Ascertainment bias in dementias: a secondary to tertiary centre analysis in Central Italy and conceptual review.

BACKGROUND AND AIMS: Ascertainment bias (AB) indicates a bias of an evaluation centre in estimating the prevalence/incidence of a disease due to the specific expertise of the centre. The aim of our study was to evaluate classification of different types of dementia in new cases appearing in secondary and tertiary centres, in order to evidence possible occurrence of AB in the various (secondary to tertiary) dementia centres. METHODS: To assess the mechanism of AB, the rates of new cases of the different forms of dementia reported by different centres were compared. The centres involved in the study were 11 hospital-based centres including a tertiary centre, located in the University Department of Clinical Neurology. The tertiary centre is endowed with state-of-the-art diagnostic facilities and its scientific production is prominently focused on dementia with Lewy bodies (DLB) thus suggesting the possible occurrence of a bias. Four main categories of dementia were identified: Alzheimer's disease (AD), DLB, fronto-temporal dementia (FTD), vascular dementia (VaD), with other forms in a category apart. The classification rate of new cases of dementia in the tertiary centre was compared with rates reported by secondary centres and rates of recoding were calculated during a follow-up of 2 years. RESULTS: The study classified 2,042 newly diagnosed cases of dementia in a population of 1,370,000 inhabitants of which 315,000 were older than 65. AD was categorized in 48-52 % of cases, DLB in 25-28 %, FTD in 2-4 % and VaD in 17-28 %. During the 2-year follow-up the diagnosis was re-classified in 40 patients (3 %). The rate of recoding was 5 % in the tertiary centre, 2-8 % in referrals from secondary to tertiary centre, 2-10 % in recodings performed in secondary centres and addressed to tertiary centre. Recoding or percentages of new cases of AD or DLB were not different in the comparison between secondary or between secondary and tertiary centres. FTD and VaD were instead significantly recoded. CONCLUSION: The results of the study suggest that in a homogeneous area, AB is not interfering with diagnosis of AD or DLB.

Activation of ethane C-H and C-C bonds by gas phase Th+ and U+: a theoretical study.

Two different approaches of density functional theory were used to analyze the C-H and C-C bond activation mechanisms during the reaction of bare Th(+) and U(+) ions with ethane. We report a complete exploration of the potential energy surfaces taking into consideration different spin states. According to B3LYP/SDD computations the double dehydrogenation of C(2)H(6) is thermodynamically favorable only in the case of Th(+). It is shown that the overall C-H and C-C bond activation processes are exothermic in the case of Th(+) and endothermic for U(+). In both cases, the C-C insertion transition state barrier exceeds the energy of the ground state reactants, preventing the observation of these species under thermal conditions.

Inducible expression of the long pentraxin PTX3 in the central nervous system.

PTX3 is a prototypic long pentraxin consisting of a C terminal 203-amino acid pentraxin-like domain coupled with an N-terminal 178-amino acid unrelated portion. PTX3 is induced by primary proinflammatory signals in various cell types, most prominently macrophages and endothelial cells. Other long pentraxins, such as murine or rat neuronal pentraxin 1 (NP1) and human neuronal pentraxin 2 (NPTX2), are expressed in the central nervous system (CNS). The present study was designed to investigate whether PTX3 is expressed in the brain and to define the structures and cells involved. Intracerebroventricular (i.c.v.), but not i.v., injection of LPS induced high levels of PTX3 mRNA in the mouse brain. In contrast NP1 is constitutively expressed in the murine CNS and is not modulated by LPS administration. I.c.v. IL-1beta was also a potent inducer of PTX3 expression in the CNS, whereas TNFalpha was substantially less effective and IL-6 induced a barely detectable signal. Central administration of LPS and IL-1 induced PTX3 also in the periphery (heart), whereas the reverse did not occur. Expression of PTX3 was also observed in the brain of mice infected with Candida albicans (C. albicans) or Cryptococcus neoformans. (C. neoformans). The kinetics of PTX3 gene induction were consistently different between C. albicans- and C. neoformans-infected mice, according to the diverse outcome of the CNS immune reaction. In situ hybridization revealed that i.c.v. injection of LPS induced a strong PTX3 expression in presumptive glial cells, in the white matter (corpus callosum, fimbria) and meningeal pia mater as well as in dentate gyrus hilus and granule cells. No constitutive expression of PTX3 was detected. Central expression of PTX3 may amplify mechanisms of innate resistance and damage in the CNS. The possibility of a direct interaction of PTX3 with neuronal cells, as suggested for NPTX2, remains to be explored.

