Transcriptomic and cellular decoding of functional brain connectivity changes reveal regional brain vulnerability to pro- and anti-inflammatory therapies.

BACKGROUND: Systemic inflammation induces acute changes in mood, motivation and cognition that closely resemble those observed in depressed individuals. However, the mechanistic pathways linking peripheral inflammation to depression-like psychopathology via intermediate effects on brain function remain incompletely understood. METHODS: We combined data from 30 patients initiating interferon-α treatment for Hepatitis-C and 20 anti-tumour necrosis factor (TNF) therapy for inflammatory arthritis and used resting-state functional magnetic resonance imaging to investigate acute effects of each treatment on regional global brain connectivity (GBC). We leveraged transcriptomic data from the Allen Human Brain Atlas to uncover potential biological and cellular pathways underpinning regional vulnerability to GBC changes induced by each treatment. RESULTS: Interferon-α and anti-TNF therapies both produced differential small-to-medium sized decreases in regional GBC. However, these were observed within distinct brain regions and the regional patterns of GBC changes induced by each treatment did not correlate suggesting independent underlying processes. Further, the spatial distribution of these differential GBC decreases could be captured by multivariate patterns of constitutive regional expression of genes respectively related to: i) neuroinflammation and glial cells; and ii) glutamatergic neurotransmission and neurons. The extent to which each participant expressed patterns of GBC changes aligning with these patterns of transcriptomic vulnerability also correlated with both acute treatment-induced changes in interleukin-6 (IL-6) and, for Interferon-α, longer-term treatment-associated changes in depressive symptoms. CONCLUSIONS: Together, we present two transcriptomic models separately linking regional vulnerability to the acute effects of interferon-α and anti-TNF treatments on brain function to glial neuroinflammation and glutamatergic neurotransmission. These findings generate hypotheses about two potential brain mechanisms through which bidirectional changes in peripheral inflammation may contribute to the development/resolution of psychopathology.

Effective prevention of post-dural puncture headache with insertion of an intrathecal catheter in parturients: a retrospective study and meta-analysis.

BACKGROUND: Accidental dural puncture is a common complication of labour analgesia. It can trigger post-dural puncture headache, with associated morbidity and increased costs. Intrathecal catheter placement is a prophylactic procedure which can reduce incidence and severity of post-dural puncture headache. METHODS: We conducted a retrospective single-centred study to define incidence and risk factors of accidental dural puncture and post-dural puncture headache in an obstetric population. We also evaluated effectiveness of intrathecal catheter placement compared to epidural catheter replacement in reducing incidence of post-dural puncture headache. We then conducted a systematic review and meta-analysis which included all studies comparing intrathecal catheter placement to epidural catheter replacement in obstetric patients with accidental dural puncture assessing the outcome of reduced incidence of post-dural puncture headache as a dichotomous variable. RESULTS: Accidental dural puncture had an incidence of 0.25% (60 cases). Of these, 66% developed post-dural puncture headache. A total of 77% (47/60) of patients with accidental dural puncture were treated with an intrathecal catheter placement, while 23% (13/60) had an epidural catheter replacement. Incidence of post-dural puncture headache was lower in the intrathecal catheter group (spinal 26/47, 60.5% epidural 11/13, 84.6%), although not reaching statistical significance (RR 0.71, CI 95%: 0.51-1.00; p = 0.049). The meta-analysis revealed that intrathecal catheter placement significantly reduced incidence of post-dural puncture headache compared to epidural catheter replacement (pooled RR 0.81, 95% CI 0.72-0.91, p < 0.001). CONCLUSIONS: Intrathecal catheter placement is a promising measure to prevent post-dural puncture headache, especially if followed by a pain management protocol and a continuous saline infusion.

Modulation of the central memory and Tr1-like regulatory T cells in multiple sclerosis patients responsive to interferon-beta therapy.

