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Bone marrow is a highly cellular connective tissue, containing both fixed elements (blood vessels, adipocytes, stromal and staminal cells) and mobile cells (blood cellular elements and their precursors). Since mature immune cells are also found in other connective tissues, especially during inflammation, the only way to identify bone marrow with certainty is by labelling its unique components, i.e. blood cell precursors and stromal components. This however creates two problems: 1-mobile cells are lost, in variable measure, during tissue sectioning, and 2-most bone marrow cells do not display unique epitopes, just unique combinations of them. Within most bones, these problems do not significantly affect marrow identification, since the latter is contained in large, well-defined bone cavities, and even after some cell loss, most of its elements are still present in sufficient quantity to be visualized by immunofluorescence. The temporal bone, however, displays unique structural complexity, and in particular the petrosal bone surrounding the inner ear is made of extremely dense bone formed by endochondral ossification (different from calvaria, which are formed by membranous ossification) and even displays cartilage remnants in the adult. Although immune cells have been observed throughout the petrosal bone, only at the petrosal apex bone marrow has been identified as such. Recently, however, in the human temporal bone, cavities compatible with bone marrow were found by synchrotron imaging to be located between the cochlear base and endolymphatic sac and connected with the latter through bone channels (Liu et al., 2024, doi: 10.3389/fneur.2024.1355785). In a similar position, by using tissue clearing, we observed in the rat temporal bone highly cellular cavities with a similar connection pattern (Perin et al., 2024, doi: 10.3389/fneur.2024.1386654). These cavities could be as small as only containing a few hundred cells only, and in previous work they had been addressed as perivascular connective. However, even the smallest cavities contained megakaryocytes, which are platelet precursor and are confined to marrow in other bones. Therefore, the small cavities within the petrosal bone can be identified as bone marrow, and may be involved in local inner ear immune responses. [ABSTRACT FROM AUTHOR]

Using tissue clearing and immunofluorescence, we have recently characterized the adult rat temporal bone marrow distribution, and in particular the distribution and connections to the inner ear of petrosal bone marrow (doi: 10.3389/ fneur.2024.1386654). Bone marrow was identified by its high cellular content and by the presence of cell populations belonging to the hemopoietic niche (e.g. megakaryocytes, see companion abstract from our group). In the cleared rat pet-rosal bone, autofluorescence allowed delineation of the otic capsule. The largest marrow island was found outside of the otic capsule, surrounding semicircular canal arms, and connecting to the dura through bone channels similar to those of calvarial bone, with only a few channels directed towards the bony labyrinth. Unexpectedly, bone marrow was also observed within the otic capsule endochondral layer, forming small clusters associated to the vestibule (VEM) and cochlear apex (CAEM). Endochondral bone marrow was connected through vascular loops to the labyrinth, and through straight channels to dural sinuses. The latter also received vascular connection from marrow located in surrounding bones, suggesting a role as immune barrier restricting pathogen spread from ear to brain. In mouse, petrosal bone marrow distribution was overall similar to the rat but displayed a few differences. The most evident difference was the volume ratio of CAEM over total petrosal bone marrow (23 ± 1% in mouse, n = 2; 2 ± 1% in rat, n = 8; p < 10E-8). Moreover, CAEM and VEM were connected by vascular bridges in mouse but not in rat. Given the importance of local bone marrow in the immune reactions of brain (doi: 10.1111/imr.13120) and middle ear (doi: 10.3389/fgene.2022.985214), this difference in CAEM volume and connectivity calls for attention in choosing a model for human inner ear immune reactions. [ABSTRACT FROM AUTHOR]