Tumor necrosis factor is a brain damaging cytokine in cerebral ischemia.

Two contrasting roles, one beneficial and the injurious, have been proposed for tumor necrosis factor (TNF) in the pathogenesis of cerebral ischemia. Reported here are results obtained in a standard model of permanent focal cortical ischemia in rats, in which the volume of cerebral infarction is measured after permanent occlusion of the middle cerebral artery. Administration of neutralizing anti-rat TNF antibodies (P114) into the brain cortex significantly reduced ischemic brain damage (85% reduced infarct volume as compared with preimmune-treated controls). Similar results were achieved by systemic administration of CNI-1493, a recently described tetravalent guanylhydrazone compound, which effectively inhibited endogenous brain TNF synthesis and conferred significant protection against the development of cerebral infarction (80% reduced infarct volume as compared with vehicle controls treated 1 h postischemia with 10 mg/kg). P114 anti-TNF and CNI-1493 were each cerebroprotective when given within a clinically relevant time window for up to 2 h after the onset of ischemia. These findings establish an important, pathophysiological role of TNF in mediating the progression of ischemic brain damage, and suggest that inhibiting TNF with CNI-1493 may be beneficial in the future treatment of stroke.

Overexpression of interleukin-6 in the central nervous system of transgenic mice increases central but not systemic proinflammatory cytokine production.

Production of inflammatory cytokines, including tumor necrosis factor (TNF), interleukin-1 (IL-1) and interleukin-6 (IL-6), in the brain is increased in various diseases. To investigate the relationships between the effect of overproduction of IL-6 in the brain on central and peripheral production of TNF, IL-1 beta and IL-6 itself, we used transgenic mice (NSE-hIL-6) where neuronal human IL-6 expression under the control of the neuronal specific enolase promoter results in astrocytosis and gliosis. These mice had higher cerebral endogenous IL-6 (12-fold), IL-1 beta (12-fold) and TNF (4-fold) production measured in brain homogenates after intracerebroventricular (i.c.v.) injection of 2.5 micrograms LPS, lipopolysaccharide (LPS) than wild-type mice (no TNF or IL-1 were detectable in saline-injected NSE or control mice). Cerebral cytokines production was also increased in NSE-hIL-6 mice treated i.p. with LPS doses that do not normally induce cytokines in the brain. The induction of peripheral (serum or spleen) TNF, IL-1 beta or IL-6 was the same in all these experiments in NSE-hIL-6 and wild-type mice. Furthermore, using microglial cell clone pretreated in vitro with IL-6, we noted an increase in LPS-induced TNF and IL-6 production and proliferation of pretreated cells than control. This study indicates that overproduction of IL-6 in the central nervous system (CNS) may ultimately result in increased central production of inflammatory cytokines, probably due to increased proliferation and activation of the cells which produce cytokine in the CNS.

Systemic interleukin 10 administration inhibits brain tumor necrosis factor production in mice.

