Combination of anti-CGRP/CGRP-R mAbs with onabotulinumtoxin A as a novel therapeutic approach for refractory chronic migraine: a retrospective study of real-world clinical evidence and a protocol for a double-blind, randomized clinical trial to establish the efficacy and safety.

Chronic migraine is a disabling neurovascular disorder that ranks amongst the top causes of years lived with disability worldwide. The duration and the frequency of migraine affect cognitive and affective domains, inducing worsening of memory, executive functions, orientation and causing anxiety. Population-based studies report a worrying level of resistance to treatments. Therefore, this study aims: 1) to assess efficacy of monoclonal antibodies (mAbs) directed towards the calcitonin gene-related peptide (CGRP) or its receptor (CGRP-R) for chronic migraine resistant to current preventatives; 2) to design a clinical trial protocol to evaluate the efficacy and safety of combination therapy utilizing anti-CGRP/CGRP-R together with onabotulinumtoxin A in patients suffering from resistant chronic migraine; 3) to provide a molecular rationale for combination therapy. A controlled trial is warranted as pooled analysis of real-world data from our group highlighted that combined treatment provides ≥50% reduction vs. baseline (onabotulinumtoxin A) of monthly headache days (MHDs) in up to 58.8% of patients, but there has been only sparse application of this combined therapy to date. The mAbs chosen are: erenumab, because its combination effect with onabotulinumtoxin A improved symptoms in 65% of patients; eptinezumab, due to its faster action. The results highlight that early diagnosis of migraine improves therapeutic outcomes with mAbs alone, confirming their effectiveness and the need for an adequately powered clinical trial evaluating the safety and potential superior effectiveness of eptinezumab/erenumab and onabotulinumtoxin A together.

NAbiximols Clinical Translation To the treatment of Pain and Agitation In Severe Dementia (NACTOPAISD): Clinical trial protocol.

Up to 80 % nursing home residents with dementia experiences chronic pain. Contextually, 97 % presents fluctuant neuropsychiatric symptoms (NPS). Among the most challenging is agitation, connected with undertreated pain and managed through neuroleptics doubling death risk. Evidence is accumulating in favor of the involvement of the endocannabinoid system in nociception and NPS. This double-blind, placebo-controlled, randomized trial (NAbiximols Clinical Translation To the treatment of Pain and Agitation In Severe Dementia [NACTOPAISD]) aims at investigating efficacy and safety of oral spray nabiximols, containing Δ9-tetrahydrocannabinol and cannabidiol (Sativex®), for pain and agitation treatment in severe dementia patients (Mini-Mental State Examination ≤ 12) over 65. The coprimary endpoints are efficacy on pain and agitation, assessed through the recently validated Italian Mobilization-Observation-Behavior-Intensity-Dementia and the Cohen-Mansfield Agitation Inventory. The secondary endpoint is the evaluation of efficacy duration after wash-out and the assessment of quality of life through the DEMQOL. Any adverse events will be reported. The results undergo statistical analysis plan. NACTOPAISD might provide rationale for a translational safer pain and agitation treatment in severe dementia. It is approved by Calabria Region Ethics Committee and follows the Standard Protocol Items: Recommendations for Interventional Trials (SPIRIT) and the Consolidated Standards of Reporting Trials (CONSORT) statements.

Pain and agitation treatment in severe dementia patients: The need for Italian Mobilization-Observation-Behavior-Intensity-Dementia (I-MOBID2) pain scale translation, adaptation and validation with psychometric testing.

The 97% of dementia patients develops fluctuant neuropsychiatric symptoms often related to under-diagnosed and unrelieved pain. Up to 80% severe demented nursing home residents experiences chronic pain due to age-related comorbidities. Patients lacking self-report skills risk not to be appropriately treated for pain. Mobilization-Observation-Behavior-Intensity-Dementia (MOBID2) is the sole pain scale to consider the frequent co-occurrence of musculoskeletal and visceral pain and to unravel concealed pain through active guided movements. Accordingly, the Italian real-world setting can benefit from its translation and validation. This clinical study provides a translated, adapted and validated version of the MOBID2, the Italian I-MOBID2. The translation, adaptation and validation of the scale for non-verbal, severe demented patients was conducted according to current guidelines in a cohort of 11 patients over 65 with mini-mental state examination ≤ 12. The I-MOBID2 proves: good face and scale content validity index (0.89); reliable internal consistency (Cronbach's α = 0.751); good to excellent inter-rater (Intraclass correlation coefficient, and test-retest (ICC = 0.902) reliability. The construct validity is high (Rho = 0.748 p < 0.05 for 11 patients, Spearman rank order correlation of the overall pain intensity score with the maximum item score of I-MOBID2 Part 1; rho=0.895 p < 0.01 for 11 patients, for the overall pain intensity score with the maximum item score of I-MOBID2 Part 2) and a good rate of inter-rater and test-retest agreement was demonstrated by Cohen's K = 0.744. The average execution time is of 5.8 min, thus making I-MOBID2 a useful tool suitable also for future development in community setting with administration by caregivers.

