The role of T and B cells in human atherosclerosis and atherothrombosis.

Far from being merely a passive cholesterol accumulation within the arterial wall, the development of atherosclerosis is currently known to imply both inflammation and immune effector mechanisms. Adaptive immunity has been implicated in the process of disease initiation and progression interwined with traditional cardiovascular risk factors. Although the body of knowledge regarding the correlation between atherosclerosis and immunity in humans is growing rapidly, a relevant proportion of it derives from studies carried out in animal models of cardiovascular disease (CVD). However, while the mouse is a well-suited model, the results obtained therein are not fully transferrable to the human setting due to intrinsic genomic and environmental differences. In the present review, we will discuss mainly human findings, obtained either by examination of post-mortem and surgical atherosclerotic material or through the analysis of the immunological profile of peripheral blood cells. In particular, we will discuss the findings supporting a pro-atherogenic role of T cell subsets, such as effector memory T cells or the potential protective function of regulatory T cells. Recent studies suggest that traditional T cell-driven B2 cell responses appear to be atherogenic, while innate B1 cells appear to exert a protective action through the secretion of naturally occurring antibodies. The insights into the immune pathogenesis of atherosclerosis can provide new targets in the quest for novel therapeutic targets to abate CVD morbidity and mortality.

Hippocampal sleep spindles preceding neocortical sleep onset in humans.

The coexistence of regionally dissociated brain activity patterns -with some brain areas being active while other already showing sleep signs- may occur throughout all vigilance states including the transition from wakefulness to sleep and may account for both physiological as well as pathological events. These dissociated electrophysiological states are often characterized by multi-domain cognitive and behavioral impairment such as amnesia for events immediately preceding sleep. By performing simultaneous intracerebral electroencephalographic recordings from hippocampal as well as from distributed neocortical sites in neurosurgical patients, we observed that sleep spindles consistently occurred in the hippocampus several minutes before sleep onset. In addition, hippocampal spindle detections consistently preceded neocortical events, with increasing delays along the cortical antero-posterior axis. Our results support the notion that wakefulness and sleep are not mutually exclusive states, but rather part of a continuum resulting from the complex interaction between diffuse neuromodulatory systems and intrinsic properties of the different thalamocortical modules. This interaction may account for the occurrence of dissociated activity across different brain structures characterizing both physiological and pathological conditions.

PARP-1 activation causes neuronal death in the hippocampal CA1 region by increasing the expression of Ca(2+)-permeable AMPA receptors.

An excessive activation of poly(ADP-ribose) polymerases (PARPs) may trigger a form of neuronal death similar to that occurring in neurodegenerative disorders. To investigate this process, we exposed organotypic hippocampal slices to N-methyl-N'-nitro-N'-nitrosoguanidine (MNNG, 100µM for 5min), an alkylating agent widely used to activate PARP-1. MNNG induced a pattern of degeneration of the CA1 pyramidal cells morphologically similar to that observed after a brief period of oxygen and glucose deprivation (OGD). MNNG exposure was also associated with a dramatic increase in PARP-activity and a robust decrease in NAD(+) and ATP content. These effects were prevented by PARP-1 but not PARP-2 inhibitors. In our experimental conditions, cell death was not mediated by AIF translocation (parthanatos) or caspase-dependent apoptotic processes. Furthermore, we found that PARP activation was followed by a significant deterioration of neuronal membrane properties. Using electrophysiological recordings we firstly investigated the suggested ability of ADP-ribose to open TRPM2 channels in MNNG-induced cells death, but the results we obtained showed that TRPM2 channels are not involved. We then studied the involvement of glutamate receptor-ion channel complex and we found that NBQX, a selective AMPA receptor antagonist, was able to effectively prevent CA1 neuronal loss while MK801, a NMDA antagonist, was not active. Moreover, we observed that MNNG treatment increased the ratio of GluA1/GluA2 AMPAR subunit expression, which was associated with an inward rectification of the IV relationship of AMPA sEPSCs in the CA1 but not in the CA3 subfield. Accordingly, 1-naphthyl acetyl spermine (NASPM), a selective blocker of Ca(2+)-permeable GluA2-lacking AMPA receptors, reduced MNNG-induced CA1 pyramidal cell death. In conclusion, our results show that activation of the nuclear enzyme PARP-1 may change the expression of membrane proteins and Ca(2+) permeability of AMPA channels, thus affecting the function and survival of CA1 pyramidal cells.