Interleukin 10 is an antiinflammatory cytokine and inhibits the production of tumor necrosis factor. We have previously found that intracerebroventricular (i.c.v.) administration of recombinant human interleukin 10 inhibits brain tumor necrosis factor production induced by an i.c.v. injection of lipopolysaccharide in mice. In view of its possible pharmacological use, we have now studied whether interleukin 10 administered peripherally could inhibit brain tumor necrosis factor production. Mice were injected with recombinant human interleukin 10 (20 microg/mouse, i.v.) 10 min-24 h before lipopolysaccharide (2.5 microg, i.c.v.). Tumor necrosis factor was measured, using a bioassay, in brain homogenates 90 min after lipopolysaccharide. Recombinant human interleukin 10 administered i.v. between 10 min and 6 h before lipopolysaccharide markedly inhibited brain tumor necrosis factor production. We also measured the production of tumor necrosis factor by whole blood of these mice, and it was also markedly inhibited by recombinant human interleukin 10 treatment. In conclusion, systemic recombinant human interleukin 10 administration inhibits brain tumor necrosis factor production. suggesting its usefulness in tumor necrosis factor-mediated pathologies of the central nervous system.

A glucocorticoid receptor-independent mechanism for neurosteroid inhibition of tumor necrosis factor production.

We investigated the effect of two neurosteroids, pregnenolone and dehydroepiandrosterone sulfate on lipopolysaccharide-induced tumor necrosis factor (TNF) production in vivo and in vitro. Dehydroepiandrosterone sulfate (0.3-30 mg/kg, i.p.) inhibited serum TNF induced by lipopolysaccharide (2.5 micrograms/mouse, i.p.), without affecting the induction of serum corticosterone. Intracerebroventricular (i.c.v.) administration of dehydroepiandrosterone sulfate (0.2-5 micrograms/mouse) also inhibited brain TNF induced by i.c.v. lipopolysaccharide (2.5 micrograms/mouse). Dehydroepiandrosterone sulfate and pregnenolone (10(-6)-10(-4) M) inhibited TNF production in vitro by lipopolysaccharide-stimulated human peripheral blood mononuclear cells or by the human THP-1 cell line, suggesting that this action might also be relevant in humans. We obtained two lines of evidence that neurosteroids do not inhibit TNF via the glucocorticoid receptor. (1) Dehydroepiandrosterone sulfate and pregnenolone did not activate the alpha 1-acid glycoprotein promoter, a typical effect of glucocorticoids mediated by the glucocorticoid receptor, while strong activation of this promoter was observed with dexamethasone. (2) The inhibitory effect of dehydroepiandrosterone sulfate and pregnenolone on TNF production was not reversed by the glucocorticoid receptor antagonist, mifepristone (RU38486). On the contrary the inhibitory effect of dexamethasone, a classical glucocorticoid and inhibitor of TNF synthesis, was completely reversed by RU38486.

MK 801 and dexamethasone reduce both tumor necrosis factor levels and infarct volume after focal cerebral ischemia in the rat brain.

Focal cerebral ischemia in rats produces elevated levels of tumor necrosis factor (TNF)alpha in the ischemic brain region. To better understand the modulation of TNF during brain ischemia processes we carried out studies in a model of permanent middle cerebral artery occlusion (MCAo) in the rat. In non-treated ischemic animals, the maximum expression of TNF was observed at 12 h (246.1+/-33 U/g) in the ischemic cortex and declined reaching near zero levels 24 h after MCAo. Given 10 min after MCAo, MK 801 (3 mg/kg, i.p.), a non-competitive NMDA receptor antagonist, exerted significant neuroprotection as measured by 47% reduction of total volume of infarction (P < 0.01 vs. ischemic-control). At the high dose of 3 mg/kg i.p., dexamethasone (DEX), which is known to reduce brain edema, decreased infarct size by 50% (P < 0.01 vs. ischemic-control). Both MK 801 and DEX reduced TNF production in the ipsilateral cortex of ischemic animals by 61 and 73%, respectively (P < 0.01 vs. ischemic-control). The data indicate that TNF levels increase after brain infarction, whereas they are reduced by neuroprotective agents, such as MK 801 and DEX, which act on different cellular levels.

Endogenous nitric oxide production by human monocytic cells regulates LPS-induced TNF production.