Is there a rational basis for cannabinoids research and development in ocular pain therapy? A systematic review of preclinical evidence.

BACKGROUND: Purpose of the present systematic review is to investigate preclinical evidence in favor of the working hypothesis of efficacy of cannabinoids in ocular pain treatment. METHODS: Literature search includes the most relevant repositories for medical scientific literature from inception until November, 24 2021. Data collection and selection of retrieved records adhere to PRISMA criteria. RESULTS: In agreement with a priori established protocol the search retrieved 2471 records leaving 479 results after duplicates removal. Eleven records result from title and abstract screening to meet the inclusion criteria; only 4 results are eligible for inclusion in the qualitative synthesis impeding meta-analysis. The qualitative analysis highlights the antinociceptive and anti-inflammatory efficacy of Δ8-tetrahydrocannabinol, cannabidiol and its derivative HU-308 and of new racemic CB1 allosteric ligand GAT211 and its enantiomers GAT228 and GAT229. Moreover, CB2R agonists RO6871304 and RO6871085 and CB2R ligand HU910 provide evidence of anti-inflammatory efficacy. CB2 agonist HU308 reduces of 241% uveitis-induced leukocyte adhesion and changes lipidome profile. Methodological and design issues raise concern of risk of bias and the amount of studies is too small for generalization. Furthermore, the ocular pain model used can resemble only inflammatory but not neuropathic pain. CONCLUSIONS: The role of the endocannabinoid system in ocular pain is underinvestigated, since only two studies assessing the effects of cannabinoid receptors modulators on pain behavior and other two on pain-related inflammatory processes are found. Preclinical studies investigating the efficacy of cannabinoids in ocular inflammatory and neuropathic pain models are needed to pave the way for clinical translation.

Eptinezumab for the treatment of migraine.

Eptinezumab (ALD-403) is a genetically engineered humanized IgG1kappa directed towards calcitonin gene-related peptide (CGRP), currently in late-stage clinical development. Eptinezumab targets the pathway of CGRP, importantly implicated in migraine pathophysiology, and may represent the first-to-market infusion therapy for migraine prevention. The background for its approval consists in preclinical data and clinical trials. Here, we provide a comprehensive review of molecular pharmacology, pharmacokinetics, metabolism, efficacy and safety investigated in the preclinical and clinical studies, with insight on possible future directions.

Modulation of pro-survival and death-associated pathways under retinal ischemia/reperfusion: effects of NMDA receptor blockade.

Loss of retinal ganglion cells occurs in a variety of pathological conditions, including central retinal artery occlusion, diabetes and glaucoma. Using an experimental model of retinal ischemia induced by transiently raise the intraocular pressure (IOP), In this study, we report the original observation that ischemic retinal ganglion cells death is associated with the transient deactivation of the pro-survival kinase Akt and activation of GSK-3beta followed, during reperfusion, by a longer lasting, PI3K-dependent, activation of Akt and phosphorylation of GSK-3beta. Under these experimental conditions, retinal ischemia induced the expression of Bad, a pro-apoptotic protein, member of the Bcl-2 family. The detrimental effects yielded by the ischemic stimulus were minimized by intravitreal administration of the NMDA receptor antagonist, MK801, that reduced the expression of Bad and significantly increased Akt phosphorylation. In conclusion, our present results contribute to unravel the mechanisms underlying retinal damage by high IOP-induced transient ischemia in rat. In addition, these data implicate the pro-survival PI3K/Akt pathway and the observed reduced expression of Bad in the neuroprotection afforded by MK801.

Brain regional and cellular localization of gelatinase activity in rat that have undergone transient middle cerebral artery occlusion.

