Cerebrospinal Fluid sTREM-2, GFAP, and ß-S100 in Symptomatic Sporadic Alzheimer's Disease: Microglial, Astrocytic, and APOE Contributions Along the Alzheimer's Disease Continuum.

BACKGROUND: Many transversal mechanisms act synergistically at different time-points in the cascade of Alzheimer's disease (AD), since amyloid-ß (Aß) deposition, tau pathology, and neuroinflammation influence each other. OBJECTIVE: We explored the contributions of microglia and astrocytes in patients with symptomatic sporadic AD stratified according to AT(N) system and APOE genotype. METHODS: We compared the cerebrospinal fluid (CSF) levels of sTREM-2 and markers of astrocytic activation (GFAP; ß-S100) from 71 patients with AD (23 A+T-,48 A+T+; 38 APOEɛ3, 33 APOEɛ4) and 30 healthy controls (HC). With multivariate analyses we investigated associations between glial biomarkers, Aß42, and p-tau in all subgroups. RESULTS: CSF sTREM-2 was higher in A+T+ [1.437 (0.264)] and A+T- [1.355 (0.213)] than in HC [1.042 (0.198); both p < 0.001]; GFAP and ß-S100 were comparable across groups. Considering all patients, sTREM-2 positively associated with Aß42 (p = 0.04) and p-tau (=0.016), with the first being present only in the A+T- subgroup (p = 0.023). GFAP positively associated with Aß42 in all patients (p = 0.020) and in the A+T+ subgroup (p = 0.04). Stratifying by APOE, a positive association of sTREM-2 and p-tau was confirmed selectively in carriers of ɛ4 (p = 0.018). Finally, sTREM-2 positively correlated with ß-S100 in all subgroups, and with GFAP in A+T+ (p = 0.042). CONCLUSION: Our results confirm the increase of CSF sTREM-2 in AD, which associates with reduced amyloidopathy in A+T- patients. Moreover, microglial activation seems to increase CSF tau levels in carriers of APOEɛ4, is associated with astrocytic reactivity (GFAP) in A+T+, and likely leads the acquisition of a more neurotoxic astrocytic phenotype (ß-S100).

New Insights Regarding Hemin Inhibition of the Purified Rat Brain 2-Oxoglutarate Carrier and Relationships with Mitochondrial Dysfunction.

A kinetic analysis of the transport assays on the purified rat brain 2-oxoglutarate/malate carrier (OGC) was performed starting from our recent results reporting about a competitive inhibitory behavior of hemin, a physiological porphyrin derivative, on the OGC reconstituted in an active form into proteoliposomes. The newly provided transport data and the elaboration of the kinetic equations show evidence that hemin exerts a mechanism of partially competitive inhibition, coupled with the formation of a ternary complex hemin-carrier substrate, when hemin targets the OGC from the matrix face. A possible interpretation of the provided kinetic analysis, which is supported by computational studies, could indicate the existence of a binding region responsible for the inhibition of the OGC and supposedly involved in the regulation of OGC activity. The proposed regulatory binding site is located on OGC mitochondrial matrix loops, where hemin could establish specific interactions with residues involved in the substrate recognition and/or conformational changes responsible for the translocation of mitochondrial carrier substrates. The regulatory binding site would be placed about 6 Å below the substrate binding site of the OGC, facing the mitochondrial matrix, and would allow the simultaneous binding of hemin and 2-oxoglutarate or malate to different regions of the carrier. Overall, the presented experimental and computational analyses help to shed light on the possible existence of the hemin-carrier substrate ternary complex, confirming the ability of the OGC to bind porphyrin derivatives, and in particular hemin, with possible consequences for the mitochondrial redox state mediated by the malate/aspartate shuttle led by the mitochondrial carriers OGC and AGC.

C57BL/6J and DBA/2J strains present opposite sex differences in flash visual evoked potential latency: A possible confusing factor in gender studies on neurological diseases' transgenic models.