Topical mannitol reduces inflammatory edema in a rat model of arthritis.

The hexahydric alcohol mannitol is widely used to shift fluids from the intracellular to the extracellular compartments, to increase diuresis and improve mucus clearance in the airways. In principle, because of its physicochemical properties, topical mannitol might also draw fluids out of epidermis or mucosa. Here, we report that topical mannitol applications on the hind paws of rats with adjuvant-induced arthritis reduced paw thickness and tissue edema without affecting the inflammatory infiltrates. Of note, the anti-edema effects of acute (4 h) mannitol application occurred earlier than those prompted by a similar treatment with classic anti-inflammatory drugs such as diclofenac or ketoprofen. Yet, the extent of edema reduction was higher with diclofenac or ketoprofen than with mannitol when the drugs were applied in a chronic (16 h) paradigm. Together, data demonstrate that topical application of mannitol exerts potent and fast anti-edema effects in a rat model of joint inflammation, suggesting a possible utilization in patients affected by osseo-arthritic disorders.

Improvement of a Cohesive Zone Model for Fatigue Delamination Rate Simulation.

The cohesive zone model (CZM) has found wide acceptance as a tool for the simulation of delamination in composites and debonding in bonded joints and various implementations of the cohesive zone model dedicated to fatigue problems have been proposed in the past decade. In previous works, the authors have developed a model based on cohesive zone to simulate the propagation of fatigue defects where damage acts on cohesive stiffness, with an initial (undamaged) stiffness representative of that of the entire thickness of an adhesive layer. In the case of a stiffness that is order of magnitude higher than the previous one (for instance, in the simulation of the ply-to-ply interface in composites), the model prediction becomes inaccurate. In this work, a new formulation of the model that overcomes this limitation is developed. Finite element simulations have been conducted on a mode I, constant bending (constant G)-loaded double cantilever beam (DCB) joint to assess the response of the new model with respect to the original one for varying initial stiffness K0 and cohesive strength σ0. The results showed that the modified model is robust with respect to changes of two orders of magnitude in initial stiffness and of a factor of two in σ0.

Psychomotor performance is not influenced by brief repeated exposures to mobile phones.

The present study investigated the presence of a cumulative effect of brief and repeated exposures to a GSM mobile phone (902.40 MHz, 217 Hz modulated; peak power of 2 W; average power of 0.25 W; SAR = 0.5 W/kg) on psychomotor functions. To this end, after each of 3 15-min exposures, both an acoustic simple reaction time task (SRTT) and a sequential finger tapping task (SFTT) were administered to 24 subjects. The present study was unable to detect the cumulative effects of brief and repeated EMF exposure on human psychomotor performance, although there was a non-statistical trend to shorter reaction times. In summary, these data show an absence of effects with these particular exposure conditions; however, possible cognitive effects induced by different signal characteristics cannot be excluded.

Visceral hypersensitivity and intolerance symptoms in lactose malabsorption.

Lactose malabsorption is not always associated with intolerance symptoms. The factors responsible for symptom onset are not yet completely known. As differences in visceral sensitivity may play a role in the pathogenesis of functional symptoms, we evaluated whether an alteration of visceral sensitivity is present in subjects with lactose intolerance. Thirty subjects, recruited regardless of whether they were aware of their capacity to absorb lactose, underwent an evaluation of intestinal hydrogen production capacity by lactulose breath test, followed by an evaluation of lactose absorption by hydrogen breath test after lactose administration and subsequently an evaluation of recto-sigmoid sensitivity threshold during fasting and after lactulose administration, to ascertain whether fermentation modifies intestinal sensitivity. The role of differences in gastrointestinal transit was excluded by gastric emptying and mouth-to-caecum transit time by (13)C-octanoic and lactulose breath tests. Lactulose administration induced a significant reduction of discomfort threshold in subjects with lactose intolerance but not in malabsorbers without intolerance symptoms or in subjects with normal lactose absorption. Perception threshold showed no changes after lactulose administration. Severity of symptoms in intolerant subjects was significantly correlated with the reduction of discomfort thresholds. Visceral hypersensitivity should be considered in the induction of intolerance symptoms in subjects with lactose malabsorption.