The ability to produce nitric oxide (NO) of human monocytes macrophages is object of debate. While studying the regulation of tumor necrosis factor (TNF) synthesis induced by endotoxin (LPS) in a human cell line of monocyte origin (THP-1) and in human peripheral blood mononuclear cells (PBMC) we found an indirect evidence of such production. We showed that L-N-monomethyl-arginine (L-NMMA), an inhibitor of NO synthase, and hemoglobin, a chelator of NO, are able to significantly reduce TNF synthesis, indicating that NO production is induced by LPS and contributes to the induction of TNF. Since NO is a known cytostatic agent, we also studied the cytostatic effect of LPS, and demonstrated that it is reverted by L-NMMA. Although we were unable to show any nitrites/nitrates accumulation in the culture media, taken together our data give an indirect evidence of a physiologically relevant LPS-induced NO production in human monocytes-macrophages.

Tumor necrosis factor is increased in the spinal cord of an animal model of motor neuron degeneration.

Autoimmunity and oxidative/excitotoxic damage are considered as possible pathogenetic mechanisms in amyotrophic lateral sclerosis (ALS). As tumor necrosis factor (TNF) is implicated in autoimmune diseases, including experimental autoimmune encephalomyelitis, and can be neurotoxic, we studied TNF production in a proposed animal model of ALS, the mnd mouse. These mice develop symptoms (progressive weakness of the limbs) as late as at 7 months of age. We measured TNF in serum, brain and spinal cord of mnd mice at 3 and 7 months of age. TNF was detectable in the brain and spinal cord (but not in the serum) at 7 months, while no TNF was detected in mnd mice at 3 months (asymptomatic) or in control mice of the same genetic background and the same age. Immunohistochemistry confirmed localization of TNF-alpha in motor neurons situated in the ventral horn of the spinal cord of 7-month old mnd mice. These results suggest the possibility of testing inhibitors of TNF production in this disease.

DHEAS inhibits TNF production in monocytes, astrocytes and microglial cells.

We previously reported that neurosteroids, including dehydroepiandrosterone sulfate (DHEAS), inhibit the production of TNF in vitro and in vivo. In this paper we evaluated the effect of DHEAS on TNF production by cultured rat astrocytes and murine glial cell clones, and compared it with the effect on monocytic THP-1 cells. We found that DHEAS at a concentration of 10(-4)-10(-7) M inhibits TNF production induced by lipopolysaccharide (LPS, 1 microgram/ml) in these cells. Since the inhibitory effect of DHEAS is not mediated by the glucocorticoid (GC) receptor and DHEAS is an allosteric antagonist of the GABAA receptor, we investigated the possible role of GABAA receptors in this effect. The results showed that the inhibitory effect of DHEAS (10(-6) M) on TNF production by THP-1 cells was completely reversed by addition of 10(-6) M GABA. However, a GABAA receptor antagonist (bicuculline) did not mimic the action of DHEAS. In conclusion, DHEAS can inhibit TNF production in astrocytic and microglial cells suggesting it could be an endogenous regulator of TNF production in the brain.

Role of the hypothalamus-pituitary-adrenal axis in the regulation of TNF production in mice. Effect of stress and inhibition of endogenous glucocorticoids.

Glucocorticoids (GC) are known inhibitors of TNF production and are increased by endotoxin (LPS) through a stimulation of the hypothalamus-pituitary-adrenal axis (HPAA), suggesting a feedback mechanism. We tried different approaches to study the role of the HPAA and endogenous GC in the regulation of TNF production. Cyanoketone, a GC synthesis inhibitor, inhibited corticosterone (CS) induction by LPS and increased LPS-induced serum TNF levels. Similar results were obtained by pretreating mice with anticorticotropin-releasing hormone Abs. Administration of adrenocorticotropic hormone increased blood CS and inhibited LPS-induced serum TNF. TNF production by mouse blood stimulated in vitro with LPS was inhibited by addition of CS. Blood from stressed or adrenocorticotropic-releasing hormone-treated mice (in which CS levels are elevated) stimulated ex vivo with LPS produced significantly less TNF than blood from control mice. Normal TNF production was restored by the addition of the GC receptor antagonist RU 38486, indicating a role for the elevated endogenous CS levels in the inhibition of TNF production. These data indicate that the HPAA is a major regulator of TNF production.