The cholinergic neurotransmitter system in the brain is crucial in processing information related to cognitive, behavioral, and motor functions. A cholinergic dysfunction has been correctly described as one of the primary causes of neurodegenerative diseases. Differences in levels of acetylcholine or expression and function of receptors in selected brain areas have been indicated as one of the causes of sexual dimorphism in neurotransmission. However, variability in results among studies based on different mice strains could affect conclusions on this topic. Visual evoked potentials (VEPs) of male and female DBA/2J and C57BL/6J mice, which are two of the most common strains backgrounds in use for developing transgenic mice models of neurological diseases, have been studied. Effects induced by a single low dose of physostigmine have also been performed to evaluate the cholinergic system involvement. VEPs responses to luminous stimuli in C57BL/6J mice have shown a consistently lower latency than in DBA/2J, confirming the previous observation of strain differences in cholinergic function. Interestingly, strains present an opposite-sex difference in VEP latency not apparently related to sensitivity to physostigmine. These findings point at paying extreme attention to the choice of the genetic background of the animal model, especially in those basic and pre-clinical experiments that involve visual functioning.

Early post-natal life stress induces permanent adrenocorticotropin-dependent hypercortisolism in male mice.

PURPOSE: It has been hypothesized that specific early-life stress (ES) procedures on CD-1 male mice produce diabetes-like alterations due to the failure of negative feedback of glucocorticoid hormone in the pituitary. The aim of this study is to investigate the possible mechanism that leads to this pathological model, framing it in a more specific clinical condition. METHODS: Metabolic and hypothalamic-pituitary-adrenal-related hormones of stressed mice (SM) have been analyzed immediately after stress procedures (21 postnatal days, PND) and after 70 days of a peaceful (unstressed) period (90 PND). These data have been compared to parameters from age-matched controls (CTR), and mice treated during ES procedures with oligonucleotide antisense for pro-opiomelanocortin (AS-POMC). RESULTS: At 21 PND, SM presented an increased secretion of hypothalamic CRH and pituitary POMC-derived peptides, as well as higher plasmatic levels of ACTH and corticosterone vs. CTR. At 90 PND, SM showed hyperglycemia, with suppression of hypothalamic CRH, while pituitary and plasmatic ACTH levels, as well as plasma corticosterone, were constantly higher than in CTR. These values are accompanied by a progressive acceleration in gaining total body weight, which became significant vs. CTR at 90 PND together with a higher pituitary weight. Treatment with AS-POMC prevented all hormonal and metabolic alterations observed in SM, both at 21 and 90 PND. CONCLUSIONS: These findings show that these specific ES procedures affect the negative glucocorticoid feedback in the pituitary, but not in the hypothalamus, suggesting a novel model of ACTH-dependent hypercortisolism that can be prevented by silencing the POMC gene.

Early-Life Stress as a Probe to Study the Opioid System in Developing Rodents.

The developmental origins of disease or fetal programming model predict that early (intrauterine and/or postnatal) exposures to external insults of sufficient length and intensity may have enduring or lifelong consequences for physical and psychological health. The method described in this chapter considers an animal model to study the pathophysiological alterations connected to an HPA axis (hypothalamic-pituitary-adrenal) hyperactivity that are induced by an early-life stressful procedure involving the opioid system.

Protective Role of Cerebrospinal Fluid Inflammatory Cytokines in Patients with Amnestic Mild Cognitive Impairment and Early Alzheimer's Disease Carrying Apolipoprotein E4 Genotype.

BACKGROUND: Neuroinflammatory cytokines can play a pivotal role in Alzheimer's disease (AD) contributing to the evolution of degenerative processes. OBJECTIVE: We aimed at evaluating the levels of cerebrospinal fluid (CSF) inflammatory cytokines, chemokines, and growth factors in subjects with diagnosis of amnestic mild cognitive impairment and mild AD. METHODS: We evaluated CSF contents of inflammatory cytokines in 66 patients divided according to the NIA-AA research framework and the APOE genotype. CSF of a group of cognitively unimpaired individuals (n = 23) was evaluated as control. All patients were evaluated for 24 months using Mini-Mental State Examination (MMSE). RESULTS: We found significant increased levels of IL-4, IL-6, IL-8, and G-CSF in the CSF of A+/T-APOE4 carriers, respect to A+/T-patients homozygous for APOE3, respect to A+/T+ patients, regardless the APOE status, and respect to controls. Over a period of 24 months, A+/T-APOE4 carriers, with increased levels of cytokines, showed a preserved cognitive evaluation when compared to the other subgroups of patients (delta MMSE at 24 months respect to baseline: 0.10±0.35; p < 0.05). CONCLUSION: Our data suggest that during early phases of AD, in APOE4 carriers, Aß pathology likely induces a specific cytokines pattern synthesis associated to cognitive preservation. These data highlight the different role that neuroinflammation can play in AD pathology based on the presence of specific CSF biomarkers and on the APOE status.