Bretylium-induced voltage-gated sodium current in human lymphocytes.

Using the whole-cell variation of the patch-clamp technique it has been determined that 0.25-3 mM bretylium tosylate (BT) exerts a repolarizing effect on partially depolarized human lymphocytes. The repolarizing effect was ouabain (40 microM)-sensitive, and was inhibited by the removal of external Na+ or by the Na(+)-channel-blocker amiloride (10-44 microM), but K(+)-channel-blockers 4-aminopyridine (0.1-5 mM) and quinine (100 microM) had no effect. The drug induced a sodium dependent, amiloride-sensitive transient inward current reaching its maximum value approx. 20-30 s after the administration of BT and lasting for 6-10 min. This current was activated by depolarization within 25 ms at around -42 mV, its inactivation took about 2 s and its reversal potential was +24 +/- 5 mV. An increase in the intracellular sodium concentration (1.8-3.2 mM) has been observed upon the addition of BT by monitoring the SBFI fluorescence of the dye-loaded cells. It has been shown that whole-cell K+ currents are significantly decreased by BT. The existence of voltage and ligand (BT)-gated sodium channels has been postulated in human lymphocytes. These channels are thought to participate in the initiation of membrane repolarization in human lymphocytes, and thereby influence mitogenic or antigen-induced cell-activation processes.

Poly(ADP-ribose) polymerase inhibitors attenuate necrotic but not apoptotic neuronal death in experimental models of cerebral ischemia.

An excessive activation of poly(ADP-ribose) polymerase (PARP) has been proposed to play a key role in post-ischemic neuronal death. We examined the neuroprotective effects of the PARP inhibitors benzamide, 6(5H)-phenanthridinone, and 3,4-dihydro-5-[4-1(1-piperidinyl)buthoxy]-1(2H)-isoquinolinone in three rodent models of cerebral ischemia. Increasing concentrations of the three PARP inhibitors attenuated neuronal injury induced by 60 min oxygen-glucose deprivation (OGD) in mixed cortical cell cultures, but were unable to reduce CA1 pyramidal cell loss in organotypic hippocampal slices exposed to 30 min OGD or in gerbils following 5 min bilateral carotid occlusion. We then examined the necrotic and apoptotic features of OGD-induced neurodegeneration in cortical cells and hippocampal slices using biochemical and morphological approaches. Cortical cells exposed to OGD released lactate dehydrogenase into the medium and displayed ultrastructural features of necrotic cell death, whereas no caspase-3 activation nor morphological characteristics of apoptosis were observed at any time point after OGD. In contrast, a marked increase in caspase-3 activity was observed in organotypic hippocampal slices after OGD, together with fluorescence and electron microscope evidence of apoptotic neuronal death in the CA1 subregion. Moreover, the caspase inhibitor Z-VAD-FMK reduced OGD-induced CA1 pyramidal cell loss. These findings suggest that PARP overactivation may be an important mechanism leading to post-ischemic neurodegeneration of the necrotic but not of the apoptotic type.

Kynurenine 3-mono-oxygenase inhibitors reduce glutamate concentration in the extracellular spaces of the basal ganglia but not in those of the cortex or hippocampus.