BACKGROUND: Interferon-beta is used to reduce disease activity in multiple sclerosis, but its action is incompletely understood, individual treatment response varies among patients, and biological markers predicting clinical benefits have yet to be identified. Since it is known that multiple sclerosis patients have a deficit of the regulatory T-cell subsets, we investigated whether interferon-beta therapy induced modifications of the two main categories of regulatory T cells (Tregs), natural and IL-10-secreting inducible Tr1 subset, in patients who are biologically responsive to the therapy. METHODS: T-cell phenotype was determined by flow cytometry, while real-time PCR was used to evaluate interferon-beta bioactivity through MxA determination, and to measure the RNA for IL-10 and CD46 molecule in peripheral blood mononuclear cells stimulated with anti-CD46 and anti-CD3 monoclonal antibodies, which are known to expand a Tr1-like population. RESULTS: Interferon-beta induced a redistribution of natural Treg subsets with a shift of naive Tregs towards the 'central memory-like' Treg population that expresses the CCR7 molecule required for the in vivo suppressive activity. Furthermore, in a subgroup of treated patients, the CD46/CD3 co-stimulation, probably through the Tr1-like subset modulation, increased the production of RNA for IL-10 and CD46. The same group showed a lower median EDSS score after two years of therapy. CONCLUSIONS: The selective increase of 'central memory-like' subset and the involvement of the Tr1-like population may be two of the mechanisms by which interferon-beta achieves its beneficial effects. The quantification of RNA for IL-10 and CD46 could be used to identify patients with a different response to interferon-beta therapy.

Effects of a Cereal Bar with a Combination of Phytoestrogens on the Climacteric Symptoms: A Placebo-Controlled, Randomized Trial.

OBJECTIVE: This study evaluated whether the consumption of a cereal bar combining different phytoestrogens could contribute to the reduction of climacteric symptoms in women. METHODS: This is a clinical, prospective, randomized, simple-blind trial. Forty-eight women, aged 40-65 years, with climacteric symptoms, from a city in southwestern Paraná, Brazil. Participants were randomly assigned into two groups; Phytoestrogens group (PHY = 24), which received for 90-day period a cereal bar containing 80.73 milligrams of soybean and flaxseed phytoestrogens, and the placebo group (PLA = 24), which consumed rice flakes biscuit. Clinical, sociodemographic and anthropometric data were collected and climacteric symptoms were assessed using the Kupperman Index (KI). RESULTS: Forty-three women were analyzed (PHY = 21 and PLA = 22). There were significant reductions in the overall KI score in both groups at the end of the intervention period (p < 0.05). However, the comparison between the groups using linear regression models presented expressively better symptom improvement in the PHY group -6.43 over time (95% CI: -11.6; -1.26; p < 0.05) KI points, with perimenopausal -15.15 (95% CI: -28.95; -1.35) and postmenopausal women -19.34 (95% CI: -33.68; -4.99) showed considerably greater reductions in symptoms at the end of the intervention period compared to premenopausal women. There was also significant reduction in symptoms of hot flushes, paresthesia, sexual complaints, insomnia and melancholy. CONCLUSION: The consumption of a cereal bar containing phytoestrogens was able to improve the symptoms of climacteric syndrome.

Intrapartum sonography for occiput posterior detection in early low dose combined spinal epidural analgesia by sufentanil and ropivacaine.

AIM: To evaluate the incidence of occiput posterior position in labour with and without combined spinal epidural analgesia (CSE) by low dose of sufentanyl and ropivacaine. MATERIAL AND METHODS: This study focused on 132 women subdivided in two groups, patients in spontaneous and in labour analgesia, administered by a low dose CSE by sufentanyl and ropivacaine; all women were evaluated by digital examinations and ultrasound till delivery. All data were collected and analyzed by an independent reviewer. RESULTS: In the second stage, 79 were persistent occiput posterior position (POPP) fetuses and 36 were translated from anterior to posterior position (TAPP) fetuses. Specifically, in spontaneous labour on 25 women in anterior position, there were 17 TAPP and in CSE analgesia on 28 women in anterior, there were 19 in TAPP, without significant differences. The number of asynclitisms was higher in the POPP group (84%) respect to the TAPP group (75%), so as the rate of caesarean section (67% versus 52.7%). CONCLUSIONS: The labour with low dose of ropivacaine and sufentanyl does not increase the occiput posterior position during fetal descent, leading to a POPP. Finally, since in the occiput anterior presentation labour analgesia significantly lengthens time to delivery, in the occiput posterior position this is significantly increased, with a prolonged second stage of labour and reduced time of descent of fetal head in obstetric pelvis.