Calvarial bone marrow has been found to be central in the brain immune response, being connected to the dura through channels which allow leukocyte trafficking. Temporal bone marrow is thought to play important roles in relation to the inner ear, but is still largely uncharacterized, given this bone complex anatomy. We characterized the geometry and connectivity of rat temporal bone marrow using lightsheet imaging of cleared samples and microCT. Bone marrow was identified in cleared tissue by cellular content (and in particular by the presence of megakaryocytes); since air-filled cavities are absent in rodents, marrow clusters could be recognized in microCT scans by their geometry. In cleared petrosal bone, autofluorescence allowed delineation of the otic capsule layers. Within the endochondral layer, bone marrow was observed in association to the cochlear base and vestibule, and to the cochlear apex. Cochlear apex endochondral marrow (CAEM) was a separated cluster from the remaining endochondral marrow, which was therefore defined as "vestibular endochondral marrow" (VEM). A much larger marrow island (petrosal non-endochondral marrow, PNEM) extended outside the otic capsule surrounding semicircular canal arms. PNEM was mainly connected to the dura, through bone channels similar to those of calvarial bone, and only a few channels were directed toward the canal periosteum. On the contrary, endochondral bone marrow was well connected to the labyrinth through vascular loops (directed to the spiral ligament for CAEM and to the bony labyrinth periosteum for VEM), and to dural sinuses. In addition, CAEM was also connected to the tensor tympani fossa of the middle ear and VEM to the endolymphatic sac. Endochondral marrow was made up of small lobules connected to each other and to other structures by channels lined by elongated macrophages, whereas PNEM displayed larger lobules connected by channels with a sparse macrophage population. Our data suggest that the rat inner ear is surrounded by bone marrow at the junctions with middle ear and brain, most likely with "customs" role, restricting pathogen spread; a second marrow network with different structural features is found within the endochondral bone layer of the otic capsule and may play different functional roles., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Perin, Cossellu, Vivado, Batti, Gantar, Voigt and Pizzala.)

Three-dimensional fluorescence microscopy is a key technology for inspecting biological samples, ranging from single cells to entire organisms. We recently proposed a novel approach called spatially modulated Selective Volume Illumination Microscopy (smSVIM) to suppress illumination artifacts and to reduce the required number of measurements using an LED source. Here, we discuss a new strategy based on smSVIM for imaging large transparent specimens or voluminous chemically cleared tissues. The strategy permits steady mounting of the sample, achieving uniform resolution over a large field of view thanks to the synchronized motion of the illumination lens and the camera rolling shutter. Aided by a tailored deconvolution method for image reconstruction, we demonstrate significant improvement of the resolution at different magnification using samples of varying sizes and spatial features.

The choroid plexus (CP) acts as a regulated gate between blood and cerebrospinal fluid (CSF). Despite its simple histology (a monostratified cuboidal epithelium overlying a vascularized stroma), this organ has remarkably complex functions several of which involve local interaction with cells located around ventricle walls. Our knowledge of CP structural organization is mainly derived from resin casts, which capture the overall features but only allow reconstruction of the vascular pattern surface, unrelated to the overlying epithelium and only loosely related to ventricular location. Recently, CP single cell atlases are starting to emerge, providing insight on local heterogeneities and interactions. So far, however, few studies have described CP spatial organization at the mesoscale level, because of its fragile nature and deep location within the brain. Here, using an iDISCO-based clearing approach and light-sheet microscopy, we have reconstructed the normal rat hindbrain CP (hCP) macro- and microstructure, using markers for epithelium, arteries, microvasculature, and macrophages, and noted its association with 4th ventricle-related neurovascular structures. The hCP is organized in domains associated to a main vessel (fronds) which carry a variable number of villi; the latter are enclosed by epithelium and may be flat (leaf-like) or rolled up to variable extent. Arteries feeding the hCP emerge from the cerebellar surface, and branch into straight arterioles terminating as small capillary anastomotic networks, which run within a single villus and terminate attaching multiple times to a large tortuous capillary (LTC) which ends into a vein. Venous outflow mostly follows arterial pathways, except for the lateral horizontal segment (LHS) and the caudal sagittal segment. The structure of fronds and villi is related to the microvascular pattern at the hCP surface: when LTCs predominate, leaflike villi are more evident and bulge from the surface; different, corkscrew-like villi are observed in association to arterioles reaching close to the CP surface with spiraling capillaries surrounding them. Both leaf-like and corkscrew-like villi may reach the 4th ventricle floor, making contact points at their tip, where no gap is seen between CP epithelium and ependyma. Contacts usually involve several adjacent villi and may harbor epiplexus macrophages. At the junction between medial (MHS) and lateral (LHS) horizontal segment, arterial supply is connected to the temporal bone subarcuate fossa, and venous outflow drains to a ventral vein which exits through the cochlear nuclei at the Luschka foramen. These vascular connections stabilize the hCP overall structure within the 4th ventricle but make MHS-LHS joint particularly fragile and very easily damaged when removing the brain from the skull. Even in damaged samples, however, CP fronds (or isolated villi) often remain strongly attached to the dorsal cochlear nucleus (DCN) surface; in these fronds, contacts are still present and connecting "bridges" may be seen, suggesting the presence of real molecular contacts rather than mere appositions., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Perin, Rossetti, Ricci, Cossellu, Lazzarini, Bethge, Voigt, Helmchen, Batti, Gantar and Pizzala.)