Peripheral effects of centrally administered interleukin-1beta in mice in relation to its clearance from the brain into the blood and tissue distribution.

Administration of interleukin IL-1 induces acute-phase response and inhibition of gastric secretion more efficiently when administered intracerebroventricularly (i.c.v.) than when the same dose of IL-1 is administered systemically. In this study we describe the pharmacokinetics of IL-1beta, administered centrally or systemically, in the serum or in peripheral tissues. IL-1beta administered i.c.v. resulted in higher peak IL-1beta concentrations, and lasted longer, than intravenous (i.v.) or intraperitoneal (i.p.) administration. Higher IL-1beta levels in the liver and heart were observed after i. c.v. administration (compared to the i.p. or i.v. route). Our data suggest that centrally injected IL-1 induces higher circulating and hepatic IL-1 levels and contributes to the fact that the i.c.v. route of administration is particularly effective in inducing a liver acute-phase response.

Differential effects of IL-6 on systemic and central production of TNF: a study with IL-6-deficient mice.

Interleukin 6 (IL-6) is known to inhibit the synthesis of tumour necrosis factor (TNF) in vitro and in vivo. In this study we investigated the possible role of IL-6 as an endogenous inhibitor of TNF production in the brain or in the periphery using IL-6-deficient mice or administering recombinant human IL-6 (rhIL-6). When IL-6-deficient mice were injected intracerebroventricularly (i.c.v.) with lipopolysaccaride (LPS), no differences were observed in the production of TNF in the brain, while in the periphery (serum or spleen) TNF levels were markedly increased (about four-fold). When normal mice were injected i.c.v. with a combination of LPS and rhIL-6, inhibition of TNF production was only slight (about 20%), while IL-6 had a stronger effect (> 80% inhibition) in the periphery. Co-administration of soluble IL-6 receptor (sIL-6R) did not enhance the effect of IL-6 on brain TNF, so this refractoriness cannot be attributed to a lack of IL-6 receptors. Interestingly, IL-6 potently inhibited LPS-induced TNF production by macrophagic cells but not by a microglial cell clone, suggesting that the defective response to IL-6 of the brain lies within the responsiveness TNF producing cells to IL-6. It thus appears that the TNF-inhibitory role of IL-6 is confined to the periphery.

Carrageenan-induced acute inflammation in the mouse air pouch synovial model. Role of tumour necrosis factor.

We used the mouse air pouch model of inflammation to study the interaction between cytokines, prostaglandin E(2) (PGE(2)) and cell migration during the various phases of acute local inflammation induced by carrageenan. In serum, the levels of interleukin 1 (IL-1), interleukin 6 (IL-6), tumour necrosis factor (TNF), serum amiloid-A (SAA) and Fe(++) were never different from controls, indicating that no systemic inflammatory changes were induced. Locally the exudate volume and the number of leukocytes recruited into the pouch increased progressively until 7 days after carrageenan. The same was true for PGE(2) production. We could not measure IL-1 but the production of IL-6 and TNF reached a maximum after 5-24 h then quickly decreased. Anti-TNF antibodies inhibited cell migration by 50% 24 h after treatment. Pretreatment with interleukin 10 (IL-10) inhibited TNF production almost completely and cell migration by 60%. Carrageenan-induced inflammation was modulated by anti-inflammatory drugs. Pretreatment with dexamethasone (DEX) or indomethacin (INDO) inhibited cell migration and reduced the concentration of TNF in the exudate. Production of PGE(2) or vascular permeability did not correlate with the number of cells in the pouch. Local TNF seems to play an important role in this model, particularly for leukocyte migration in the first phase of the inflammatory process. In conclusion, the air pouch seems to be a good model for studying the regulation of the early events of local inflammation, particularly the role of cytokines and cell migration.

IL-13 inhibits TNF production but potentiates that of IL-6 in vivo and ex vivo in mice.

