Glutamate modulation of human lymphocyte growth: in vitro studies

Peripheral blood mononuclear cell (PBMC) proliferation induced by phytohemagglutinin, or by anti-CD3 alone or plus anti-CD28 monoclonal antibodies (mAb) was inhibited by glutamate (Glu) in a concentration-dependent manner. This inhibition was not reproduced by selective ionotropic Glu receptor agonists, whereas it was potentiated by l-buthionine-(S,R)-sulfoximine, which depletes glutathione (GSH) stores, and counteracted by 2-mercaptoethanol, a preserver of cell thiols. The inhibitory effects of Glu were related to depletion of intracellular GSH stores, since it decreased GSH levels in a concentration-dependent manner. Furthermore, Glu modulated cytokine secretion by anti-CD3 mAb activated PBMC: it increased IFN-gamma (+44.3+/-8.2%) and IL-10 (+31.6+/-9.7%) secretion, whereas that of IL-2, IL-4, IL-5, and TNF-alpha was not affected. These data suggest that high levels of Glu, which can be reached in damaged tissues, modulate lymphocyte responses to activating stimuli by favouring polarization of the T helper effector response.     

Role of caspases in the regulation of apoptotic pancreatic islet beta-cells death

The homeostatic control of beta-cell mass in normal and pathological conditions is based on the balance of proliferation, differentiation, and death of the insulin-secreting cells. A considerable body of evidence, accumulated during the last decade, has emphasized the significance of the disregulation of the mechanisms regulating the apoptosis of beta-cells in the sequence of events that lead to the development of diabetes. The identification of agents capable of interfering with this process needs to be based on a better understanding of the beta-cell specific pathways that are activated during apoptosis. The aim of this article is fivefold: (1) a review of the evidence for beta-cell apoptosis in Type I diabetes, Type II diabetes, and islet transplantation, (2) to review the common stimuli and their mechanisms in pancreatic beta-cell apoptosis, (3) to review the role of caspases and their activation pathway in beta-cell apoptosis, (4) to review the caspase cascade and morphological cellular changes in apoptotic beta-cells, and (5) to highlight the putative strategies for preventing pancreatic beta-cells from apoptosis.     

Four year incidence of respiratory syncytial virus infection in infants and young children referred to emergency departments for lower respiratory tract diseases in Italy: the "Osservatorio VRS" Study (2000-2004)

Respiratory Syncytial Virus (RSV) is a frequent cause of hospital admission in young children and high risk babies such as premature newborns, or babies with underlying cardiac or pulmonary disease, or immunodeficiency. Outbreaks occur most frequently in the cold season in areas with temperate and Mediterranean climates. Aim of the "Osservatorio VRS" Study was to describe the time-related pattern of RSV epidemics in Italy, across four consecutive epidemics, from 2000 to 2004. Nasal specimens for RSV detection were obtained and tested by an immunoenzymatic test. A total of 2110 children were tested for RSV determination, the rate of children with RSV infection was 21%, and that of children hospitalized for RSV disease was 49%. Considering the whole study period, the RSV epidemics started in October-November and ended in May, showing a peak incidence in February, with a median of 28.1% and a maximum of 48.9%. Analysis of monthly distribution of each year of the study showed a biennial trend for an earlier appearance. A different epidemiological pattern of the infection was observed among the three national areas. In conclusion, even though the mechanism governing RSV infection periodicity remains unknown, its awareness in the absence of an RSV surveillance system as in Italy, may be useful for scheduling RSV prophylaxis and for hospital resource management.     

Modeling the euglycemic hyperinsulinemic clamp by stochastic differential equations

The Euglycemic Hyperinsulinemic Clamp (EHC) is the most widely used experimental procedure for the determination of insulin sensitivity. In the present study, 16 subjects with BMI between 18.5 and 63.6 kg/m² have been studied with a long-duration (5 hours) EHC. In order to explain the oscillations of glycemia occurring in response to the hyperinsulinization and to the continuous glucose infusion at varying speeds, we first hypothesized a system of ordinary differential equations (ODEs), with limited success. We then extended the model and represented the experiment using a system of stochastic differential equations (SDEs). The latter allow for distinction between (i) random variation imputable to observation error and (ii) system noise (intrinsic variability of the metabolic system), due to a variety of influences which change over time. The stochastic model of the EHC was fitted to data and the system noise was estimated by means of a (simulated) maximum likelihood procedure, for a series of different hypothetical measurement error values. We showed that, for the whole range of reasonable measurement error values: (i) the system noise estimates are non-negligible; and (ii) these estimates are robust to changes in the likely value of the measurement error. Explicit expression of system noise is physiologically relevant in this case, since glucose uptake rate is known to be affected by a host of additive influences, usually neglected when modeling metabolism. While in some of the studied subjects system noise appeared to only marginally affect the dynamics, in others the system appeared to be driven more by the erratic oscillations in tissue glucose transport rather than by the overall glucose-insulin control system. It is possible that the quantitative relevance of the unexpressed effects (system noise) should be considered in other physiological situations, represented so far only with deterministic models.The work was supported by grants from the Danish Medical Research Council and the Lundbeck Foundation to S. Ditlevsen.     