Cannabis has been used for centuries for recreational and therapeutic purposes. Whereas, the recreative uses are based on the psychotropic effect of some of its compounds, its therapeutic effects range over a wide spectrum of actions, most of which target the brain or the immune system. Several studies have found cannabinoid receptors in the auditory system, both at peripheral and central levels, thus raising the interest in cannabinoid signaling in hearing, and especially in tinnitus, which is affected also by anxiety, memory, and attention circuits where cannabinoid effects are well described. Available studies on animal models of tinnitus suggest that cannabinoids are not likely to be helpful in tinnitus treatment and could even be harmful. However, the pharmacology of cannabinoids is very complex, and most studies focused on neural CB1R-based responses. Cannabinoid effects on the immune system (where CB2Rs predominate) are increasingly recognized as essential in understanding nervous system pathological responses, and data on immune cannabinoid targets have emerged in the auditory system as well. In addition, nonclassical cannabinoid targets (such as TRP channels) appear to play an important role in the auditory system as well. This review will focus on neuroimmunological mechanisms for cannabinoid effects and their possible use as protective and therapeutic agents in the ear and auditory system, especially in tinnitus., (Copyright © 2020 Perin, Mabou Tagne, Enrico, Marino, Cosentino, Pizzala and Boselli.)

Light-sheet microscopy is an ideal technique for imaging large cleared samples; however, the community is still lacking instruments capable of producing volumetric images of centimeter-sized cleared samples with near-isotropic resolution within minutes. Here, we introduce the mesoscale selective plane-illumination microscopy initiative, an open-hardware project for building and operating a light-sheet microscope that addresses these challenges and is compatible with any type of cleared or expanded sample ( www.mesospim.org ).

The lower stations of the auditory system display a complex anatomy. The inner ear labyrinth is composed of several interconnecting membranous structures encased in cavities of the temporal bone, and the cerebellopontine angle contains fragile structures such as meningeal folds, the choroid plexus (CP), and highly variable vascular formations. For this reason, most histological studies of the auditory system have either focused on the inner ear or the CNS by physically detaching the temporal bone from the brainstem. However, several studies of neuroimmune interactions have pinpointed the importance of structures such as meninges and CP; in the auditory system, an immune function has also been suggested for inner ear structures such as the endolymphatic duct (ED) and sac. All these structures are thin, fragile, and have complex 3D shapes. In order to study the immune cell populations located on these structures and their relevance to the inner ear and auditory brainstem in health and disease, we obtained a clarified-decalcified preparation of the rat hindbrain still attached to the intact temporal bone. This preparation may be immunolabeled using a clearing protocol (based on iDISCO+) to show location and functional state of immune cells. The observed macrophage distribution suggests the presence of CP-mediated communication pathways between the inner ear and the cochlear nuclei.

The aim of this study was to investigate the ability of the sulfate metabolites of hydroxytyrosol (HT) and tyrosol (TYR) to act as antioxidants counteracting the pro-oxidant effect of oxidized cholesterol in intestinal cells. For this purpose, we synthesized sulfate metabolites of HT and TYR using a chemical methodology and examined their antioxidant activity in Caco-2 monolayers in comparison with the parent compounds. Exposure to oxidized cholesterol led to ROS production, oxidative damage, as indicated by the MDA increase, a decrease of reduced glutathione concentration and an enhancement of glutathione peroxidase activity. All the tested compounds were able to counteract the oxidizing action of oxidized cholesterol; HT and TYR sulfate metabolites showed an efficiency in protecting intestinal cells comparable to that of the parent compounds, strengthening the assumption that the potential beneficial effect of the parent compounds is retained, although extensive metabolisation occurs, the resulting metabolites being able to exert a biological action themselves.

