz-50 fiber immunoreactivity was observed in the dorsal roots, in the dorsal and dorsolateral funiculi, and in restricted regions of the dorsal horn. Alz-50 immunoreactivity was also demonstrated in the dorsal root ganglion neurons. In the adult spinal cord a consistent pattern of Alz-50 fiber immunoreactivity was localized in the superficial layers of the dorsal horn (lamina I and II) but not in dorsal and dorsolateral funiculi and in the dorsal root ganglion. Comparable results in fetal specimens have been obtained employing PHF-1, a monoclonal antibody generated against paired helical filament proteins from Alzheimer brains, while no significant immunostaining for PHF-1 was observed in the adult spinal cord. In addition, the staining with monoclonal and polyclonal anti-tau antibodies overlapped with that of Alz-50. The transient, selective pattern of Alz-50 and PHF-1 immunoreactivity may disclose some relevant functions of tau proteins during somatosensory pathway development.

A polymerase chain reaction-derived method was used to identify and quantitate the relative abundance of the different mRNAs encoding various isoforms of the guanine nucleotide regulatory protein Gs, Gi, and Go alpha subunits in the striata of rats unilaterally injected with 6-hydroxydopamine in the substantia nigra. Thirty days after the lesion, the mRNA levels of the G(o) and of the Gi 1 alpha subunits were increased by about 2-3 times, those of the Gi 3 decreased by about 60% and those of Gi 2 and Gs unmodified. The pattern of expression of the G(o) alpha subunits mRNA changed in a time-dependent fashion, being significant 20 days after the lesion. The decrease in Gi 3 alpha subunit mRNA levels was maximum 10 days after the lesion and tended to be reduced in magnitude with time while the changes in Gi 1 alpha subunit mRNA showed a byphasic behaviour being reduced at 10 days and increased at 30 days after the lesion. These data suggest that the expression of the various G protein alpha subunits in the striatum are continuously regulated by factors originating from afferent neurons and surrounding cells.

GH3 cells are a clonal strain from a rat pituitary tumor that synthesizes and secretes both PRL and GH. The peculiarity of these cells is that they do not express receptors for dopamine; thus the hormone release is insensitive to the inhibitory effect of dopamine and D2 receptor agonists. Exposure of GH3 cells to epidermal growth factor for 4 consecutive days markedly altered the cell morphology, from a spherical appearance to an elongated flattened shape, and increased the cell size. These morphological changes were accompanied by the functional expression of D2 dopamine receptors as shown by the presence of a specific, saturable, and stereoselective high affinity binding for [3H]spiroperidol in epidermal growth factor-treated cells and by the fact that the selective D2 agonist quinpirole recovered the property to inhibit PRL secretion in the cell cultures exposed to the neurotrophic factor. The effect of EGF on the functional expression of D2 receptors was dose dependent (EC50 = 8 pM) and reversible. These data suggest that EGF elicits major effects on the expression of specific genes leading to the differentiation of GH3 cells into lactotroph-like cells endowed with dopamine D2 receptors.

Vasoactive intestinal peptide (VIP) is a prolactin (PRL)-releasing factor which has been proposed to exert its secreting property by activating the adenylate cyclase enzyme. The present study shows that the omission of external Ca2+ did not affect the ability of VIP to induce PRL release while it completely abolished the VIP stimulatory effect on adenylate cyclase. We found that VIP (500 nM) stimulated PRL secretion in a time-dependent manner reaching a plateau at 3 min. This pattern was not changed when Ca2+ was omitted from the incubation medium. When tested at different concentrations, VIP stimulated PRL release with EC50 values of 1.3 nM in the presence of Ca2+ and 30 nM in the absence of Ca2+. On the other hand, Ca2+ removal completely suppressed the VIP-induced cAMP formation. VIP (200 nM) was also found to activate Ca2+ influx into pituitary cells. The increase in Ca2+ permeability showed a peak at 5 s and remained significantly higher than control values until 1 min. In conclusion, in an experimental condition where Ca2+ was omitted from the medium, VIP was found to induce PRL release without stimulating cAMP production. This cAMP-independent PRL release was blocked by preincubation of the cells with 1 microgram/ml pertussis toxin. An additional mechanism other than adenylate cyclase activation or Ca2+ entry is proposed to sustain VIP-induced PRL release.

Clinical and pharmacological evidence suggested that dopamine is involved in the control of esophageal motility. The present study was designed to determine whether or not dopamine receptors are present in human esophagus. With this aim we measured adenylate cyclase activity as a biochemical index of dopamine receptor function in esophageal specimens taken from five patients during surgery for upper esophageal carcinoma. The selective D-1 agonist fenoldopam stimulated cAMP formation in the lower esophageal sphincter, but not in the esophageal body; this effect was prevented by the selective D-1 antagonist SCH 23390 and by d-butaclamol. Bromocriptine, a selective D-2 stimulator, inhibited adenylate cyclase activity in the lower esophageal sphincter, an effect blocked by the D-2 antagonist (-)sulpiride. No effects of bromocriptine were found in the esophageal body. These data indicate that both D-1 and D-2 receptors are present in the lower esophageal sphincter, but not in esophageal body and emphasize the role of dopamine in the regulation of esophageal function.