Pierluigi Navarra

The mTOR kinase inhibitor rapamycin decreases iNOS mRNA stability in astrocytes

BackgroundReactive astrocytes are capable of producing a variety of pro-inflammatory mediators and potentially neurotoxic compounds, including nitric oxide (NO). High amounts of NO are synthesized following up-regulation of inducible NO synthase (iNOS). The expression of iNOS is tightly regulated by complex molecular mechanisms, involving both transcriptional and post-transcriptional processes. The mammalian target of rapamycin (mTOR) kinase modulates the activity of some proteins directly involved in post-transcriptional processes of mRNA degradation. mTOR is a serine-threonine kinase that plays an evolutionarily conserved role in the regulation of cell growth, proliferation, survival, and metabolism. It is also a key regulator of intracellular processes in glial cells. However, with respect to iNOS expression, both stimulatory and inhibitory actions involving the mTOR pathway have been described. In this study the effects of mTOR inhibition on iNOS regulation were evaluated in astrocytes.MethodsPrimary cultures of rat cortical astrocytes were activated with different proinflammatory stimuli, namely a mixture of cytokines (TNFα, IFNγ, and IL-1β) or by LPS plus IFNγ. Rapamycin was used at nM concentrations to block mTOR activity and under these conditions we measured its effects on the iNOS promoter, mRNA and protein levels. Functional experiments to evaluate iNOS activity were also included.ResultsIn this experimental paradigm mTOR activation did not significantly affect astrocyte iNOS activity, but mTOR pathway was involved in the regulation of iNOS expression. Rapamycin did not display any significant effects under basal conditions, on either iNOS activity or its expression. However, the drug significantly increased iNOS mRNA levels after 4 h incubation in presence of pro-inflammatory stimuli. This stimulatory effect was transient, since no differences in either iNOS mRNA or protein levels were detected after 24 h. Interestingly, reduced levels of iNOS mRNA were detected after 48 hours, suggesting that rapamycin can modify iNOS mRNA stability. In this regard, we found that rapamycin significantly reduced the half-life of iNOS mRNA, from 4 h to 50 min when cells were co-incubated with cytokine mixture and 10 nM rapamycin. Similarly, rapamycin induced a significant up-regulation of tristetraprolin (TTP), a protein involved in the regulation of iNOS mRNA stability.ConclusionThe present findings show that mTOR controls the rate of iNOS mRNA degradation in astrocytes. Together with the marked anti-inflammatory effects that we previously observed in microglial cells, these data suggest possible beneficial effects of mTOR inhibitors in the treatment of inflammatory-based CNS pathologies.

Erythropoietin exerts anti‐apoptotic effects on rat microglial cells in vitro

Erythropoietin (EPO), a renal cytokine regulating haematopoiesis, is also produced by different cell types within the central nervous system, where it acts via the activation of specific receptors. Current evidence shows that EPO exerts neurotrophic and neuroprotective activities in different in vivo and in vitro models of brain damage. In the present study we investigated the effects of EPO on primary cultures of rat cortical microglia and astrocytes. We found that: (i) EPO exerted a marked stimulatory effect on microglial cell viability, assessed through the MTS assay, whereas astrocytes were almost unaffected; (ii) the cytokine increased microglial cell population size in a concentration‐dependent manner; however, as microglia cultures undergo spontaneous apoptosis after separation from astrocytes, the apparent effect on cell proliferation could be attributed to EPO antagonism of normal apoptosis; (iii) subsequent flow cytometry analysis on microglial cells demonstrated both the trophic role of factor(s) released by astrocytes in mixed cultures, and the putative anti‐apoptotic action of EPO; (iv) the latter was further confirmed through the assessment of gene expression of anti‐ and pro‐apoptotic factors, which showed that EPO is able to shift the Bcl : Bax ratio towards a net anti‐apoptotic effect; (v) EPO did not affect the pro‐inflammatory function of microglial cells.

Involvement of mTOR kinase in cytokine-dependent microglial activation and cell proliferation.