The daily-practiced post-partum hemorrhage management: an Italian multidisciplinary attended protocol.

Postpartum hemorrhage (PPH) is one of the most frequent causes of mortality and morbidity in the obstetric population globally, causing about a quarter of maternal deaths yearly, and is the leading cause of maternal death worldwide. The management of PPH remains a topic of great debate, even in view of new diagnostic and therapeutic possibilities in recent years, for which, however, the body of evidence available thus far is still scarce, as the standard values are lacking. The protocol hereby presented was developed after a literature review and during several meetings of an Italian multidisciplinary task group of specialists adopting a modified Delphi method, and is the result of the synthesis of therapeutic operational protocols for the treatment of PPH applied by the different specialties within the team. This protocol is intended to represent a practical proposal to support clinicians in the management of a particularly complex event that requires the intervention of a multidisciplinary team and the implementation of dedicated management protocols.

Limited proteolysis of bovine alpha-lactalbumin: isolation and characterization of protein domains.

The partly folded states of alpha-lactalbumin (alpha-LA) exposed to acid solution at pH 2.0 (A-state) or at neutral pH upon EDTA-mediated removal of the single protein-bound calcium ion (apo form) have been probed by limited proteolysis experiments. These states are nowadays commonly considered to be molten globules and thus protein-folding intermediates. Pepsin was used for proteolysis at acid pH, while proteinase K and chymotrypsin at neutral pH. The expectations were that these proteolytic probes would detect sites and/or chain regions in the partly folded states of alpha-LA sufficiently dynamic, or even unfolded, capable of binding and adaptation to the specific stereochemistry of the protease's active site. A time-course analysis of the proteolytic events revealed that the fast, initial proteolytic cuts of the 123-residue chain of alpha-LA in its A-state or apo form by the three proteases occur at the same chain region 39-54, the actual site(s) of cleavage depending upon the protease employed. This region in native alpha-LA encompasses the beta-sheets of the protein. Subsequent cleavages occur mostly at chain regions 31-35 and 95-105. Four fragment species of alpha-LA have been isolated by reverse-phase high-performance liquid chromatography, and their conformational properties examined by circular dichroism and fluorescence emission spectroscopy. The single chain fragment 53-103, containing all the binding sites for calcium in native alpha-LA and cross-linked by two disulfide bridges, maintains in aqueous buffer and in the presence of calcium ions a folded structure characterized by the same content of alpha-helix of the corresponding chain segment in native alpha-LA. Evidence for some structure was also obtained for the two-chain species 1-40 and 104-123, as well as 1-31 and 105-123, both systems being covalently linked by two disulfide bonds. In contrast, the protein species given by fragment 1-34 connected to fragment 54-123 or 57-123 via four disulfide bridges adopts in solution a folded structure with the helical content expected for a native-like conformation. Of interest, the proteolytic fragment species herewith isolated correspond to the structural domains and subdomains of alpha-LA that can be identified by computational analysis of the three-dimensional structure of native alpha-LA (Siddiqui AS, Barton GI, 1995, Protein Sci 4:872-884). The fast, initial cleavages at the level of the beta-sheet region of native alpha-LA indicate that this region is highly mobile or even unfolded in the alpha-LA molten globule(s), while the rest of the protein chain maintains sufficient structure and rigidity to prevent extensive proteolysis. The subsequent cleavages at chain segment 95-105 indicate that also this region is somewhat mobile in the A-state or apo form of the protein. It is concluded that the overall domain topology of native alpha-LA is maintained in acid or at neutral pH upon calcium depletion. Moreover, the molecular properties of the partly folded states of alpha-LA deduced here from proteolysis experiments do correlate with those derived from previous NMR and other physicochemical measurements.

