Biancamaria Longoni

Melatonin as an antioxidant in retinal photoreceptors

Abstract: Dark‐adapted, single photoreceptors isolated from the frog retina produce reactive oxygen species (ROS) after about 1 min of illumination with saturating light that we verified by their oxidation of preloaded dihydrorhodamine 123 (DHR) into the fluorescent rhodamine 123 (RHO). In this preparation we tested the antioxidant effects of vitamin E and of melatonin. Melatonin at picomolar and low nanomolar concentrations was determined to be 100 times more potent in inhibiting the light‐induced oxidative processes than was vitamin E. On the contrary, both compounds exerted potent prooxidant effects at micromolar concentrations that is above the physiological levels of melatonin. This provides evidence that physiological concentrations of melatonin in a living cell may exert protective actions against a natural oxidant stimulus (light). This helps to define the functional role of endogenous melatonin in photoreceptors, which by their physiological characteristics, are among the marked producers of ROS in the organism.

Properties and functional roles of hyperpolarization‐gated currents in guinea‐pig retinal rods

1 The inward rectification induced by membrane hyperpolarization was studied in adult guinea‐pig rods by the perforated‐patch‐clamp technique. 2 CsCl blocked the rectification observed in both voltage‐ and current‐clamp recordings at voltages negative to −60 mV, while BaCl2 blocked the inward relaxation observed at voltages positive to −60 mV. The current activated at −90 mV had a low selectivity between sodium and potassium and reversed at −31.0 mV. 3 These observations suggest that two inward rectifiers are present in guinea‐pig rods: a hyperpolarization‐activated (Ih) and a hyperpolarization‐deactivated (Ikx) current. The functional roles of Ih and Ikx were evaluated by stimulating rods with currents sinusoidally modulated in time. 4 Rods behave like bandpass amplifiers, with a peak amplification of 1.5 at about 2 Hz. For hyperpolarizations that mainly gate Ikx, amplification and phase shifts are fully accounted for by a rod membrane analogue model that includes an inductance. For hyperpolarizations that also gate Ih, a harmonic distortion became apparent. 5 Bandpass filtering and amplification of rod signals, associated with Ih and Ikx gating by membrane hyperpolarization, are strategically located to extend, beyond the limits imposed by the slow phototransductive cascade, the temporal resolution of signals spreading to the rod synapse.

Oxidative Stress and Inflammatory Reaction Modulation by White Wine

Abstract: Wine and olive oil, essential components of the Mediterranean diet, are considered important factors for a healthy life style. Tyrosol (T) and caffeic acid (CA) are found in both extra virgin olive oil and in white wine. Three white wines from the northeast Italy and four white wines from Germany were analyzed for their content of T and CA. These compounds were tested for their antioxidant activity and their capacity to modulate three different cytokines: IL‐1β, IL‐6, and TNF‐α, which are currently considered to be the major cytokines influencing the acute phase of the inflammatory response. Furthermore, the antioxidant activity of T and CA was analyzed by monitoring the oxidation of a redox‐sensitive probe by using laser scanning confocal microscopy. T and CA, applied at nanomolar range, were found to significantly reduce the generation of oxidants induced by azobis‐amidinopropanedihydrochloride. Peripheral blood mononuclear cells (PBMC) from healthy volunteers were incubated at 37°C for 12 hours with 100 ng LPS (E. coli and P. maltofilia). Increasing doses of T and CA (150 nM to 300 μM) were added and cell‐associated IL‐1β and TNF‐α were determined by immunoreactive tests after three freeze‐thaw cycles. IL‐6 release was also determined in cell surnatants. LPS‐stimulated PBMC showed a significant increase in cytokine release, while T and CA, used at nanomolar concentrations, were able to modulate their expression. Taken together, these results suggest a remarkable effect of white wine non‐alcoholic compounds on oxidative stress and inflammatory reaction.

Apoptosis and adaptive responses to oxidative stress in human endothelial cells exposed to cyclosporin A correlate with BCL-2 expression levels.