Cnf1 Variants Endowed with the Ability to Cross the Blood-Brain Barrier: A New Potential Therapeutic Strategy for Glioblastoma.

Among gliomas, primary tumors originating from glial cells, glioblastoma (GBM) identified as WHO grade IV glioma, is the most common and aggressive malignant brain tumor. We have previously shown that the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1) is remarkably effective as an anti-neoplastic agent in a mouse model of glioma, reducing the tumor volume, increasing survival, and maintaining the functional properties of peritumoral neurons. However, being unable to cross the blood-brain barrier (BBB), CNF1 requires injection directly into the brain, which is a very invasive administration route. Thus, to overcome this pitfall, we designed a CNF1 variant characterized by the presence of an N-terminal BBB-crossing tag. The variant was produced and we verified whether its activity was comparable to that of wild-type CNF1 in GBM cells. We investigated the signaling pathways engaged in the cell response to CNF1 variants to provide preliminary data to the subsequent studies in experimental animals. CNF1 may represent a novel avenue for GBM therapy, particularly because, besides blocking tumor growth, it also preserves the healthy surrounding tissue, maintaining its architecture and functionality. This renders CNF1 the most interesting candidate for the treatment of brain tumors, among other potentially effective bacterial toxins.

The Bacterial Toxin CNF1 Protects Human Neuroblastoma SH-SY5Y Cells against 6-Hydroxydopamine-Induced Cell Damage: The Hypothesis of CNF1-Promoted Autophagy as an Antioxidant Strategy.

Several chronic neuroinflammatory diseases, including Parkinson's disease (PD), have the so-called 'redox imbalance' in common, a dynamic system modulated by various factors. Among them, alteration of the mitochondrial functionality can cause overproduction of reactive oxygen species (ROS) with the consequent induction of oxidative DNA damage and apoptosis. Considering the failure of clinical trials with drugs that eliminate ROS directly, research currently focuses on approaches that counteract redox imbalance, thus restoring normal physiology in a neuroinflammatory condition. Herein, we used SH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA), a neurotoxin broadly employed to generate experimental models of PD. Cells were pre-treated with the Rho-modulating Escherichia coli cytotoxic necrotizing factor 1 (CNF1), before the addition of 6-OHDA. Then, cell viability, mitochondrial morphology and dynamics, redox profile as well as autophagic markers expression were assessed. We found that CNF1 preserves cell viability and counteracts oxidative stress induced by 6-OHDA. These effects are accompanied by modulation of the mitochondrial network and an increase in macroautophagic markers. Our results confirm the Rho GTPases as suitable pharmacological targets to counteract neuroinflammatory diseases and evidence the potentiality of CNF1, whose beneficial effects on pathological animal models have been already proven to act against oxidative stress through an autophagic strategy.

Eye Drop Instillation of the Rac1 Modulator CNF1 Attenuates Retinal Gliosis and Ameliorates Visual Performance in a Rat Model of Hypertensive Retinopathy.

In hypertensive retinopathy, the retinal damage due to high blood pressure is accompanied by increased expression of Glial Fibrillary Acidic Protein (GFAP), which indicates a role of neuroinflammatory processes in such a retinopathy. Proteins belonging to the Rho GTPase family, particularly Rac1, are involved in the activation of Müller glia and in the progression of photoreceptor degeneration, and may thus represent a novel candidate for therapeutic intervention following central nervous system inflammation. In this paper, we have observed that topical administration as eye drops of Cytotoxic Necrotizing Factor 1 (CNF1), a Rho GTPase modulator, surprisingly improves electrophysiological and behavioral visual performances in aged spontaneously hypertensive rats. Furthermore, such functional improvement is accompanied by a reduction of Rac1 activity and retinal GFAP expression. Our results suggest that Rac1 inhibition through CNF1 topical administration may represent a new strategy to target retinal gliosis.