Enhanced Protein Thermostability by Ala --> Aib Replacement

The introduction into peptide chains of alpha-aminoisobutyric acid (Aib) has proven to stabilize the helical structure in short peptides by restricting the available range of polypeptide backbone conformations. In order to evaluate the potential stabilizing effect of Aib at the protein level, we have studied the conformational and stability properties of Aib-containing analogs of the carboxy-terminal subdomain 255-316 of thermolysin. Previous NMR studies have shown that this disulfide-free 62-residue fragment forms a dimer in solution and that the global 3D structure of each monomer (3 alpha-helices encompassing residues 260-274, 281-295, and 301-311) is largely coincident with that of the corresponding region in the X-ray structure of intact thermolysin. The Aib analogs of fragment 255-316 were prepared by a semisynthetic approach in which the natural fragment 255-316 was coupled to synthetic analogs of peptide 303-316 using V8-protease in 50% (v/v) aqueous glycerol [De Filippis, V., and Fontana, A. (1990) Int. J. Pept. Protein Res. 35, 219-227]. The Ala residue in position 304, 309, or 312 of fragment 255-316 was replaced by Aib, leading to the singly substituted fragments Ala304Aib, Ala309Aib, and Ala312Aib. Moreover, fragment Ala304Aib/Ala309Aib with a double Ala --> Aib exchange in positions 304 and 309 was produced. Far- and near-UV circular dichroism measurements demonstrated that both secondary and tertiary structures of the natural fragment 255-316 are fully retained upon Ala --> Aib substitution(s). Thermal unfolding measurements, carried out by recording the ellipticity at 222 nm upon heating, showed that the melting temperatures (Tm) of analogs Ala304Aib and Ala309Aib were 2.2 and 5.4 °C higher than that of the Ala-containing natural species (Tm = 63.5 °C), respectively, whereas the Tm of the Ala312Aib analog was lowered by -0.6 °C. The enhanced stability of the Ala304Aib analog can be quantitatively explained on the basis of a reduced backbone entropy of unfolding due to the restriction of the conformational space allowed to Aib in respect to Ala, while the larger stabilization observed for the Ala309Aib analog can be accounted for by both entropic and hydrophobic effects. In fact, whereas Ala304 is a surface residue, Ala309 is shielded from the solvent, and thus the enhanced stability of fragment Ala309Aib is also due to the burial of an additional -CH3 group with respect to the natural fragment. The slightly destabilizing effect of the Ala --> Aib exchange in position 312 appears to derive from unfavorable strain energy effects, since phi and psi values for Ala312 are out of the allowed angles for Aib. Of interest, the simultaneous incorporation of Aib at positions 304 and 309 leads to a significant and additive increase of +8 °C in Tm. The results of this study indicate that the rational incorporation of Aib into a polypeptide chain can be a general procedure to significantly stabilize proteins.

Increased cytokine release from peripheral blood cells after acute stroke.

Cytokines interleukin (IL)-6 and tumor necrosis factor (TNF)-alpha can play pathogenetic or protective roles in stroke. They are increased in the brain after experimental ischemia and in the CSF of patients with stroke. However, their presence in the periphery is still controversial. To determine the source and time-course of cytokines in blood of stroke patients, IL-6 and TNF-alpha release from blood cells and serum levels were determined in 40 patients on days 1 through 2, 4, 10, 30, and 90 after stroke. Twenty healthy age-matched volunteers were used as controls. IL-6 and TNF-alpha release from stimulated blood cells was increased in stroke patients, compared to controls. A peak response (+224%) was observed at day 4 for IL-6, while TNF-alpha release was largely and significantly increased (about three-fold compared to controls) from day 1 to 2 until day 90 after stroke. The increase in IL-6 release was significantly higher in ischemic, compared to hemorrhagic strokes, at days 1 and 4. Circulating IL-6 was increased at each time point. The ischemic processes in the CNS induces a long-lasting activation of IL-6 and TNF-alpha production in peripheral blood cells, which are a major source of serum cytokines after stroke.