Matrix metalloproteinases (MMPs) have been implicated in the pathophysiology of ischemic stroke. In particular, the gelatinases MMP-2 and MMP-9 contribute to disruption of the blood-brain barrier and hemorrhagic transformation following ischemic injury. In addition to extracellular matrix degradation, MMPs may directly regulate neuronal cell death through mechanisms that are not completely understood. Here we describe the spatio-temporal distribution of activated MMP-2 and MMP-9 in the brain of rats subjected to 2 h middle cerebral artery occlusion (MCAo) followed by different periods of reperfusion (15 min, 2 h, 6 h and 22 h). By in situ zymography we have observed that gelatinases become activated 15 min and 2 h after the beginning of reperfusion in the ischemic core and penumbra, respectively. In situ zymography signal broadly co-localized with NeuN-positive cells, thus suggesting that proteolysis mainly occurs in neurons. Gelatinolytic activity was mainly detected in cell nuclei, marginally appearing in the cytosol only at later stages following the insult; we did not detect variations in gelatinolysis in the extracellular matrix. Finally, we report that pharmacological inhibition of MMPs by N-[(2R)-2-(hydroxamidocarbonyl-methyl)-4-methylpenthanoyl]-L-tryptophan methylamide (GM6001) significantly reduces brain infarct volume induced by transient MCAo. Taken together our data underscore the crucial role of gelatinases during the early stages of reperfusion and further extend previous observations documenting the detrimental role of these enzymes in the pathophysiology of brain ischemia.

Cell signaling pathways in the mechanisms of neuroprotection afforded by bergamot essential oil against NMDA-induced cell death in vitro.

BACKGROUND AND PURPOSE: The effects of bergamot essential oil (BEO; Citrus bergamia, Risso) on excitotoxic neuronal damage was investigated in vitro. EXPERIMENTAL APPROACH: The study was performed in human SH-SY5Y neuroblastoma cells exposed to N-methyl-D-aspartate (NMDA). Cell viability was measured by dye exclusion. Reactive oxygen species (ROS) and caspase-3 activity were measured fluorimetrically. Calpain I activity and the activation (phosphorylation) of Akt and glycogen synthase kinase-3beta (GSK-3beta) were assayed by Western blotting. KEY RESULTS: NMDA induced concentration-dependent, receptor-mediated, death of SH-SY5Y cells, ranging from 11 to 25% (0.25-5 mM). Cell death induced by 1 mM NMDA (21%) was preceded by a significant accumulation of intracellular ROS and by a rapid activation of the calcium-activated protease calpain I. In addition, NMDA caused a rapid deactivation of Akt kinase and this preceded the detrimental activation of the downstream kinase, GSK-3beta. BEO (0.0005-0.01%) concentration dependently reduced death of SH-SY5Y cells caused by 1 mM NMDA. In addition to preventing ROS accumulation and activation of calpain, BEO (0.01%) counteracted the deactivation of Akt and the consequent activation of GSK-3beta, induced by NMDA. Results obtained by using specific fractions of BEO, suggested that monoterpene hydrocarbons were responsible for neuroprotection afforded by BEO against NMDA-induced cell death. CONCLUSIONS AND IMPLICATIONS: Our data demonstrate that BEO reduces neuronal damage caused in vitro by excitotoxic stimuli and that this neuroprotection was associated with prevention of injury-induced engagement of critical death pathways.

Rational Basis for the Use of Bergamot Essential Oil in Complementary Medicine to Treat Chronic Pain.

In complementary medicine, aromatherapy uses essential oils to improve agitation and aggression observed in dementia, mood, depression, anxiety and chronic pain. Preclinical research studies have reported that the essential oil obtained from bergamot (BEO) fruit (Citrus bergamia, Risso) modifies normal and pathological synaptic plasticity implicated, for instance, in nociceptive and neuropathic pain. Interestingly, recent results indicated that BEO modulates sensitive perception of pain in different models of nociceptive, inflammatory and neuropathic pain modulating endogenous systems. Thus, local administration of BEO inhibited the nociceptive behavioral effect induced by intraplantar injection of capsaicin or formalin in mice. Similar effects were observed with linalool and linalyl acetate, major volatile components of the phytocomplex, Pharmacological studies showed that the latter effects are reversed by local or systemic pretreatment with the opioid antagonist naloxone hydrochloride alike with naloxone methiodide, high affinity peripheral µ-opioid receptor antagonist. These results and the synergistic effect observed following systemic or intrathecal injection of an inactive dose of morphine with BEO or linalool indicated an activation of peripheral opioid system. Recently, in neuropathic pain models systemic or local administration of BEO or linalool induced antiallodynic effects. In particular, in partial sciatic nerve ligation (PSNL) model, intraplantar injection of the phytocomplex or linalool in the ipsilateral hindpaw, but not in the contralateral, reduced PSNL-induced extracellularsignal- regulated kinase (ERK) activation and mechanical allodynia. In neuropathic pain high doses of morphine are needed to reduce pain. Interestingly, combination of inactive doses of BEO or linalool with a low dose of morphine induced antiallodynic effects in mice. Peripheral cannabinoid and opioid systems appear to be involved in the antinociception produced by intraplantar injection of ß -caryophyllene, present in different essential oils including BEO. The data gathered so far indicate that the essential oil of bergamot is endowed with antinociceptive and antiallodynic effects and contribute to form the rational basis for rigorous testing of its efficacy in complementary medicine.