Neuroinflammation plays a prominent role in the pathophysiology of several neurodegenerative disorders, including Multiple Sclerosis. Reactive microglial cells are always found in areas of active demyelination as well as in normal-appearing white matter. Microglia contribute to initiating and maintaining brain inflammation, and once activated release pro-inflammatory mediators potentially cytotoxic, like nitric oxide (NO). It is now evident that the mTOR signaling pathway regulates different functions in the innate immune system, contributing to macrophage activation. More recently, mTOR has been found to enhance the survival of EOC2 microglia during oxygen-glucose deprivation and increase NO synthase 2 (NOS2) expression during hypoxia in BV2 microglial cell line, thus suggesting an involvement in microglial pro-inflammatory activation. In the present study, we detected mTOR activation in response to two different stimuli, namely LPS and a mixture of cytokines, in primary cultures of rat cortical microglia. Moreover, mTOR inhibitors reduced NOS activity and NOS2 expression induced by cytokines, but not those induced by LPS. The mTOR inhibitor RAD001, in combination with cytokines, also reduced microglial proliferation and the intracellular levels of cyclooxygenase. Under basal conditions mTOR inhibition significantly reduced microglial viability. Interestingly, mTOR inhibitors did not display any relevant effect on astrocyte NOS2 activity or cell viability. In conclusion, mTOR selectively controls microglial activation in response to pro-inflammatory cytokines and appears to play a crucial role in microglial viability; thus these drugs may be a useful pharmacological tool to reduce neuroinflammation.

Interleukin-1β and Interleukin-6 Specifically Increase the Release of Prostaglandin E2 from Rat Hypothalamic Explants in vitro

It has previously been shown that the cytokines interleukin-1 beta and interleukin-6 (IL-1 beta and IL-6) stimulate directly the release of corticotrophin-releasing-hormone-41 from the rat hypothalamus in vitro, while IL-1 beta can also stimulate the release of somatostatin. These effects can be antagonized by drugs which block prostaglandin (PG) synthesis. PGs are also involved in the control of hypothalamic neuropeptides by other neurotransmitters. In the present study, we have characterized the production of PGs from the rat hypothalamus in vitro, and investigated the effects of IL-1 beta and IL-6, as well as the neurotransmitters norepinephrine, acetylcholine and 5-hydroxytryptamine, on the acute release of PGs, using a well-validated acute hypothalamic incubation system. The rate of release of PGs [PGE2, PGF2 alpha, 6-keto-PGF1 alpha (6KPGF1 alpha) and thromboxane B2 (TXB2) in the medium was found to stabilize after 60 min of preincubation and thereafter remain constant, with TXB2 being the predominant species. Twenty-minute incubation in the presence of human recombinant IL-1 beta or IL-6, in the dose range 1-100 U/ml, had no effect on the release of PGF2 alpha, 6KPGF1 alpha or TXB2; however, the release of PGE2 was significantly increased by both IL-1 beta and IL-6. The effect of IL-1 beta was antagonized by both indomethacin and dexamethasone. None of the other neurotransmitters tested had any effect on the release of any of the PGs.(ABSTRACT TRUNCATED AT 250 WORDS)

The role of small intestinal bacterial overgrowth in Parkinson's disease

Parkinsons disease is associated with gastrointestinal motility abnormalities favoring the occurrence of local infections. The aim of this study was to investigate whether small intestinal bacterial overgrowth contributes to the pathophysiology of motor fluctuations. Thirty‐three patients and 30 controls underwent glucose, lactulose, and urea breath tests to detect small intestinal bacterial overgrowth and Helicobacter pylori infection. Patients also underwent ultrasonography to evaluate gastric emptying. The clinical status and plasma concentration of levodopa were assessed after an acute drug challenge with a standard dose of levodopa, and motor complications were assessed by Unified Parkinsons Disease Rating Scale–IV and by 1‐week diaries of motor conditions. Patients with small intestinal bacterial overgrowth were treated with rifaximin and were clinically and instrumentally reevaluated 1 and 6 months later. The prevalence of small intestinal bacterial overgrowth was significantly higher in patients than in controls (54.5% vs. 20.0%; P = .01), whereas the prevalence of Helicobacter pylori infection was not (33.3% vs. 26.7%). Compared with patients without any infection, the prevalence of unpredictable fluctuations was significantly higher in patients with both infections (8.3% vs. 87.5%; P = .008). Gastric half‐emptying time was significantly longer in patients than in healthy controls but did not differ in patients based on their infective status. Compared with patients without isolated small intestinal bacterial overgrowth, patients with isolated small intestinal bacterial overgrowth had longer off time daily and more episodes of delayed‐on and no‐on. The eradication of small intestinal bacterial overgrowth resulted in improvement in motor fluctuations without affecting the pharmacokinetics of levodopa. The relapse rate of small intestinal bacterial overgrowth at 6 months was 43%.