Treatment of transplanted patients with cyclosporin A (CSA) may cause adverse effects such as nephrotoxicity and hypertension. As CSA is known to induce oxidative stress in several tissues, it may cause vascular problems by triggering oxidative stress in endothelial cells (EC). However, oxidative stress has been reported for acute exposure to CSA concentra¬tions exceeding its clinical range, whereas immunosuppression requires life‐long treatment with therapeutic concentrations. We therefore compared the effects of 21 h pharmacological (200 μM) vs. 8 days clinical (0.5–2.5 μM) doses of CSA on cultured human EC. Pharmacological doses of CSA cause a decrease in cell density via apoptosis and a down‐regulation of the antiapoptotic protein Bcl‐2. However, these effects are independent of CSA‐induced oxidative stress. In con¬trast, therapeutic concentrations of CSA cause Bcl‐2 up‐regulation and modification of EC morphology, both effects blocked by antioxidants. Therefore, a low level of oxidants may act in EC as second messengers that up‐regulate Bcl‐2, thus promoting survival of im¬paired EC. Our data suggest that the oxidative stress induced by clinical concentrations of CSA may be involved in the adverse effects of the drug on the vascular system of transplanted patients via an adaptive response involving Bcl‐2 up‐regulation rather than an apoptotic process.—Longoni, B., Boschi, E., Demontis, G. C., Ratto, G. M., Mosca, F. Apoptosis and adaptive responses to oxidative stress in human endothelial cells exposed to cyclosporin A correlate with BCL‐2 expres¬sion levels. FASEB J. 15, 731‐740 (2001)

Mesenchymal Stem Cells Prevent Acute Rejection and Prolong Graft Function in Pancreatic Islet Transplantation

BACKGROUND Pancreatic islet transplantation is a promising cell-based therapy for type 1 diabetes (insulin-dependent diabetes mellitus), a disease triggered by the immune response against autoantigens of beta-cells. However, the recurrence of immune response after transplantation and the diabetogenic and growth-stunting side effects of immunosuppressants are major challenges to the application of islet transplantation. Mesenchymal stem cells (MSCs) have recently been reported to modulate the immune response in allogeneic transplantation. METHODS The ability of MSCs, either syngeneic or allogeneic to recipients, to prevent acute rejection and improve glycemic control was investigated in rats with diabetes given a marginal mass of pancreatic islets through the portal vein. RESULTS Reduced glucose levels and low-grade rejections were observed up to 15 days after transplantation upon triple-dose administration of MSCs, indicating that MSCs prolong graft function by preventing acute rejection. The efficacy of MSCs was associated with a reduction of pro-inflammatory cytokines and was independent of the administration route. Efficacy was similar for MSCs whether syngeneic or allogeneic to recipients and comparable to that of immunosuppressive therapy. CONCLUSIONS The results show that MSCs modulate the immune response through a down-regulation of pro-inflammatory cytokines, suggesting that MSCs may prevent acute rejection and improve graft function in portal vein pancreatic islet transplantation.

Functional characterisation and subcellular localisation of HCN1 channels in rabbit retinal rod photoreceptors

Gating of voltage‐dependent conductances in retinal photoreceptors is the first step of a process leading to the enhancement of the temporal performance of the visual system. The molecular components underlying voltage‐dependent gating in rods are presently poorly defined. In the present work we have investigated the isoform composition and the functional characteristics of hyperpolarisation‐activated cyclic nucleotide‐gated channels (HCN) in rabbit rods. Using immunocytochemistry we show the expression in the inner segment and cell body of the isoform 1 (HCN1). Electrophysiological investigations show that hyperpolarisation‐activated currents (Ih) can be measured only from the cell regions where HCN1 is expressed. Half‐activation voltage (–75.0 ± 0.3 mV) and kinetics (t1/2 of 101 ± 8 ms at –110 mV and 20 °C) of the Ih in rods are similar to those of the macroscopic current carried by homomeric rabbit HCN1 channels expressed in HEK 293 cells. The homomeric nature of HCN1 channels in rods is compatible with the observation that cAMP induces a small shift (2.3 ± 0.8 mV) in the half‐activation voltage of Ih. In addition, the observation that within the physiological range of membrane potentials, cAMP does not significantly affect the gain of the current‐to‐voltage conversion, may reflect the need to protect the first step in the processing of visual signals from changes in cAMP turnover.

Angiogenesis and nerve regeneration in a model of human skin equivalent transplant

The angiogenesis and reinnervation were studied in a porcine model of human skin equivalent (SE) graft and the relationship between the two processes was investigated. Confocal laser scanning microscopy was used to monitor, during the healing process, the pattern of vascularization and reinnervation at different time points. The SE was obtained by co-culturing fibroblasts and keratinocytes on a collagen-glycosaminoglycan-chitosan biopolymer and grafted on dorsal wounds generated by full-thickness resection in 25/30 Kg Large white pigs. Frozen sections were obtained from biopsies performed in autograft and xenograft, then were immunolabeled by using the endothelial marker lectin Lactifolia and with the neuronal marker gene product PGP9.5. Cajal staining was also used to visualize the nerve fibers. The results show that the vascularization precedes the innervation process. These data are consistent with the view that the development of nervous tissue is driven by nutritional and trophic factors provided by the vascular system. The arborization of the two systems observed during the third week from the graft might play a key role in maintaining the healing process and the graft survival.