17beta-estradiol reduces neuronal apoptosis induced by HIV-1 gp120 in the neocortex of rat.

The human immunodeficiency virus type 1 (HIV-1) coat glycoprotein gp120 represents a likely contributor to the development of HIV-1 associated dementia (HAD), a neurological syndrome often observed in AIDS patients and characterised by significant neuronal loss in the neocortex. Since recent studies have highlighted that female sex hormones represent potential neuroprotective agents against damage produced by acute and chronic injuries in the adult brain, we have investigated whether estrogens exert protection in a rat model of gp120 neurotoxicity. Our results demonstrate that systemic administration of 17beta-estradiol (E2, 0.02-0.2 mg/kg) significantly reduces apoptotic cell death observed in the neocortex of rat following subchronic i.c.v. administration of gp120 (100 ng/rat/day). Furthermore, both tamoxifen and ICI182,780, two selective antagonists of estrogen receptors (ER) in the brain, reverted the neuroprotective effect of E2. The molecular mechanism of estrogenic neuroprotection does not appear to involve modulation of the antiapoptotic Bcl-2 or the proapoptotic Bax since we failed to observe changes in the levels of the two proteins in the neocortical tissue after gp120 and/or E2 treatment. However, we detected increased levels of IL-1beta in the neocortex of rats injected with gp120, as early as 6h after drug administration, and this effect was potentiated following pretreatment with E2. Taken together, our results demonstrate that E2 exerts neuroprotection against gp120 neurotoxicity in vivo through a mechanism involving ER activation and, possibly, via modulation of neocortical levels of IL-1beta.

Age-related changes in Cu,Zn superoxide dismutase, Se-dependent and -independent glutathione peroxidase and catalase activities in specific areas of rat brain.

Oxidative injury of tissues involves both accumulation of damage due to persistent oxidative stress and loss of the proper balance of antioxidative enzymes. These events may produce a faster rate of tissue senescence. In this regard, we have assayed the antioxidative enzyme activities (Cu,Zn superoxide dismutase, glutathione peroxidase and catalase), in various areas of rat brain (prefrontal cortex, parietal cortex, hippocampus, hypothalamus, caudate nucleus, mesencephalon and lower brain stem) for the age groups of 3, 6, 12, 24 months. The results obtained show that the levels of antioxidant enzyme activities differed considerably in the various brain parts studied. Furthermore, changes in the specific activities of superoxide dismutase, catalase, and glutathione peroxidase did not follow the same pattern as a function of aging. In particular, in prefrontal cortex and caudate nucleus, superoxide dismutase and glutathione peroxidase activities did not change, while catalase activity decreased. In parietal cortex and mesencephalon, superoxide dismutase and glutathione peroxidase activities increased, but the catalase activity decreased in parietal cortex and did not change in mesencephalon. In lower brain stem, the activities of glutathione peroxidase and catalase decreased in 3-12-month-old rats. The activity of glutathione peroxidase was increased in the hippocampus and was decreased in hypothalamus during aging. In this area the catalase activity was also significantly diminished.

High vulnerability of dentate granule cells to the neuropathological effects induced by intrahippocampal injection of tetanus toxin.