Ipilimumab: A novel immunostimulatory monoclonal antibody for the treatment of cancer

Ipilimumab (Yervoy, developed by Medarex and Bristol-Myers Squibb) is a fully human monoclonal IgG1κ antibody against the cytotoxic T-lymphocyte antigen-4 (CTLA-4), an immune-inhibitory molecule expressed in activated T cells and in suppressor T regulatory cells. Interaction of the monoclonal antibody with CTLA-4 blocks inhibitory signals generated through this receptor and enhances T cell activation, leading to increased antitumor responses. Ipilimumab has been approved by FDA in March 2011 as monotherapy (3mg/kg every 3 weeks for 4 doses) for the treatment of advanced (unresectable or metastatic) melanoma both in pre-treated or chemotherapy naïve patients. Four months later, ipilimumab has received a rapid approval by the European Commission, after a positive opinion from the Committee for Medicinal Products for Human Use. However, the indication in the EU is limited to previously-treated patients with advanced melanoma. Ipilimumab is the first agent that has demonstrated to improve overall survival in patients with metastatic melanoma, which has a very poor prognosis, in randomized phase III clinical trials. The patterns of tumour response to ipilimumab differ from those observed with cytotoxic chemotherapeutic agents, since patients may have a delayed yet durable response and obtain long-term survival benefit despite an initial tumour growth. The major draw-back of ipilimumab is the induction of immune-related adverse effects; the latter can be life-threatening, unless promptly managed with immunosuppressive agents (most frequently corticosteroids) according to specific guidelines. Further development of ipilimumab includes its use in the neoadjuvant or adjuvant high-risk melanoma setting and for the treatment of other refractory and advanced solid tumours, either as single agent or in combination with additional immunostimulating agents or molecularly targeted therapies.

Electrophysiological and molecular evidence of L-(Cav1), N- (Cav2.2), and R- (Cav2.3) type Ca2+ channels in rat cortical astrocytes

Changes in intracellular Ca2+ levels are an important signal underlying neuron‐glia cross‐talk, but little is known about the possible role of voltage‐gated Ca2+ channels (VGCCs) in controlling glial cell Ca2+ influx. We investigated the pharmacological and biophysical features of VGCCs in cultured rat cortical astrocytes. In whole‐cell patch‐clamp experiments, L‐channel blockade (5 μM nifedipine) reduced Ba2+ current amplitude by 28% of controls, and further decrease (32%) was produced by N‐channel blockade (3 μM ω‐conotoxin‐GVIA). No significant additional changes were observed after P/Q channel blockade (3 μM ω‐conotoxin‐MVIIC). Residual current (36% of controls) amounted to roughly the same percentage (34%) that was abolished by R‐channel blockade (100 nM SNX‐482). Electrophysiological evidence of L‐, N‐, and R‐channels was associated with RT‐PCR detection of mRNA transcripts for VGCC subunits α1C (L‐type), α1B (N‐type), and α1E (R‐type). In cell‐attached recordings, single‐channel properties (L‐currents: amplitude, −1.21 ± 0.02 pA at 10 mV; slope conductance, 22.0 ± 1.1 pS; mean open time, 5.95 ± 0.24 ms; N‐currents: amplitude, −1.09 ± 0.02 pA at 10 mV; slope conductance, 18.0 ± 1.1 pS; mean open time, 1.14 ± 0.02 ms; R‐currents: amplitude, −0.81 ± 0.01 pA at 20 mV; slope conductance, 10.5 ± 0.3 pS; mean open time, 0.88 ± 0.02 ms) resembled those of corresponding VGCCs in neurons. These novel findings indicate that VGCC expression by cortical astrocytes may be more varied than previously thought, suggesting that these channels may indeed play substantial roles in the regulation of astrocyte Ca2+ influx, which influences neuron‐glia cross‐talk and numerous other calcium‐mediated glial‐cell functions.

The effect of growth hormone secretagogues and neuropeptide Y on hypothalamic hormone release from acute rat hypothalamic explants.