Kynurenine 3-mono-oxygenase (KMO, kynurenine hydroxylase) inhibitors increase brain kynurenic acid (KYNA) synthesis and cause pharmacological actions possibly mediated by a reduced activity of excitatory synapses. We used in vivo microdialysis and passive avoidance to study the effects of local KYNA or systemic KMO inhibitor administration on glutamate (GLU) neurotransmission. Local application of KYNA (30-100 nM) through reverse microdialysis reduced GLU content in caudate and cortical dialysates by 75 and 55%, respectively. No changes were found in the hippocampus. Systemic administration of Ro 61-8048 (4-40 mg/kg) increased KYNA levels in dialysates obtained from the cortex (from 10.3 +/- 1.9 to 45.5 +/- 15 nM), caudate (from 2.4 +/- 0.8 to 9.5 +/- 0.9 nM) and hippocampus (from 7.7 +/- 1.7 to 19.2 +/- 3.5 nM). It also caused a parallel robust decrease in GLU levels in the dialysates collected from the caudate (from 2.2 +/- 0.5 to 0.63 +/- 0.05 microM) but not in those collected from the parietal cortex or the hippocampus. In a passive avoidance paradigm, the administration of the NMDA receptor antagonist MK-801 (0.1 mg/kg) reduced, while Ro 61-8048 (4-80 mg/kg) did not change the latency time of entering into the dark compartment on the recall trial. Our data show that KMO inhibitors increase brain KYNA synthesis and selectively reduce GLU extracellular concentration in the basal ganglia.

Is the brain influenced by a phone call? An EEG study of resting wakefulness.

We recorded the resting electroencephalogram of 20 healthy subjects in order to investigate the effect of electromagnetic field (EMF) exposure on EEG waking activity and its temporal development. The subjects were randomly assigned to two groups and exposed, in double-blind conditions, to a typical mobile phone signal (902.40 MHz, modulated at 217 Hz, with an average power of 0.25 W) before or during the EEG recording session. The results show that, under real exposure as compared to baseline and sham conditions, EEG spectral power was influenced in some bins of the alpha band. This effect was greater when the EMF was on during the EEG recording session than before it. The present data lend further support to the idea that pulsed high-frequency electromagnetic fields can affect normal brain functioning, also if no conclusions can be drawn about the possible health effects.

Combined inhibition of indoleamine 2,3-dioxygenase and nitric oxide synthase modulates neurotoxin release by interferon-gamma-activated macrophages.

We evaluated the synthesis of nitric oxide (NO) and of the neurotoxic kynurenine metabolites 3OH-kynurenine and quinolinic acid (QUIN) in interferon-gamma (IFN-gamma)-activated macrophages of the murine BACl.2F5 cell line with the aim of investigating the roles of mononuclear phagocytes in inflammatory neurological disorders. IFN-gamma induced indoleamine 2,3-dioxygenase (IDO) and NO synthase (NOS) and increased the synthesis of 3OH-kynurenine, QUIN, and NO that accumulated in the incubation medium where they reached neurotoxic levels. Macrophage exposure to norharmane, an IDO inhibitor, resulted in a decreased formation of not only the kynurenine metabolites but also NO. The inhibition of NO synthesis could not be ascribed to reduced NADPH availability or decreased NOS induction. Norharmane inhibited NOS activity also in coronary vascular endothelial cells and in isolated aortic rings. Our findings suggest that activated macrophages release large amounts of neurotoxic molecules and that norharmane may represent a prototype compound to study macrophage involvement in inflammatory brain damage.

A sodium channel opener inhibits stimulation of human peripheral blood mononuclear cells.

The role of membrane potential changes in T cell activation was studied on human peripheral blood lymphocytes stimulated with phytohemagglutinin. Addition of bretylium tosylate, a sodium channels opener, to PHA treated lymphocytes modified the membrane potential and consequently blocked cell activation in a dose-dependent fashion. BT was non-toxic even in long-term (72 hr) incubations. It was reversibly removable, and the removal restored the stimulatory effect of PHA. 3H-thymidine incorporation was blocked if BT was present during the first 20-24 hr of the mitogenic activation. The later BT was added after PHA, the less inhibition of proliferation was observed. BT hyperpolarized the lymphocytes also in the presence of PHA. BT hindered the depolarizing effect of high extracellular potassium concns. The sustained polarized state of the lymphocytes did not influence the intracellular calcium increase upon PHA treatment. IL-2 and transferrin receptor expression was not hindered by BT during PHA stimulation of lymphocytes. Addition of rIL-2 did not abolish the inhibitory effect of BT. According to cell-cycle analysis BT arrested the majority of the cells in G1 phase. It is suggested that cell activation demands the flexible maintenance of a relatively narrow membrane potential "window". Any sustained and significant hyper-, or depolarization, may dramatically decrease the effectivity of transmembrane signalling.