The behavioural and neuropathological effects of tetanus toxin, injected into the dentate gyrus, were studied in rats. The monolateral injection of a single dose (1000 mouse minimum lethal doses, MLDs; n = 14 rats) of tetanus toxin produced time-dependent behavioural stimulation. Wet-dog shakes and facial stereotypy were observed 3-4 days after the injection, culminating 4-5 days after treatment, in "limbic motor seizures". Ten days after injection, histological examination revealed death of dentate granule cells in the tetanus toxin-treated side but not in the contralateral, control side (treated with neutralized toxin). This effect was observed in all rats (n = 5) receiving tetanus toxin in the dentate gyrus and no damage was reported in other sectors (e.g. CA1 and CA3 pyramidal cell layers) of the hippocampus. Quantification of the neuronal damage yielded an approximately 70% reduction (P less than 0.01) in the number of granule cells in the toxin-injected dentate gyrus, compared with the control side. This was greater than that previously reported (30% reduction) in the CA1 pyramidal cell layer of rats receiving the same dose of toxin, into the regio superior of the hippocampus. In conclusion, the present experiments have shown that the focal injection of tetanus toxin into the dentate gyrus produced behavioural excitation and selective death of dentate granule cells.

Behavioural and electrocortical changes induced by muscimol in rats withdrawn from chronic treatment with diazepam.

In rats withdrawn from a chronic treatment with diazepam, the effects of muscimol, given into the III cerebral ventricle, on behaviour and spectrum power of activity in the electrocorticogram (ECoG) were studied. In comparison to control rats which received only muscimol, in rats pretreated with diazepam (1 mg/kg/day for 30 consecutive days) the behavioural and ECoG effects of muscimol were significantly reduced or abolished. In fact, in rats pretreated with diazepam a small dose (50 ng) of muscimol did not affect behaviour or ECoG activity, in contrast to control animals in which the same dose produced, after a period of locomotor stimulation and ECoG desynchronization, typical and long-lasting behavioural sedation or sleep accompanied by a significant increase in total voltage power and in the lower frequency bands in the ECoG. In addition, larger doses (100 and 200 ng) of muscimol, which in control rats produced a typical biphasic pattern of ECoG and behavioural changes, i.e. an initial period of ECoG desynchronization and behavioural stimulation, followed by a second period of behavioural and ECoG sleep, in animals pretreated with diazepam, produced only an increase in total voltage power and in the lower frequency bands in the ECoG resembling the effects of the smaller (50 ng) dose. The present experiments suggest that, after chronic stimulation of benzodiazepine receptors a decrease in sensitivity of receptors for gamma-aminobutyric acid (GABA) occurs, since the effects of muscimol on behaviour and spectrum power were significantly reduced or abolished.

Behavioural and neuropathological effects produced by tetanus toxin injected into the hippocampus of rats.

The behavioural effects of tetanus toxin, injected into the rostral hippocampus, have been studied in rats. A single dose (1000 mouse minimum lethal doses; n = 10) of the toxin produced tail rigidity, hunched back and sound- and touch-evoked stimuli, 48 hr after the injection in all rats treated and these culminated in generalized convulsions 5-7 days later. Seizures were also observed 4 days after the injection of 2000 MLDs (n = 10), whereas a dose of 500 MLDs (n = 10) was ineffective. Similarly, dose- and time-dependent lethal effects were observed. In comparison to the contralateral (untreated) hippocampus, tetanus toxin (1000 MLDs; n = 3) produced a statistically significantly reduction in the number of cells in the CA1 pyramidal cell layer of the injected side, 7 and 10 days after the injection. No changes were observed in other sectors (CA2 and CA3 areas) of the hippocampus. In conclusion, the present experiments have shown that the focal injection of tetanus toxin into the hippocampus produced dose- and time-dependent behavioural stimulation and lethal effects in rats.

N-methyl-D-aspartate-induced excessive formation of nitric oxide in CHP100 neuroblastoma cells produces death of BMEL melanoma cells in co-culture.