Growth hormone (GH) secretagogues (GH‐releasing peptides and their non‐peptide analogues) stimulate growth hormone release via specific G‐protein coupled receptors both directly from the pituitary gland and through stimulation of the hypothalamus. The exact mechanism of action in the hypothalamus is not known. The presence of endogenous GH releasing hormone (GHRH) seems to be necessary for the in‐vivo actions of growth hormone secretagogues (GHSs), but data suggest that further factors must be involved as well. The effect of GHSs is not entirely specific for the GH axis; they release prolactin and stimulate the hypothalamo‐pituitary‐adrenal axis causing elevations in circulating ACTH and cortisol levels in both animal and human studies. Recently, it has also been suggested that GHSs stimulate hypothalamic neuropeptide Y (NPY) neurones. In the present study, we have therefore investigated the direct effect of several GHSs (GHRP‐6, hexarelin and the non‐peptide analogues L‐692, 429 and L‐692, 585) on GHRH, somatostatin (SS), corticotrophin‐releasing hormone (CRH) and arginine vasopressin (AVP) release in vitro in an acute rat hypothalamic incubation system. We also assessed the effect of NPY on GHRH, SS and AVP release. Freshly removed hypothalami were incubated in control media for 20 min and then in 1–4 consecutive 20‐min periods in each of the test substances at different concentrations. There was no significant change in either the basal or potassium‐stimulated release of GHRH or SS at low concentrations of any of the secretagogues; however, at millimolar doses a paradoxical inhibition of GHRH was observed with GHRP‐6, hexarelin and L‐692 585 (data are expressed as the ratio of treated to preceding basal release; at 20 min control group: 0.97±0.02, GHRP‐6: 0.55±0.04, P<0.001 compared to control group; hexarelin: 0.56±0.06, P<0.001, L‐692,585: 0.70±0.03, P<0.001), while SS was stimulated after 60 or 80 min (at 80 min control: 0.80±0.03, hexarelin: 1.23±0.07, P<0.05 and L‐692,585: 1.37±0.11, P<0.05). GHSs stimulated hypothalamic AVP release (at 20 min control: 0.99±0.06 ratio to basal release, 10−4 M concentration of GHRP‐6: 6.31±1, P<0.001, hexarelin: 1.88±0.4, P<0.01, L‐692,429: 1.90±0.5, P<0.05 and L‐692,585: 2.34±0.96, P<0.01), while no stimulatory effect was found on CRH release. NPY significantly stimulated SS and inhibited basal and potassium‐stimulated GHRH release, while potentiating potassium‐evoked AVP secretion. The Y1 receptor antagonist BIBP 3226 did not inhibit the effects of NPY on SS, GHRH or AVP release. We therefore conclude that, in this in‐vitro rat hypothalamic incubation model, growth hormone secretagogues stimulate the release of AVP but have no effect on either GHRH, SS or CRH at low doses; at high doses paradoxically they inhibit the hypothalamic GH axis similar to in‐vivo data in the rat. We speculate that these effects might be mediated by NPY.

Pharmacokinetic variability of antiretroviral drugs and correlation with virological outcome: 2 years of experience in routine clinical practice

OBJECTIVES To assess the inter-individual and intra-individual plasma concentration variabilities of non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs) in routine clinical practice and to investigate their relationships with virological failure. METHODS We retrospectively enrolled HIV-infected patients undergoing therapeutic drug monitoring (TDM) of NNRTIs and PIs during routine outpatient visits. Plasma drug concentrations were measured by HPLC-UV and were considered therapeutic if above the proposed minimum efficacy trough concentration. Inter-individual and intra-individual variabilities were evaluated through the coefficient of variation (CV). RESULTS A total of 457 PI and 172 NNRTI plasma concentrations were measured from 363 patients (HIV-RNA <50 copies/mL in 70.8%, median CD4 count 434 cells/mm(3)). NNRTIs showed less inter-individual (CV(inter) 54.8% versus 84.3%) and intra-individual (CV(intra) 19.0% versus 38.1%) pharmacokinetic variabilities than PIs. Intra-individual variability was constantly lower than inter-individual variability for each drug. Subtherapeutic drug concentrations were observed in 106 samples (16.9%). Older age (P = 0.020) and higher viral load (P = 0.013) were associated with subtherapeutic levels. Patients with therapeutic levels had a viral load of <50 copies/mL more frequently than those with subtherapeutic levels (74.8% versus 63.2%, P = 0.020). The estimated proportion with virological failure at 24 weeks was 0.21 in patients with suboptimal baseline drug levels and 0.08 in those with optimal levels (P < 0.001). In the multivariate analysis, therapeutic drug levels showed an independent negative association with virological failure (P = 0.004). CONCLUSIONS A wide inter-individual and limited intra-individual pharmacokinetic variabilities, together with the demonstration of a concentration-response relationship, suggest that TDM is a useful tool for the clinical management of patients treated with NNRTIs or PIs.

Roles of nitric oxide, carbon monoxide, and hydrogen sulfide in the regulation of the hypothalamic–pituitary–adrenal axis

J. Neurochem. (2010) 113, 563–575.