Kynurenine hydroxylase inhibitors reduce ischemic brain damage: studies with (m-nitrobenzoyl)-alanine (mNBA) and 3,4-dimethoxy-[-N-4-(nitrophenyl)thiazol-2yl]-benzenesulfonamide (Ro 61-8048) in models of focal or global brain ischemia.

Two kynurenine hydroxylase inhibitors, (m-nitrobenzoyl)-alanine (mNBA) and 3,4-dimethoxy-[-N-4-(nitrophenyl)thiazol-2yl]-benzenesulfona mide (Ro 61-8048), have been tested as neuroprotective agents on brain lesions induced by bilateral carotid occlusion in gerbils or by middle cerebral artery occlusion in rats. The percentage of lesioned pyramidal neurones found in the hippocampal CA1 region of gerbils subjected to bilateral carotid occlusion for 5 minutes decreased from 92+/-10% in vehicle-treated animals to 7+/-6% after mNBA (400 mg/kg intraperitoneally, three times at 1, 30, and 180 minutes after occlusion) or to 10+/-11% after Ro 61-8048 (40 mg/kg intraperitoneally, three times). A significant reduction in infarct volumes also was found when the kynurenine hydroxylase inhibitors were given to rats after permanent middle cerebral artery occlusion (from 207+/-111 mm3 in vehicle-treated rats to 82+/-18 and to 62+/-57 mm3 in rats treated with mNBA, 400 mg/kg intraperitoneally, or with Ro 61-8048, 40 mg/kg intraperitoneally, respectively). The administration of mNBA (400 mg/kg intraperitoneally) or Ro 61-8048 (40 mg/kg intraperitoneally) to gerbils with a dialysis probe in their dorsal hippocampus or to rats with a dialysis probe in their parietal cortex significantly increased kynurenic acid concentration in the dialysates. The data suggest that inhibition of kynurenine hydroxylase could be a new avenue to reduce neuronal loss in brain ischemia.

Senile dementia and Alzheimer's disease: lack of changes of the cortical content of quinolinic acid.

The content of Quinolinic Acid (QUIN) was fragmentographically measured in the frontal, parietal and temporal cortex obtained at autopsy from patients affected by Alzheimer's disease-senile dementia Alzheimer type (AD/SDAT) or matched controls. The density of large cholinergic neurons in the nucleus basalis magnocellularis and the density of plaques in the hippocampal formation, parietal and frontal cortex of these patients was also evaluated in order to obtain a quantitative estimation of the Alzheimer type changes. In the three cortical areas studied, the content of QUIN was similar in AD/SDAT patients and age matched controls. The AD/SDAT patients had an important reduction of the number of large cholinergic neurons in the nucleus basalis magnocellularis and a much higher density of plaques in cortex and in hippocampus than age matched controls. The data reported here do not support the possibility than an accumulation of QUIN plays a role in the neuronal degeneration occurring in the cortex of patients affected by AD/SDAT.

The kynurenine metabolic pathway in the eye: studies on 3-hydroxykynurenine, a putative cataractogenic compound.

The rabbit lens has an elevated content of 3-hydroxykynurenine (30HKYN) in spite of a very low activity of the enzymes leading to its synthesis. The iris/ciliary body, on the contrary, has very high activity of 30HKYN synthesizing enzymes but a content of 30HKYN lower than that of the lens. These observations suggest that 30HKYN is formed in the iris/ ciliary body, released into the aqueous humor and then taken up into the lens where it may be used for the synthesis of UV filtering products. An excessive accumulation of 30HKYN in the lens has been associated with cataract formation. We found that available selective inhibitors of kynurenine hydroxylase reduced 30HKYN synthesis in both the lens and the iris/ciliary body.

Vitamin E deficiency impairs the modifications of mitochondrial membrane potential and mass in rat splenocytes stimulated to proliferate.