The bacterial protein CNF1 as a new strategy against Plasmodium falciparum cytoadherence.

Plasmodium falciparum severe malaria causes more than 400,000 deaths every year. One feature of P. falciparum-parasitized erythrocytes (pRBC) leading to cerebral malaria (CM), the most dangerous form of severe malaria, is cytoadherence to endothelium and blockage of the brain microvasculature. Preventing ligand-receptor interactions involved in this process could inhibit pRBC sequestration and insurgence of severe disease whilst reversing existing cytoadherence could be a saving life adjunct therapy. Increasing evidence indicate the endothelial Rho signaling as a crucial player in malaria parasite cytoadherence. Therefore, we have used the cytotoxic necrotizing factor 1 (CNF1), an Escherichia coli protein able to modulate the activity of Cdc42, Rac, and Rho, three subfamilies of the Rho GTPases family, to study interactions between infected erythrocytes and cerebral endothelium in co-culture models. The main results are that CNF1 not only prevents cytoadherence but, more importantly, induces the detachment of pRBCs from endothelia monolayers. We first observed that CNF1 does affect neither parasite growth, nor the morphology and concentration of knobs that characterize the parasitized erythrocyte surface, as viewed by scanning electron microscopy. On the other hand, flow cytometry experiments show that cytoadherence reversion induced by CNF1 occurs in parallel with a decreased ICAM-1 receptor expression on the cell surface, suggesting the involvement of a toxin-promoted endocytic activity in such a response. Furthermore, since the endothelial barrier functionality is compromised by P. falciparum, we conducted a permeability assay on endothelial cells, revealing the CNF1 capacity to restore the brain endothelial barrier integrity. Then, using pull-down assays and inhibitory studies, we demonstrated, for the first time, that CNF1 is able not only to prevent but also to cause the parasite detachment by simultaneously activating Rho, Rac and Cdc42 in endothelial cells. All in all our findings indicate that CNF1 may represent a potential novel therapeutic strategy for preventing neurological complications of CM.

The Bacterial Protein CNF1 as a Potential Therapeutic Strategy against Mitochondrial Diseases: A Pilot Study.

The Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1), which acts on the Rho GTPases that are key regulators of the actin cytoskeleton, is emerging as a potential therapeutic tool against certain neurological diseases characterized by cellular energy homeostasis impairment. In this brief communication, we show explorative results on the toxin's effect on fibroblasts derived from a patient affected by myoclonic epilepsy with ragged-red fibers (MERRF) that carries a mutation in the m.8344A>G gene of mitochondrial DNA. We found that, in the patient's cells, besides rescuing the wild-type-like mitochondrial morphology, CNF1 administration is able to trigger a significant increase in cellular content of ATP and of the mitochondrial outer membrane marker Tom20. These results were accompanied by a profound F-actin reorganization in MERRF fibroblasts, which is a typical CNF1-induced effect on cell cytoskeleton. These results point at a possible role of the actin organization in preventing or limiting the cell damage due to mitochondrial impairment and at CNF1 treatment as a possible novel strategy against mitochondrial diseases still without cure.

New therapeutics from Nature: The odd case of the bacterial cytotoxic necrotizing factor 1.

Natural products may represent a rich source of new drugs. The enthusiasm toward this topic has recently been fueled by the 2015 Nobel Prize in Physiology or Medicine, awarded for the discovery of avermectin and artemisinin, natural products from Bacteria and Plantae, respectively, which have targeted one of the major global health issues, the parasitic diseases. Specifically, bacteria either living in the environment or colonizing our body may produce compounds of unexpected biomedical value with the potentiality to be employed as therapeutic drugs. In this review, the fascinating history of CNF1, a protein toxin produced by pathogenic strains of Escherichia coli, is divulged. Even if produced by bacteria responsible for a variety of diseases, CNF1 can behave as a promising benefactor to mankind. By modulating the Rho GTPases, this bacterial product plays a key role in organizing the actin cytoskeleton, enhancing synaptic plasticity and brain energy level, rescuing cognitive deficits, reducing glioma growth in experimental animals. These abilities strongly suggest the need to proceed with the studies on this odd drug in order to pave the way toward clinical trials.