The photobehavior of ciprofloxacin, lomefloxacin and ofloxacin fluoroquinolones was investigated using several in vitro methods to assess their cytotoxic, antiproliferative, and genotoxic potential against two human cancer cell lines. We focused our attention on the possible relationship between their chemical structure, O₂ partial pressure and photobiological activity on cancer cells. The three molecules share the main features of most fluoroquinolones, a fluorine in 6 and a piperazino group in 7, but differ at the key position 8, unsubstituted in ciprofloxacin, a fluorine in lomefloxacin and an alkoxy group in ofloxacin. Studies in solution show that ofloxacin has a low photoreactivity; lomefloxacin reacts via aryl cation, ciprofloxacin reacts but not via the cation. In our experiments, ciprofloxacin and lomefloxacin showed a high and comparable potential for photodamaging cells and DNA. Lomefloxacin appeared the most efficient molecule in hypoxia, acting mainly against tumour cell proliferation and generating DNA plasmid photocleavage. Although our results do not directly provide evidence that a carbocation is involved in photodamage induced by lomefloxacin, our data strongly support this hypothesis. This may lead to new and more efficient anti-tumour drugs involving a cation in their mechanism of action. This latter acting independently of oxygen, can target hypoxic tumour tissue., (Copyright © 2014 Elsevier B.V. All rights reserved.)

In experimental cerebral ischemia, melanocortin MC4 receptor agonists induce neuroprotection and neurogenesis with subsequent long-lasting functional recovery. Here we investigated the molecular mechanisms underlying melanocortin-induced neurogenesis. Gerbils were subjected to transient global cerebral ischemia, then they were treated every 12 h, and until sacrifice, with 5-bromo-2'-deoxyuridine (BrdU; to label proliferating cells), and the melanocortin analog [Nle(4),d-Phe(7)]α-melanocyte-stimulating hormone (NDP-α-MSH) or saline. NDP-α-MSH increased hippocampus dentate gyrus (DG) expression of Wnt-3A, β-catenin, Sonic hedgehog (Shh), Zif268, interleukin-10 (IL-10) and doublecortin (DCX), as detected at days 3, 6 and 10 after the ischemic insult. Further, an elevated number of BrdU immunoreactive cells was found at days 3 and 10, and an improved histological picture with reduced neuronal loss at day 10, associated with learning and memory recovery. Pharmacological blockade of the Wnt-3A/β-catenin and Shh pathways, as well as of melanocortin MC4 receptors, prevented all effects of NDP-α-MSH. These data indicate that, in experimental brain ischemia, treatment with melanocortins acting at MC4 receptors induces neural stem/progenitor cell proliferation in the DG by promptly and effectively triggering the canonical Wnt-3A/β-catenin and Shh signaling pathways. Activation of these pathways is associated with up-regulation of the repair factor Zif268 and the neurogenesis facilitating factor IL-10, and it seems to address mainly toward a neuronal fate, as indicated by the increase in DCX positive cells., (Copyright © 2013 Elsevier B.V. All rights reserved.)

Resveratrol inhibits endothelin-1, a vascular tension regulator. We synthesized the resveratrol analogue 4,4'-dihydroxy-trans-stilbene with 2 hydroxyl groups in the 4 and 4' position to obtain a molecule more active than resveratrol (3,4',5-trihydroxy-trans-stilbene). The results demonstrate that 4,4'-dihydroxy-trans-stilbene led to a significant decrease in total endothelin-1 secretion and in endothelin-1 messenger RNA (mRNA) levels in human endothelial cells. In addition, resveratrol and its analogue decreased endothelin-converting enzyme-1 mRNA levels and further reduced the activity of the enzyme. 4,4'-dihydroxy-trans-stilbene was more active than resveratrol because the new molecule exerted greater activity at the level of endothelin synthesis and conversion, even at a lower concentration. Although 4,4'-dihydroxy-trans-stilbene and resveratrol inhibited formation of reactive oxygen species and lipid peroxidation, the treatment of cells with different oxidant agents did not modify the endothelin-1 release. This finding suggests that the inhibition of endothelin-1 secretion is independent of the antioxidant properties of the 2 compounds. On the basis of these results, the resveratrol analogue 4,4'-dihydroxy-trans-stilbene could be a promising chemopreventive agent against cardiovascular diseases.