Distribution of a putative endogenous modulator of the GABAergic system in human brain.

Diazepam binding inhibitor (DBI) is a novel neuropeptide purified from rat, cow, and human brain that allosterically modulates GABAergic transmission by binding to benzodiazepine (BDZ)-recognition sites. Using a specific radioimmunoassay for human DBI, we investigated the distribution of this peptide in different brain areas. We characterized with high-pressure liquid chromatography the DBI immunoreactivity in brain tissue obtained by biopsy and autopsy; we detected one molecular species of DBI in both instances. The regional distribution of DBI in the human brain is similar to that observed in rat brain: high concentrations in cortical and limbic areas, cerebellum, and brainstem, and low concentrations in the basal ganglia. These data suggest a modulatory role for DBI in human brain.

Cerebrospinal fluid levels of diazepam-binding inhibitor in neurodegenerative disorders with dementia.

We investigated CSF levels of diazepam-binding inhibitor (DBI), a recently discovered neuropeptide that allosterically modulates GABAergic transmission, in various neurodegenerative disorders with dementia (28 patients with Parkinson's disease, 10 with Alzheimer's disease, 7 with Huntington's chorea). We applied a battery of neuropsychological tests to determine the degree of dementia and to exclude the presence of mood alterations. CSF DBI levels were elevated in parkinsonian subjects with dementia and in patients with Alzheimer's disease, but decreased in Huntington's chorea patients. We hypothesize that modifications of CSF DBI levels may be related to a functional or structural alteration of the GABAergic system.

Creutzfeldt-Jakob disease with a novel four extra-repeat insertional mutation in the PrP gene.

OBJECTIVE: To investigate the role of a short insertional mutation in the prion protein (PrP) gene (PRNP) in prion disease pathogenesis. BACKGROUND: The genetic forms of Creutzfeldt-Jakob disease (CJD) are associated with point or insertional mutations in PRNP. Whereas patients with five, six, seven, eight, and nine extra octapeptide repeats show an autosomal dominant pattern of inheritance and features of CJD, Gerstmann-Sträussler-Scheinker disease, or atypical dementia, patients with one, two, or four extra repeats have typical CJD and lack a family history of neurologic disorder. METHODS: A genetic, neuropathologic, and biochemical study was carried out in a 65-year-old patient with clinical features of sporadic CJD. RESULTS: A novel four extra-repeat insertional mutation of PRNP was found in the patient and in his 59-year-old healthy sister. The patient showed spongiosis, nerve cell loss, and gliosis associated with diffuse PrP immunoreactivity in the cerebral cortex, subcortical gray structures, and cerebellum. A peculiar aspect was the presence of focal PrP deposits in the basal ganglia and hypothalamus, superimposed to diffuse PrP immunoreactivity. The biochemical analysis revealed that both mutant and wild-type PrP participated in the pathologic process, and that the protease-resistant core of the altered PrP isoforms was distinct from that observed in sporadic, acquired, and other genetic forms of CJD. CONCLUSION: These findings support the view that the four extra-repeat insertion in PRNP is a pathogenic mutation with low penetrance rather than a benign polymorphism, and suggest that this mutation results in the formation of a distinct PrP conformer.

Characterization of peripheral benzodiazepine receptors in human blood mononuclear cells.