Etiopathogenesis and Pharmacological Prevention of a Type-2 Diabetes Model in Male Mice.

We describe a stress-derived type-2 diabetes model in male mice, and formulate new hypotheses on how the model was induced, how diabetes-like alterations were prevented through specific pharmacological treatments, and how its possible neuroendocrine pathogenesis could be hypothesized. Pregnant females arrived in our laboratory on their 14th day of conceptional age. After birth, control mice never showed any apparent behavioral-metabolic-endocrine alterations. However, application of postnatal stress (brief mother deprivation, plus sham injection, daily from birth to weaning), was followed in adult male mice by two series of diabetes-like alterations. Some alterations (e.g., body overweight, immune, neurophysiologic, neurobehavioral alterations) were selectively prevented by opioid antagonist naloxone daily administered during nursing period. The aforementioned alterations plus several others (e.g., hyperglycemia, neuroendocrine alterations) were prevented by administration of specific antisense oligodeoxinucleotide, which modulated synthesis-hyperfunction of proopiomelanocortin-derived corticotropin (ACTH)-corticosterone and endorphins in the pituitary. Surprisingly, together with metabolic alterations, enduring increment of neurophysiologic/neurobehavioral brain performances were observed, accompanied by energy compensative reactions, and brain mitochondria hyperfunction. Thus, increased glycemia/lipidemia appeared to furnish fuel necessary to cope with increased request of energy. Diabetes-like alterations were accompanied by enduring hyperfunction of opioid- and ACTH-corticosterone-endogenous structures in the brain, which were apparently due to failure of negative feedback hormone mechanisms in the pituitary, for the control of the hypothalamus-pituitary-adrenal axis. In conclusion, for the first time we can hypothesize that a diabetes-like syndrome is produced by enduring hyperfunction of two proopiomelanocortin-dependent endogenous systems (brain opioid- and ACTH-corticosterone systems), following failure of pituitary feedback hormonal control, after complex stress procedures.

CSF tau is associated with impaired cortical plasticity, cognitive decline and astrocyte survival only in APOE4-positive Alzheimer's disease.

In Alzheimer's disease (AD) patients, apopoliprotein (APOE) polymorphism is the main genetic factor associated with more aggressive clinical course. However, the interaction between cerebrospinal fluid (CSF) tau protein levels and APOE genotype has been scarcely investigated. A possible key mechanism invokes the dysfunction of synaptic plasticity. We investigated how CSF tau interacts with APOE genotype in AD patients. We firstly explored whether CSF tau levels and APOE genotype influence disease progression and long-term potentiation (LTP)-like cortical plasticity as measured by transcranial magnetic stimulation (TMS) in AD patients. Then, we incubated normal human astrocytes (NHAs) with CSF collected from sub-groups of AD patients to determine whether APOE genotype and CSF biomarkers influence astrocytes survival. LTP-like cortical plasticity differed between AD patients with apolipoprotein E4 (APOE4) and apolipoprotein E3 (APOE3) genotype. Higher CSF tau levels were associated with more impaired LTP-like cortical plasticity and faster disease progression in AD patients with APOE4 but not APOE3 genotype. Apoptotic activity was higher when cells were incubated with CSF from AD patients with APOE4 and high tau levels. CSF tau is detrimental on cortical plasticity, disease progression and astrocyte survival only when associated with APOE4 genotype. This is relevant for new therapeutic approaches targeting tau.

Cell-to-cell propagation of the bacterial toxin CNF1 via extracellular vesicles: potential impact on the therapeutic use of the toxin.