Grape waste management is one of the main problems of winery industries, but, conversely, grape waste contains a high amount of polyphenols that might protect against human diseases related to oxidative stress, such as colorectal cancer. Therefore, the aim of this work was to investigate the antioxidant and antiproliferative activities of a grape waste extract obtained by supercritical fluid extraction. Because the beneficial effect of grape is related to its content of polyphenolic molecules, the extract was chemically characterized by high-performance liquid chromatography in order to assess its major bioactive components. The antioxidant activity of the grape extract was determined. The results showed that the grape extract presents a strong antiradical activity in the in vitro 2,2-diphenyl-1-picrylhydrazyl radical assay and protects against reactive oxygen species production in human colon adenocarcinoma cells (Caco-2). In contrast, the extract did not protect in the citronellal thermooxidation system and showed a weak protective action against lipid peroxidation in Caco-2 cells. The clonogenic assay and the cell cycle distribution analysis showed that the grape extract has a significant antiproliferative effect in a tumor cell line. These data indicate that grape extract is a promising product to be used as an anti-free radical agent and could exert a chemopreventive action.

Ischemic stroke is one of the main causes of death and disability. We investigated whether melanocortin peptides, which have protective effects in severe hypoxic conditions, also produce neuroprotection in a gerbil model of ischemic stroke. A 10-min period of global cerebral ischemia, induced by occluding both common carotid arteries, caused impairment in spatial learning and memory that was associated with activation of inflammatory and apoptotic pathways, including severe DNA damage and delayed neuronal death, in the hippocampus. Treatment with nanomolar doses of the melanocortin analog [Nle4, D-Phe7] alpha-MSH [which activates the melanocortin receptor subtypes (MC) mainly expressed in central nervous system, namely MC3 and MC4] modulated the inflammatory and apoptotic cascades and reduced hippocampus injuries even when delayed up to 9 h after ischemia, with consequent dose-dependent improvement in subsequent functional recovery. The selective MC3 receptor agonist gamma2-MSH had no protective effects. Pharmacological blockade of MC4 receptors prevented the neuroprotective effects of [Nle4, D-Phe7] alpha-MSH and worsened some ischemia outcomes. Together, our findings suggest that MC4 receptor-stimulating melanocortins might provide potential to develop a class of drugs with a broad treatment window for a novel approach to neuroprotection in ischemic stroke.

Epidemiological and intervention studies correlate anthocyanin-rich beverages and a low incidence of coronary heart diseases. Since endothelin-1 (ET-1) and nitric oxide (NO) produced by endothelial NO synthase (eNOS) are vascular tension regulators secreted by endothelial cells, we studied the influence of two anthocyanidins, namely cyanidin (CY) and delphinidin (DP), on the regulation of ET-1 and eNOS in cultured human umbilical vein endothelial cells (HUVECs). Aglycon anthocyanidin forms, such as CY and DP, may be present in vivo after the first deglycosylation step occurring in the jejunum and in the liver. DP showed a major action compared to CY inducing a significant dose-dependent inhibitory effect on both protein and mRNA levels of ET-1. CY and DP both increased the protein level of eNOS, but DP showed the major effect raising eNOS protein in a dose-dependent manner. To correlate the vasoprotective effect of CY and DP with their antioxidant activity, we analysed also the antioxidant effect of anthocyanidins both in vitro and in HUVECs. In particular, we examined the effect of anthocyanidins on endothelial heme oxygenase-1 (HO-1), an inducible stress protein. In all tests, DP showed a higher antioxidant activity than CY. Finally, the antiproliferative effect induced by DP was detected in HUVECs. DP and CY differ in the number and position of hydroxyl groups in their structure; therefore, the greater biological activity by DP, compared with CY, seems to be due to the presence of the three hydroxyl groups on the B ring in the molecular structure of DP.

To investigate the mechanistic basis for the biological properties of anthocyanins, two aglycone anthocyanins [delphinidin (DY) and cyanidin (CY)] were used to examine their effects on cell cycle progression and on induction of apoptosis in human cancer cells (uterine carcinoma and colon adenocarcinoma cells) and in normal human fibroblasts. These compounds differ in the number and position of hydroxyl groups on the beta ring in the molecular structure. Cellular uptake of anthocyanins was confirmed by HPLC analysis and no metabolites were detected. The clonogenic assay showed that CY induces a dose-dependent growth inhibitory effect only in fibroblasts. This effect was confirmed by flow cytometric analysis, showing a significant reduction of cells in S phase. In contrast, DP inhibited cell growth in normal and tumour cell lines. This event is accompanied in fibroblasts by an accumulation of cells in the S phase suggesting a block in the transition from S to G2 phase. On the other hand, in tumour cell lines we observed a reduction of cells in G1 phase, paralleled by the appearance of a fraction of cells with a hypodiploid DNA content, thus demonstrating an apoptotic effect by DP. The occurrence of apoptosis induced by DP was confirmed by morphological and biochemical features, including nuclear condensation and fragmentation, annexin V staining, DNA laddering and poly(ADP-ribose) polymerase-1-proteolysis. Furthermore, the mitochondrial membrane potential of apoptotic cells after treatment with DP was significantly lost. The different effects exerted by DP as compared with CY suggest that the presence of the three hydroxyl groups on the beta ring in the molecular structure of DP may be important for its greater biological activity.