In the present experiments we planned to ascertain whether an abnormal production of nitric oxide (NO) by human CHP100 neuroblastoma cells in culture following stimulation of N-methyl-D-aspartate (NMDA) receptors, produced lethal effects in co-cultured human BMEL melanoma cells. Human BMEL melanoma cells in culture were found to be positive to the nicotinamide adenine dinucleotide phosphate diaphorase (NADPH diaphorase) histochemical reaction and produced NO as revealed by measurements of nitrite under basal culture conditions. Exposure for 50 min to aspartate (1-2 mM) or to NMDA (0.5-1.5 mM) did not evoke significant melanoma cell death. The dose of 1.0 mM NMDA applied for 1 min to BMEL cell cultures did not increase significantly nitrite concentrations in comparison to controls. Incubation for 50 min of human CHP100 neuroblastoma cells with NMDA (0.5-1.5 mM) elicited dose-dependent death of BMEL melanoma cells co-cultured in trans-wells. Under these experimental conditions, nitrite levels in cell culture-inserts containing melanoma cells increased by 120% 1 min after application of the excitotoxin (1 mM) to CHP100 neuroblastoma cultures. The lethal effects produced in BMEL cell culture-inserts by application of NMDA (1.0 mM) to CHP100 cultures were prevented by pretreatment of neuroblastoma cultures with MK801 (200 nM). Similar protection was also afforded by N omega-nitro-L-arginine methyl ester (L-NAME; 0.2 mM) and N omega-monomethyl-L-arginine (L-NMMA; 0.2 mM), two inhibitors of nitric oxide synthase, and by haemoglobin (10 microM), a nitric oxide trapping agent.(ABSTRACT TRUNCATED AT 250 WORDS)

Behavioural and ECoG spectrum power effects after intraventricular injection of drugs altering dopaminergic transmission in rats.

In rats with cannulae permanently implanted into the third cerebral ventricle, the effects of different pharmacological manipulations affecting dopaminergic mechanisms, were studied on behaviour and electrocorticographic (ECoG) activity, continuously quantified in its spectrum power. The intraventricular injection (0.1-1 nmol) of (-)3PPP[3-(3-hydroxyphenyl) N-n-propylpiperidine], a specific agonist at dopamine (DA) autoreceptors, produced dose-dependent behavioural sedation or sleep and an increase in ECoG spectrum power, with a predominant increase in the lower frequency bands. Short episodes of stereotyped movements, wet-dog syndrome, penile grooming and erection were also observed. Similar behavioural and ECoG effects were elicited by the intraventricular injection of R-(+)-8-chloro-2,3,4,5-tetrohydro-3-methyl-5-phenyl-1H-3-benzazepi ne-7-ol (SCH 23390), a selective antagonist at D1 postsynaptic receptors, although these were preceded by a short period of behavioural and sexual stimulation. In addition, the intraventricular administration of some neuroleptics, chloropromazine and haloperidol, produced behavioural and ECoG slow wave sleep. No significant changes were observed with a neuroleptic drug, 1-sulpiride, which is reputed to act selectively as an antagonist at dopamine D2 receptors. In conclusion, the present experiments add new evidence in favour of the idea that dopaminergic mechanisms are involved in mammalian species in the control of arousal and that both post-synaptic D1 and D2 receptors may take part in such a control.

Involvement of interleukin-1beta in the mechanism of human immunodeficiency virus type 1 (HIV-1) recombinant protein gp120-induced apoptosis in the neocortex of rat.

The effect of subchronic intracerebroventricular injection of the human immunodeficiency virus type 1 (HIV-1) recombinant protein gp120 (100 ng, given daily for up to seven consecutive days) on interleukin-1beta expression was studied by immunohistochemistry in the brain of adult rats. In comparison to control, bovine serum albumin (300 ng, given intracerebroventricularly for up to seven days) -treated animals (n=6), interleukin-1beta immunoreactivity increased in the brain cortex and hippocampus of rats (n=6) receiving a single injection of the viral protein 24 h before analysis with more substantial increases being observed in these regions of the brain (n=6) after seven days treatment. Double-labelling immunofluorescence experiments support a neuronal and, possibly, a microglial cell origin for gp120-enhanced interleukin-1beta expression. Transmission electron microscopy analysis of brain tissue sections revealed that combination treatments (given intracerebroventricularly daily for seven days) with gp120 (100 ng) and interleukin-1 receptor antagonist (80 ng) or with the interleukin converting enzyme inhibitor II (100 pmol), but not with leupeptin (100 pmol), prevented apoptotic death of rat (n=6/group) brain cortical cells typically elicited by the viral protein. These data demonstrate that gp120 enhances interleukin-1beta expression in the brain and this may be involved in the mechanism underlying apoptosis induced by gp120 in the brain cortex of rat. Further support to this hypothesis comes from the evidence that intracerebroventricular injection of murine recombinant interleukin-1beta (200 U, given daily for seven consecutive days) produces DNA fragmentation in the brain cortex of rat (n=6). Interestingly, the latter treatment enhanced nerve growth factor level in the hippocampus but not in the cerebral cortex and this coincides with a similar effect recently reported in identical brain areas of rats treated likewise with gp120. In conclusion, the present data demonstrate that treatment with gp120 enhances interleukin-1beta expression and this participates in the mechanism of apoptotic cell death in the brain cortex of rat. By contrast, in the hippocampus, gp120-enhanced interleukin-1beta expression elevates nerve growth factor that may prevent or delay apoptosis in this plastic region of the rat brain.