In the present study, peripheral-type benzodiazepine receptors in human circulating mononuclear cells were characterized, using [3H]PK 11195 as specific ligand. The specific binding was saturable, with a Bmax of 14 pmol/mg protein and a Kd of 7 nM. The pharmacological characterization, using different displacing drugs, indicated a mitochondrial type of peripheral benzodiazepine receptor since it was not coupled to the GABA receptor and was displaced by protoporphyrin IX. These data indicate that human circulating mononuclear cells possess benzodiazepine recognition sites, similar to non-neuronal receptors. The role of these receptors and possible modifications in different diseases need to be investigated.

Diazepam binding inhibitor (DBI) increases after acute stress in rat.

Diazepam binding inhibitor (DBI) acts in brain by binding to GABAA/benzodiazepine receptors (GBR) and to mitochondrial benzodiazepine receptors (MBR). Because DBI acting at MBR, has been shown to be an effector of ACTH-induced steroidogenesis and stress is known to change the level of GBR and MBR, the model of acute noise stress in rats was used to study modifications of DBI and GRB or the content of MBR in various areas of the brain and adrenal gland. It was found that, in the brain of stressed rats, DBI and its processing products (ODN-like immunoreactivity), increased selectively in the hippocampus. This increase in the content of DBI was preceded and followed by a net decrease of GBR and an increase of MBR. Similarly, in adrenal cortex, the content of DBI and MBR increased during the first hour, following acute stress and this increase paralleled the increase in plasma corticosterone. These data suggest that DBI, acting on MBR may regulate steroidogenic function in stress.

Pixantrone (BBR2778) reduces the severity of experimental allergic encephalomyelitis.

Pixantrone is less cardiotoxic and is similarly effective to mitoxantrone (MTX) as an antineoplastic drug. In our study, pixantrone reduced the severity of acute and decreased the relapse rate of chronic relapsing experimental allergic encephalomyelitis (EAE) in rats. A marked and long-lasting decrease in CD3+, CD4+, CD8+ and CD45RA+ blood cells and reduced anti-MBP titers were observed with both pixantrone and MTX. In vitro mitogen- and antigen-induced T-cell proliferation tests of human and rodents cells evidenced that pixantrone was effective at concentrations which can be effectively obtained after i.v. administration in humans. Cardiotoxicity was present only in MTX-treated rats. The effectiveness and the favorable safety profile makes pixantrone a most promising immunosuppressant agent for clinical use in multiple sclerosis (MS).

Benzodiazepine receptors and diazepam binding inhibitor: a possible link between stress, anxiety and the immune system.

This review summarizes the evidence available on the involvement in stress of different classes of benzodiazepine receptors and their putative endogenous ligand, diazepam binding inhibitor (DBI), with particular reference to their role in modifications of the immune response. The presented data from in vitro, experimental, and clinical studies suggest that benzodiazepine receptors and DBI play a major role in regulating steroid production in both the adrenals and central nervous system, and may be involved in the activation of the hypothalamic-pituitary-adrenal axis in stress response.

Acute noise stress in rats increases the levels of diazepam binding inhibitor (DBI) in hippocampus and adrenal gland.

We investigated the effect of acute noise-induced stress on the concentrations of diazepam binding inhibitor (DBI) and its processing products in brain regions and adrenal glands of rats. DBI levels in hippocampus began to increase at 15 and 30 min and became significantly higher (+100%) at 90 and 120 min after stress; they returned to normal values at 360 min. While basal DBI levels were similar in the left and right hippocampus, the stress-induced increase of DBI levels was significantly higher in the left compared to the right side. A significant increase was also detected in the adrenals; here, the time course of DBI increase paralleled that of previously reported plasma corticosterone in stressed rats, being significantly higher 30 min after stress, and recovering to normal values at 60 and 90 min. After acute noise-induced stress, no significant change of DBI levels was detectable in cerebral cortex, striatum, hypothalamus and cerebellum. The present study reports for the first time the occurrence of a modification of DBI and its processing products (ODN-like immunoreactivity) in an experimental model of stress, and suggests a role for these neuropeptides in emotional responses.

Diazepam binding inhibitor and total cholesterol plasma levels in cirrhosis and hepatocellular carcinoma.