Eukaryotic cells secrete extracellular vesicles (EVs), either constitutively or in a regulated manner, which represent an important mode of intercellular communication. EVs serve as vehicles for transfer between cells of membrane and cytosolic proteins, lipids and RNA. Furthermore, certain bacterial protein toxins, or possibly their derived messages, can be transferred cell to cell via EVs. We have herein demonstrated that eukaryotic EVs represent an additional route of cell-to-cell propagation for the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1). Our results prove that EVs from CNF1 pre-infected epithelial cells can induce cytoskeleton changes, Rac1 and NF-κB activation comparable to that triggered by CNF1. The observation that the toxin is detectable inside EVs derived from CNF1-intoxicated cells strongly supports the hypothesis that extracellular vesicles can offer to the toxin a novel route to travel from cell to cell. Since anthrax and tetanus toxins have also been reported to engage in the same process, we can hypothesize that EVs represent a common mechanism exploited by bacterial toxins to enhance their pathogenicity.

CNF1 Enhances Brain Energy Content and Counteracts Spontaneous Epileptiform Phenomena in Aged DBA/2J Mice.

Epilepsy, one of the most common conditions affecting the brain, is characterized by neuroplasticity and brain cell energy defects. In this work, we demonstrate the ability of the Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1) to counteract epileptiform phenomena in inbred DBA/2J mice, an animal model displaying genetic background with an high susceptibility to induced- and spontaneous seizures. Via modulation of the Rho GTPases, CNF1 regulates actin dynamics with a consequent increase in spine density and length in pyramidal neurons of rat visual cortex, and influences the mitochondrial homeostasis with remarkable changes in the mitochondrial network architecture. In addition, CNF1 improves cognitive performances and increases ATP brain content in mouse models of Rett syndrome and Alzheimer's disease. The results herein reported show that a single dose of CNF1 induces a remarkable amelioration of the seizure phenotype, with a significant augmentation in neuroplasticity markers and in cortex mitochondrial ATP content. This latter effect is accompanied by a decrease in the expression of mitochondrial fission proteins, suggesting a role of mitochondrial dynamics in the CNF1-induced beneficial effects on this epileptiform phenotype. Our results strongly support the crucial role of brain energy homeostasis in the pathogenesis of certain neurological diseases, and suggest that CNF1 could represent a putative new therapeutic tool for epilepsy.

Visual evoked potentials of Niemann-Pick type C1 mice reveal an impairment of the visual pathway that is rescued by 2-hydroxypropyl-ß-cyclodextrin.

BACKGROUND: The lysosomal storage disorder, Niemann Pick type C1 (NPC1), presents a variable phenotype including neurovisceral and neurological symptoms. 2-Hydroxypropyl-ß-cyclodextrin (HPßCD)-based therapies are presently the most promising route of intervention. While severe cerebellar dysfunction remains the main disabling feature of NPC1, sensory functions including auditory and olfactory ones are also affected. Morphological and functional anomalies of Npc1 (-/-) mouse retina have also been observed, although the functional integrity of the visual pathway from retina to visual cortex is still unsettled. We have addressed this issue by characterizing the visual evoked potential (VEP) response of Npc1 (-/-) mice and determining if/how HPßCD administration influences the VEPs of both Npc1 (-/-) and Npc1 (+/+) mice. METHODS: VEP elicited by a brief visual stimulus were recorded from the scalp overlying the visual cortex of adult (PN, postnatal days 60, 75, 85 and 100) Npc1 (+/+) and Npc1 (-/-) mice that had received repeated injections of either HPßCD or plain vehicle. The first injection was given at PN4 and was followed by a second one at PN7 and thereafter by weekly injections up to PN49. Cholesterol accumulation and myelin loss were finally assessed by filipin staining and myelin basic protein immunohistochemistry, respectively. RESULTS AND DISCUSSION: We have found that the transmission of visual signals from retina to visual cortex is negatively influenced by the loss of Npc1 function. In fact, the VEP response of Npc1 (-/-) mice displayed a highly significant increase in the latency compared to that of Npc1 (+/+) mice. HPßCD administration fully rescued this defect and counteracted the cholesterol accumulation in retinal ganglion cells and dorsal lateral geniculate nucleus neurons, as well as the myelin loss in optic nerve fibers and axons projecting to the visual cortex observed in of Npc1 (-/-) mice. By contrast, HPßCD administration had no effect on the VEP response of Npc1 (+/+) mice, further strengthening the treatment efficacy. CONCLUSIONS: This study pinpoints the analysis of VEP response as a potentially accurate and non-invasive approach to assess neural activity and visual information processing in NPC1 patients, as well as for monitoring the progression of the disease and assessing the efficacy of potential therapies.