We investigated the molecular relationships between lipid peroxidation and mitochondrial DNA (mtDNA) single strand breaks (ssb) in isolated rat hepatocytes and mitochondria exposed to tert-butylhydroperoxide (TBH). Our results show that mtDNA ssb induced by TBH are independent of lipid peroxidation and dependent on the presence of iron and of hydroxyl free radicals. These data contribute to the definition of the mechanisms whereby mtDNA ssb are induced and provide possible molecular targets for the prevention of this kind of damage in vivo.

Anthocyanins are flavonoids present in a variety of pigmented food and, like other flavonoids, seem to play a role in preventing human pathologies related to oxidative stress. In fact, anthocyanins have been shown to exert antiproliferative effects in cell cultures and exhibit antiinflammatory and vasoprotective activities in animal models. Although these biological activities have been related to their antioxidant properties, little is known on the molecular mechanism of action of anthocyanins. The effects of pretreatment with the anthocyanins delphinidin, cyanidin, and their glycoside and rutinoside derivatives against induction of DNA damage induced by tert-butyl-hydroperoxide (TBHP) were evaluated in rat smooth muscle and in rat hepatoma cell lines using alkaline single cell gel electrophoresis (Comet test). In addition, a possible protection exerted by anthocyanins on cell killing, lipid peroxidation, and redox state alterations induced by TBHP was also investigated. It was found that the treatment with TBHP induces the formation of DNA single strand breaks (SSB) and oxidised bases, along with cell killing, lipid peroxidation and redox state alteration. Our data demonstrate that anthocyanins are effective against cytotoxicity, DNA SSB formation and lipid peroxidation induced by TBHP, but they do not have any detectable effect against impairment by TBHP of cellular redox state and on protection against DNA bases oxidation. The presence of a sugar moiety in anthocyanin derivatives reduced this protective effect, mainly in rat hepatoma cells. The different activity of anthocyanins and their derivatives may be explained taking into account a structure/function relationship that could also influence anthocyanin intracellular localisation., (Copyright 2002 Elsevier Science B.V.)

In normal human fibroblasts, beta-carotene induces a cell-cycle delay in the G1 phase independent of its provitamin A activity via a mechanism not yet elucidated. In this study we provide biochemical evidence showing that delayed progression through the G1 phase occurs concomitantly with: an increase in both nuclear-bound and total p21waf1/cip1 protein levels; an increase in the amount of p21waf1/cip1 associated with cdk4; the inhibition of cyclin D1-associated cdk4 kinase activity; and a reduction in the levels of hyperphosphorylated forms of retinoblastoma protein, and particularly, in phosphorylated Ser780. The role of p21waf1/cip1 in the antiproliferative effect of the carotenoid was further supported by genetic evidence that neither changes in cell-cycle progression nor in the phosphorylation status of retinoblastoma protein were observed in p21waf1/cip1-deficient human fibroblasts treated with beta-carotene. These results clearly demonstrate that p21waf1/cip1 is involved directly in the molecular pathway by which beta-carotene inhibits cell-cycle progression.

Familial hypercholesterolemia (FH) is an autosomal dominant genetic disorder characterized by a lifelong elevation in the concentration of low-density lipoprotein (LDL) bound cholesterol in blood by cholesterol deposits and by early coronary artery disease. The LDL apheresis technique has been introduced with the goal of reducing LDL cholesterol levels, thereby preventing the development of atherosclerosis. The literature on LDL apheresis reports 2 different facets, the therapeutic aspect associated with the lessening of LDL concentration and the initiation of a peroxidation process associated with the biocompatibility of the artificial membrane. Lipid and protein peroxidation gives rise to toxic and atherogenic hydroperoxide, mostly lipid hydroperoxides, and derivative compounds, which may offset the benefit of the procedure. In this paper, plasma hydroperoxide levels are determined along with the elevation of the serum and LDL antioxidant status in hypercholesterolemic patients before and following repeated LDL apheresis sessions. Hydroperoxide concentration has been expressed both in terms of plasma volume and LDL concentration. A highly significant increase in LDL lipid hydroperoxides is demonstrated when expressed in terms of LDL concentration and is associated with the LDL apheresis procedure. The usefulness of antioxidant supplementation in LDL apheresis is discussed.