Cholesterol is used by cells for biosynthetic processes and for steroid synthesis. Although the role of cholesterol in tumorigenesis is not clear it is known that steroids are important factors in human carcinogenesis. A polypeptide, diazepam binding inhibitor (DBI), which is an endogenous ligand for peripheral benzodiazepine receptors enhances steroidigenesis by promoting cholesterol delivery to the inner mitochondrial membrane which represents the rate-limiting step of steroid biosynthesis. We have assayed the total cholesterol (TC) and the DBI plasma concentrations in patients with liver cirrhosis complicated by hepatocellular carcinoma (HCC) in comparison with those of uncomplicated liver cirrhosis. TC and DBI levels have been studied in 73 cirrhotic patients and in 23 patients with HCC. Both TC and DBI levels were higher in HCC patients when compared with age, sex and Child-Pugh class matched cirrhotic controls. The values (mean+/-S.D.) in patients in Child-Pugh class B and C with and without HCC were respectively 128+/-30 mg/dl vs. 106+/-27 mg/dl (P < 0.01) and 2.05+/-0.78 pmol/ml vs. 0.78+/-0.84 pmol/ml (P < 0.0001). The data may be the result of the metabolic influence of tumors that enhances steroid biosynthesis during tumor proliferation.

Up-regulation of peripheral benzodiazepine receptor system in hepatocellular carcinoma.

Increased number of peripheral benzodiazepine receptors (PBRs) have been found in some tumors outside the liver. The present study was to verify whether the PBR system is altered in hepatocellular carcinoma (HCC). The levels of endogenous benzodiazepine-like compounds (BZDs), measured by radioreceptor binding technique after HPLC purification and the endogenous ligand for PBRs, termed diazepam binding inhibitor (DBI), measured by radioimmunoassay utilizing a specific antibody for human DBI, were studied in the blood of 15 normal subjects, 12 liver cirrhosis and 10 patients with HCC. The levels of BZDs in serum were increased hundred fold in liver cirrhosis patients and slightly elevated in HCC patients. DBI was found to be increased in HCC patients. The binding recognition sites for PBRs (Bmax) were increased 4 to 7 fold in HCC tissue in comparison with that found in non-tumoral liver tissue (NTLT). On the contrary the concentrations of DBI were found to be significantly decreased in HCC tissue in comparison with the respective NTLT. These results seem to suggest an implication of PBRs and of their putative endogenous ligands in the metabolism of these neoplastic cells and possibly in their proliferation. The up-regulation of PBRs found in HCC tissue seems to indicate an increased functional activity of these receptors and opens up the possibility of new pharmacological and diagnostic approaches while the changes in the circulating endogenous ligands for the above receptors might be envisaged as early markers of tumorigenesis in liver cirrhosis.

Glutamate and multiple sclerosis.

Multiple sclerosis (MS) has been considered for a long time a typical inflammatory demyelinating disease of the central nervous system due to autoimmunity targeting oligodendrocytes with sparing of axons until advanced stages of the disease. For this reason, most of the earliest experimental studies focused on the role of cytokines and chemokines at the site of oligodendrocytes loss and on the importance in MS pathogenesis of classical inflammatory mechanisms. As a result, several attempts to treat MS through reduction of the local inflammatory milieau have been performed, leading to the current "immunomodulatory" treatment of the disease. However, more recently the importance of axonal loss and neurodegeneration even in the earliest stages of MS has been also recognized, and additional or concomitant players have been therefore searched. Evidence is now increasing that excessive glutamate is released at the site of demyelination and axonal degeneration in MS plaques, and the most probable candidates for this cellular release are infiltrating leukocytes and activated microglia. These observations are no longer simply preclinical results obtained in the MS animal model, i.e. experimental allergic encephalomyelitis, but have already been partially confirmed by post-mortem studies and in vivo analysis in MS patients, thus raising the possibility that modulation of glutamate release and transport as well as receptors blockade might be relevant targets for the development of future therapeutic interventions.