HIV-1 coat protein gp120 stimulates interleukin-1beta secretion from human neuroblastoma cells: evidence for a role in the mechanism of cell death.

1. The role of the pro-inflammatory cytokine interleukin-1beta (IL-1beta) in the mechanism of cell death induced by the human immunodeficiency virus type 1 (HIV-1) recombinant coat glycoprotein, gp120 IIIB, has been studied in the human CHP100 neuroblastoma cell line maintained in culture. 2. Death of neuroblastoma cells typically elicited by 10 pM gp120 or by human recombinant IL-1beta (10 ng x ml(-1)) has been minimized by the antagonist of IL-1 receptor, i.e. IL-1ra (0.5 and 50 ng x ml(-1), respectively), an endogenous molecule that antagonizes most of the biological actions of IL-1beta, or by an antibody (5 and 50 ng x ml(-1)) which blocks the human IL-1 receptor type I (IL-1RI). 3. ELISA experiments have established that gp120 enhances immunoreactive IL-1beta levels in the culture medium and this is prevented by exposure to the IL-1 converting enzyme (ICE) inhibitor t-butoxycarbonyl-L-aspartic acid benzyl ester-chloromethylketone [Boc-Asp(OBzl)-CMK] used at a concentration (2.5 microM) which significantly (P<0.001) reduces cell death. 4. Death of CHP100 cells induced by gp120 is also prevented by acetyl-Tyr-Val-Ala-Asp-chloromethylketone (Ac-YVAD-CMK; 10-100 microM), a second inhibitor of ICE, supporting the concept that the viral protein stimulates the conversion of the 31 kDa pro-IL-1beta in to the 17 kDa mature cytokine which is then secreted to cause death. 5. In conclusion, our present data demonstrate that gp120 stimulates the secretion of IL-1beta which then triggers CHP100 neuroblastoma cell death via stimulation of IL-1 receptor type I.

The HIV envelope protein gp120 in the nervous system: interactions with nitric oxide, interleukin-1beta and nerve growth factor signalling, with pathological implications in vivo and in vitro.

The neuronal loss often described at post-mortem in the brain neocortex of patients suffering from AIDS has been proposed to be responsible for the development of the AIDS dementia complex. Neuroinvasive strains of the HIV virus infect macrophages, microglial cells, and multinucleated giant cells, but not neurones. Processing of the virus by cells of the myelomonocytic lineage yields viral products known to initiate a complex network of events that may lead to the death of neurones and to the development of AIDS-associated neurological syndrome. The HIV-1 coat protein gp120, in particular, has been proposed as a likely etiologic agent of the described neuronal loss because it causes the death of neurones in culture. More recently, it has been shown that brain cortical cell death caused in rats by intracerebroventricular injection of gp120 occurs via apoptosis. This observation broadens our knowledge of the pathophysiology of the reported neuronal cell loss and opens a new avenue of experimental research for the development of novel therapeutic strategies for the treatment of patients suffering from AIDS-associated neurological syndrome.

The HIV-1 gp120 causes ultrastructural changes typical of apoptosis in the rat cerebral cortex.

We used transmission electron microscopy (TEM) and terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labelling (TUNEL) techniques to study the neuropathological effects of intracerebroventricular (i.c.v.) injection of recombinant HIV-1 gp 120 in rats. In brain cortical tissue sections from rats treated with a single daily dose of gp120 (100 ng day-1 for 7 or 14 consecutive days) TEM analysis showed chromatin compaction and marginalization along the inner surface of the nuclear envelope followed by masses of condensed chromatin, ultrastructural signs demonstrating the occurrence of apoptotic cell death. These effects were paralleled by in situ DNA fragmentation, as revealed by application of TUNEL technique to cryostat brain tissue sections from rats treated likewise with the viral coat protein. In no instance was apoptosis seen in the brain cortex of control rats. The present data demonstrate that gp120 given i.c.v. produces apoptosis in the neocortex of rats.