The protective effect of beta-carotene (beta-C) and alpha-tocopherol (alpha-T), singularly and in equimolar mixtures, toward the photomutagenicity induced by 8-methoxypsoralen (8-MOP), at different oxygen partial pressure (pO2), was evaluated in two different experimental models: Salmonella typhimurium TA102 and Saccharomyces cerevisiae D7. After phototreatment with 8-MOP, the results show a lethal effect under hypoxic conditions in both experimental model systems, an increase in revertants associated to the pO2 increase in S. typhimurium TA102, and a decrease in revertants and convertants associated to the pO2 increase in S. cerevisiae D7. In S. typhimurium TA102, in atmospheric condition, beta-C and alpha-T (1.86 or 18.6 microM) show a protective effect only at the higher dosage. Alpha-T was more protective than beta-C. The equimolar mixtures show an antimutagenic effect at both dosage used with a synergistic effect at lower dosage and an additive antimutagenic activity at higher dosage. An inhibition of the spontaneous mutagenicity by mixtures at higher dosage was also observed. The results obtained in S. typhimurium TA102 show an antimutagenic effects of beta-C, alpha-T and their mixture at 190 mmHg pO2, confirming the data obtained in air condition. At 380 mmHg pO2, alpha-T and the mixture show a significant antimutagenic activity; at 570 mmHg pO2, only alpha-T is protective. At 760 mmHg pO2, no protective effect was observed by the two antioxidants, and beta-C increases the photomutagenicity induced by 8-MOP. In S. cerevisiae D7 a protective effect was only observed at 380 mmHg pO2 with the mixture. No antigenotoxic effect was found in the other experimental conditions, even if the uptake of the two antioxidants was confirmed by HPLC. Our results underline the role of oxygen in the photomutagenicity induced by 8-MOP and in the antimutagenic activity of beta-C and alpha-T. This is the first report confirming in a cellular experimental model the data obtained in some chemical systems: the protective effect of beta-C only at low pO2 and the synergistic effect of mixture of beta-C and alpha-T.

The supplementation with beta-carotene and canthaxanthin, two carotenoids with and without pro-vitamin A activity, reduced in vitro the number of micronuclei induced in human cultured lymphocytes by the chemotherapeutic radiomimetic drug bleomycin. The genotoxic activity of this substance is supposed to be mediated by a free radical mechanism. The reduction of micronucleated cells was in correlation with donors' carotenoid blood levels. It has been observed that 20 weeks are needed, following the end of carotenoid supplementation, in order to return to the high levels of micronuclei induced by bleomycin. It is suggested that this protective effect is due to the antioxidant properties of carotenoids acting against free radical-mediated genotoxic damage.

421 strains of Pseudomonas aeruginosa were isolated from patients admitted to intensive care units and tested with automated systems for sensitivity to 21 antimicrobial agents. Data were collected in a database for evaluation and monitoring of resistance development. Results showed that assisted monitoring of antimicrobial resistance gives continuously updated information, with particular attention to the different local therapeutical schedules. It is therefore advisable that clinicians constantly exchange information with the microbiology laboratory through a hospital information system in which data from different laboratories are pooled in real time.

In a one year-study, 9 healthy human donors were being supplemented with beta-carotene (BC) plus canthaxanthin (CX), to determine the effect of carotenoids on chromosomal damage (micronuclei) induced in the donors' lymphocyte cell cultures by exposure to bleomycin (BLM), an antineoplastic drug that has been shown to produce chromosomal aberrations through the production of free radicals. The first four months monitoring data, including determination of carotenoid blood levels, are here reported. These data show that carotenoid supplementation significantly decrease (up to 50%) the formation of micronuclei induced by BLM in human lymphocyte cell cultures. This decrease is in correlation with carotenoid blood levels.