Antisense versus proopiomelanocortin mRNA reduces vascular risk in a murine model of type-2 diabetes following stress exposure in early post-natal life.

Mechanisms of vascular complications in type-2 diabetes patients and animal models are matter of debate. We previously demonstrated that a double-stress model applied to male mice during nursing period produces enduring hyperfunction of endogenous opioid and adrenocorticotropin (ACTH)-corticosteroid systems, accompanied by type-2 diabetes-like alterations in adult animals. Administration of the opioid receptor antagonist naloxone, or of an antisense oligodeoxynucleotide versus proopiomelanocortin mRNA, capable to block the pro-opiomelanocortin-derived peptides ß-endorphin and ACTH, selectively prevent these alterations. Here, we investigated alterations produced by our stress model on aorta endothelium-dependent relaxation and contractile responses. Mice, stressed during nursing period, showed in the adulthood hormonal and metabolic type-2 diabetes-like alterations, including hyperglycemia, increased body weight and increased plasma ACTH and corticosterone levels. Ex vivo isolated aorta rings, gathered from stressed mice, were less sensitive to noradrenaline-induced contractions versus controls. This effect was blocked by nitric-oxide synthase-inhibitor l-N(G)-nitroarginine added to bath organ solution. Aorta rings relaxation caused by acetylcholine was enhanced in stressed mice versus controls, but following treatment with the nitric-oxide donor sodium nitroprusside, concentration-relaxation curves in aorta from stressed groups were similar to controls. Therefore, vascular response alterations to physiologic-pharmacologic stimuli were apparently due to nitric-oxide hyperfunction-dependent mechanisms. Aorta functional alterations, and plasma stress hormones enhancement, were prevented in mice stressed and treated with antisense oligodeoxinucleotide, addressed to reduce ACTH- and corticosteroid-mediated hyperfunction. This study demonstrates the key role of ACTH-corticosteroid axis hyperfunction for the triggering of vascular conditions in male adult rodents following postnatal stress in a type-2 diabetes model.

Enhancement of mitochondrial ATP production by the Escherichia coli cytotoxic necrotizing factor 1.

Mitochondria are dynamic organelles that constantly change shape and structure in response to different stimuli and metabolic demands of the cell. The Escherichia coli protein toxin cytotoxic necrotizing factor 1 (CNF1) has recently been reported to influence mitochondrial activity in a mouse model of Rett syndrome and to increase ATP content in the brain tissue of an Alzheimer's disease mouse model. In the present work, the ability of CNF1 to influence mitochondrial activity was investigated in IEC-6 normal intestinal crypt cells. In these cells, the toxin was able to induce an increase in cellular ATP content, probably due to an increment of the mitochondrial electron transport chain. In addition, the CNF1-induced Rho GTPase activity also caused changes in the mitochondrial architecture that mainly consisted in the formation of a complex network of elongated mitochondria. The involvement of the cAMP-dependent protein kinase A signaling pathway was postulated. Our results demonstrate that CNF1 positively affects mitochondria by bursting their energetic function and modifying their morphology.

Mode of action of fibrous amphiboles: the case of Biancavilla (Sicily, Italy).

BACKGROUND: The inhalation of fibrous amphiboles can result in pulmonary fibrosis, lung cancer and mesothelioma. Although these fibres have the same disease-causing potential, their different morphologies and chemical composition can determine different biological activities. An unusual cluster of mesothelioma was evidenced in Biancavilla (Sicily) where no inhabitant had been significantly exposed to asbestos. OBJECTIVE: We herein discuss the mechanism of action of amphiboles, focusing on the fibres identified in the study area. RESULTS: Human lung carcinoma cells have been exposed to two different materials: prismatic fluoro-edenite and fibres with fluoro-edenitic composition. Only in the second case, they exhibit features typical of transformed cells, such as multinucleation, prosurvival activity and pro-inflammatory cytokine release. Accordingly, in vivo studies demonstrated that the fibrous sample only could induce a mesotheliomatogenic effect. CONCLUSIONS: Fibres with fluoro-edenitic composition behave similarly to the asbestos crocidolite, whose connection with inflammation and lung cancer is well established.