Magnetic carbon nanotubes: a new tool for shepherding mesenchymal stem cells by magnetic fields.

AIMS We investigated the interaction between magnetic carbon nanotubes (CNTs) and mesenchymal stem cells (MSCs), and their ability to guide these intravenously injected cells in living rats by using an external magnetic field. MATERIALS & METHODS Multiwalled CNTs were used to treat MSCs derived from rat bone marrow. Cytotoxicity induced by nanotubes was studied using the WST-1 proliferation and Hoechest 33258 apoptosis assays. The effects of nanotubes on MSCs were evaluated by monitoring the effects on cellular growth rates, immunophenotyping and differentiation, and on the arrangement of cytoskeletal actin. MSCs loaded with nanotubes were injected in vivo in the portal vein of rats driving their localization in the liver by magnetic field. An histological analysis was performed on the liver, lungs and kidneys of all animals. RESULTS CNTs did not affect cell viability and their ability to differentiate in osteocytes and adipocytes. Both the CNTs and the magnetic field did not alter the cell growth rate, phenotype and cytoskeletal conformation. CNTs, when exposed to magnetic fields, are able to shepherd MSCs towards the magnetic source in vitro. Moreover, the application of a magnetic field alters the biodistribution of CNT-labelled MSCs after intravenous injection into rats, increasing the accumulation of cells into the target organ (liver). CONCLUSION Multiwalled CNTs hold the potential for use as nanodevices to improve therapeutic protocols for transplantation and homing of stem cells in vivo. This could pave the way for the development of new strategies for the manipulation/guidance of MSCs in regenerative medicine and cell transplantation.

In vivo visualization of transplanted pancreatic islets by MRI: comparison between in vivo, histological and electron microscopy findings

The aim of the work was to compare in vivo MRI visualization of pancreatic islets labeled with clinical-grade superparamagnetic iron oxide (SPIOs) contrast agents with ex vivo examination of liver tissue in an experimental model of marginal mass transplantation in rats. Seven hundred IEq (Islet Equivalent) from Wistar rats, labeled by incubation with Endorem or Resovist, were transplanted into Sprague-Dawley rats through the portal vein. Liver MR images of recipient rats were acquired at different time points (3-42 days) after transplantation. Animals were sacrificed during this period and their livers were excised and prepared for histology and electron microscopy. Hypointense spots originating from iron particles were observed in MR images. The number of separate spots was counted. Three days after transplantation one spot for every three or four transplanted islets was observed. Seven days after transplantation, histological sections showed the presence of iron within pancreatic islets. The time course of MR images showed a decrease in the number of spots, at 42 days, amounting to 65 and 22% of the initial value, for Resovist and Endorem respectively, while no immunopositive endocrine cells were detected in histological slices. The present work shows that pancreatic islets can be labeled using clinically approved SPIO contrast agents and visualized using in vivo MRI with high sensitivity, consistently with findings in the literature. Differently from reports in the literature, our findings indicate that iron particles could last in the liver for long periods, independently of the presence of intact pancreatic islets.

Melatonin prevents cyclosporine-induced nephrotoxicity in isolated and perfused rat kidney

Cyclosporine A (CsA) is a potent and effective immunosuppressive agent, but its action is frequently accompanied by severe renal toxicity. The precise mechanism by which CsA causes renal injury is not known. Reactive oxygen species (ROS) have been shown to play a role, since CsA-induced renal lipid peroxidation is attenuated in vivo and in vitro by the concomitant administration of antioxidants such as vitamin E. We show here the effect of the antioxidant melatonin (MLT), a hormone produced by the pineal gland during the dark phase of the circadian cycle, in a model of CsA nephrotoxicity in the isolated and perfused rat kidney. Kidneys isolated from rats were divided into seven groups. At the end of perfusion, malondialdehyde and 4-hydroxyalkenals (MDA+4-HDA), metabolites of nitric oxide N O 2 m +N O 3 m were measured and histopathological examination was performed. CsA treatment induced a significant increase in MDA+4-HDA while not affecting the nitric oxide metabolite level. MLT remarkably prevented glomerular collapse and tubular damage as revealed by morphometric analysis. Our study suggests that lipid peroxidation is an early important event in the pathogenesis of CsA nephrotoxicity and that MLT is able to protect kidneys from CsA at a relatively low concentration.