Non-random, individual-specific methylation profiles are present at the sixth CTCF binding site in the human H19/IGF2 imprinting control region

Expression of imprinted genes is classically associated with differential methylation of specific CpG-rich DNA regions (DMRs). The H19/IGF2 locus is considered a paradigm for epigenetic regulation. In mice, as in humans, the essential H19 DMR--target of the CTCF insulator--is located between the two genes. Here, we performed a pyrosequencing-based quantitative analysis of its CpG methylation in normal human tissues. The quantitative analysis of the methylation level in the H19 DMR revealed three unexpected discrete, individual-specific methylation states. This epigenetic polymorphism was confined to the sixth CTCF binding site while a unique median-methylated profile was found at the third CTCF binding site as well as in the H19 promoter. Monoallelic expression of H19 and IGF2 was maintained independently of the methylation status at the sixth CTCF binding site and the IGF2 DMR2 displayed a median-methylated profile in all individuals and tissues analyzed. Interestingly, the methylation profile was genetically transmitted. Transgenerational inheritance of the H19 methylation profile was compatible with a simple model involving one gene with three alleles. The existence of three individual-specific epigenotypes in the H19 DMR in a non-pathological situation means it is important to reconsider the diagnostic value and functional importance of the sixth CTCF binding site.     

Multiscale modeling of cellular actin filaments: from atomistic molecular to coarse-grained dynamics

In this article, we present a computational multiscale model for the characterization of subcellular proteins. The model is encoded inside a simulation tool that builds coarse-grained (CG) force fields from atomistic simulations. Equilibrium molecular dynamics simulations on an all-atom model of the actin filament are performed. Then, using the statistical distribution of the distances between pairs of selected groups of atoms at the output of the MD simulations, the force field is parameterized using the Boltzmann inversion approach. This CG force field is further used to characterize the dynamics of the protein via Brownian dynamics simulations. This combination of methods into a single computational tool flow enables the simulation of actin filaments with length up to 400 nm, extending the time and length scales compared to state-of-the-art approaches. Moreover, the proposed multiscale modeling approach allows to investigate the relationship between atomistic structure and changes on the overall dynamics and mechanics of the filament and can be easily (i) extended to the characterization of other subcellular structures and (ii) used to investigate the cellular effects of molecular alterations due to pathological conditions.     

Resistance of neuroblastoma GI-ME-N cell line to glutathione depletion involves Nrf2 and heme oxygenase-1

Cancer cell survival is known to be related to the ability to counteract oxidative stress, and glutathione (GSH) depletion has been proposed as a mechanism to sensitize cells to anticancer therapy. However, we observed that GI-ME-N cells, a neuroblastoma cell line without MYCN amplification, are able to survive even if GSH-depleted by l-buthionine-(S,R)-sulfoximine (BSO). Here, we show that in GI-ME-N cells, BSO activates Nrf2 and up-regulates heme oxygenase-1 (HO-1). Silencing of Nrf2 restrained HO-1 induction by BSO. Inhibition of HO-1 and silencing of Nrf2 or HO-1 sensitized GI-ME-N cells to BSO, leading to reactive oxygen/nitrogen species overproduction and decreasing viability. Moreover, targeting the Nrf2/HO-1 axis sensitized GI-ME-N cells to etoposide more than GSH depletion. Therefore, we have provided evidence that in GI-ME-N cells, the Nrf2/HO-1 axis plays a crucial role as a protective factor against cellular stress, and we suggest that the inhibition of Nfr2/HO-1 signaling should be considered as a central target in the clinical battle against neuroblastoma.     

The switch from pRb/p105 to Rb2/p130 in DNA damage and cellular senescence

Cellular senescence is a response to genotoxic stress that results in an irreversible cell cycle arrest. Activation of this pathway relies on the activity of the retinoblastoma proteins and proteins of the DNA damage response cascade. Here, we discuss the functional relevance of the switch from pRb/p105 to Rb2/p130 that becomes apparent when cells enter senescent arrest.     

Rho-kinase inhibition improves vasodilator responsiveness during hyperinsulinemia in the metabolic syndrome

In patients with the metabolic syndrome (MetS), the facilitatory effect of insulin on forearm vasodilator responsiveness to different stimuli is impaired. Whether the RhoA/Rho kinase (ROCK) pathway is involved in this abnormality is unknown. We tested the hypotheses that, in MetS patients, ROCK inhibition with fasudil restores insulin-stimulated vasodilator reactivity and that oxidative stress plays a role in this mechanism. Endothelium-dependent and -independent forearm blood flow responses to acetylcholine (ACh) and sodium nitroprusside (SNP), respectively, were assessed in MetS patients (n = 8) and healthy controls (n = 5) before and after the addition of fasudil (200 μg/min) to an intra-arterial infusion of insulin (0.1 mU/kg/min). In MetS patients (n = 5), fasudil was also infused without hyperinsulinemia. The possible involvement of oxidative stress in the effect of fasudil during hyperinsulinemia was investigated in MetS patients (n = 5) by infusing vitamin C (25 mg/min). In MetS patients, compared with saline, fasudil enhanced endothelium-dependent and -independent vasodilator responses during insulin infusion (P < 0.001 and P = 0.008, respectively), but not in the absence of hyperinsulinemia (P = 0.25 and P = 0.13, respectively). By contrast, fasudil did not affect vasoreactivity to ACh and SNP during hyperinsulinemia in controls (P = 0.11 and P = 0.56, respectively). In MetS patients, fasudil added to insulin and vitamin C did not further enhance vasodilation to ACh and SNP (P = 0.15 and P = 0.43, respectively). In the forearm circulation of patients with the MetS, ROCK inhibition by fasudil improves endothelium-dependent and -independent vasodilator responsiveness during hyperinsulinemia; increased oxidative stress seems to be involved in the pathophysiology of this phenomenon.