This study was designed to evaluate the time-dependent changes of mitochondrial membrane potential and mass during Con-A-induced proliferation of splenic lymphocytes from rat fed a normal or a vitamin E deficient diet. Rhodamine 123 and Nonyl Acridine Orange were used as specific probes to monitor the membrane potential and mass of mitochondria, respectively, by means of flow cytometry. The results demonstrate that the increase of Rh-123 and NAO uptake observed in cells from normally fed rats was prevented by vitamin E deficiency, at any time considered. After 72 h from Con A stimulation, 62% of cells from controls, as against 16% of cells from vitamin E deficient rats, showed hyperpolarized mitochondria. At the same time, in this last group, 60% of cells had depolarized organelles. The same pattern was observed considering the changes of mitochondrial mass, measured using NAO as a probe. These data support that mitogenic stimulation induced an increase of the respiratory activity of mitochondria with subsequent production of superoxide radicals. This resulted in depolarization and loss of mass of the organelles if the intracellular level of vitamin E is not adequate.

Age-dependent modifications of mitochondrial trans-membrane potential and mass in rat splenic lymphocytes during proliferation.

The specific fluorescent probes, Rhodamine 123 (Rh-123) and Nonyl-Acridine Orange (NAO) were, respectively, used to monitor the changes in membrane potential and mass of lymphocyte mitochondria during aging and proliferation. An age-dependent increase of the uptake of both fluorochromes was observed in resting cells; however, NAO fluorescence increased to a greater extent when compared with the Rh-123 probe. This resulted in a lower respiratory activity per unit of mitochondrial mass in old cells than in the young ones. Following mitogenic stimulation, most of the lymphocytes from young rats showed an increase in their membrane potential and mass. On the contrary about 50% of cells from old rats had depolarized mitochondria after 72 h from the stimulation. Present data support that mitochondria of lymphocytes from old rats are extremely sensitive to the stressing conditions resulting from mitogenic stimulation.

Food restriction increases the protection of erythrocytes against the hemolysis induced by peroxyl radicals.

We have investigated the susceptibility to peroxidation of erythrocytes from young, adult and old ad libitum (AL) fed, as well as from adult and old food restricted rats, measuring the rate of hemolysis under controlled peroxidative condition. Food restriction has been applied on an every-other-day (EOD) schedule starting from the age of 3.5 months. The oxidation of red blood cells by molecular oxygen was performed in an aqueous suspension using the azo-compound 2-2'-azo-bis-(2-amidinopropane)hydrochloride (AAPH) as the free radical initiator. Several parameters were calculated from the time-dependent curve of AAPH induced hemolysis. The time required to achieve 50% hemolysis decreased with aging and this decrease was prevented by food restriction. The lag time, which reflects the capacity of the cell to buffer free radicals, was longer in young than in old AL fed animals also this impairment was almost completely prevented in EOD fed animals. The same beneficial effect of food restriction was observed considering the maximal amount of hemolysis attained with the dose of AAPH applied and the time necessary to reach this level. The general picture emerging from the present study is that erythrocyte membranes from EOD fed rats are better protected, than those from AL fed ones, against damages caused by peroxidation. This effect may be due to a difference in the chemical composition of the erythrocyte membranes as it was found in other organs.

Phytohemagglutinin induced changes of membrane lipid packing, c-myc and c-myb encoded protein expression in human lymphocytes during aging.

Three parameters which signal different stages of cell activation were analyzed in lymphocytes from young and old subjects. Merocyanine 540 (MC-540) incorporation into the membrane lipid phase was used as a very early marker of activation and was measured after 1 h of phytohemagglutinin (PHA) stimulation. The proteins coded by c-myc and c-myb protooncogenes were determined by appropriate antibodies and were taken as markers of the G0/G1 and G1/S phase transition, respectively. The number of cells which increased the uptake of MC-540 following PHA stimulation did not differ when comparing young and old individuals. Both the number of the responding cells and the size of the response were decreased during aging when the presence of the c-myc protein was taken into account. A consistent decrease of the percentage of lymphocytes able to express the c-myb protein was observed in the cells from old donors as compared to those from the young ones, but the amount of detectable protein per cell remained unchanged. Our data suggest that the deficiency of responsiveness which accompanies aging is due to impairments at different points of the cell cycle. The very low number of cells expressing the c-myb protein is likely the result of step by step elimination of those cells not able to fulfill the requirements to progress along the cell cycle.