IL-13 was reported to inhibit the synthesis of various cytokines in vitro, including that of TNF. It has divergent effects on IL-6 production, which is increased in endothelial cells and decreased in monocytes. We studied the effect of IL-13 administration on TNF and IL-6 production in vivo in mice. IL-13 (1 microg/mouse, i.v., 10 min to 6 h before LPS) decreased LPS (100 ng/mouse, i.v.)-induced serum TNF levels by 50%, while it increased the levels of IL-6 by fourfold. IL-13 potentiated IL-1beta (100 ng/mouse, i.v.)-induced serum IL-6 levels as well as IL-1- or LPS-induced serum amyloid A. When blood from IL-13-treated mice was stimulated with LPS in vitro, TNF production was decreased fivefold, and that of IL-6 was slightly decreased. We also cultured in vitro the aorta obtained from IL-13-pretreated mice and found that they produce more IL-6 (up to sevenfold) than aorta from control mice. Little or no TNF could be detected in these samples. Thus, IL-13 in vivo inhibits serum TNF but up-regulates serum IL-6. The differential regulation of IL-6 and TNF together with the results of ex vivo experiments could be explained by hypothesizing that the cellular origins of the two cytokines are different.

Hyperresponsive febrile reactions to interleukin (IL) 1alpha and IL-1beta, and altered brain cytokine mRNA and serum cytokine levels, in IL-1beta-deficient mice.

IL-1beta is an endogenous pyrogen that is induced during systemic lipopolysaccharide (LPS)- or IL-1-induced fever. We have examined the fever and cytokine responses following i.p. injection of IL-1 agonists, IL-1alpha and IL-1beta, and compared these with response to LPS (i.p.) in wild-type and IL-1beta-deficient mice. The IL-1beta deficient mice appear to have elevated body temperature but exhibit a normal circadian temperature cycle. Exogenously injected IL-1beta, IL-1alpha, or LPS induced hyperresponsive fevers in the IL-1beta-deficient mice. We also observed phenotypic differences between wild-type and IL-1beta-deficient mice in hypothalamic basal mRNA levels for IL-1alpha and IL-6, but not for IL-1beta-converting enzyme or IL-1 receptor type I or type II. The IL-1alpha mRNA levels were down-regulated, whereas the IL-6 mRNA levels were up-regulated in the hypothalamus of IL-1beta-deficient mice as compared with wild-type mice. The IL-1beta-deficient mice also responded to LPS challenge with significantly higher serum corticosterone and with lower serum tumor necrosis factor type alpha levels than the wild-type mice. The data suggest that, in the redundant cascade of proinflammatory cytokines, IL-1beta plays an important but not obligatory role in fever induction by LPS or IL-1alpha, as well as in the induction of serum tumor necrosis factor type alpha and corticosterone responses either by LPS or by IL-1alpha or IL-1beta.

Interleukin-10 inhibits lipopolysaccharide-induced tumor necrosis factor and interleukin-1 beta production in the brain without affecting the activation of the hypothalamus-pituitary-adrenal axis.

Interleukin (IL) 10 inhibits endotoxin (lipopolysaccharide; LPS) induced tumor necrosis factor (TNF) production in vivo and in vitro. In turn, IL-10 is induced by LPS and acts as a negative feedback to limit TNF production. We investigated the effects of IL-10 on brain TNF and IL-1 beta production induced by a central LPS administration in mice. Because central LPS also induces peripheral TNF, we also measured the serum TNF levels. A single intracerebroventricular injection of murine recombinant IL-10 (75 ng/mouse) simultaneously with LPS (2.5 micrograms/mouse) almost completely inhibited brain TNF production. The brain IL-1 beta production was also inhibited, as was the serum concentration of the acute-phase protein serum amyloid A. On the other hand, intracerebroventricular administration of an anti-IL-10 monoclonal antibody (JES5-2A5; 60 micrograms/mouse) potentiated brain TNF and IL-1 beta production. Identical results were obtained when the serum TNF levels were measured. IL-10 did not affect the LPS-induced increase of serum corticosterone, the main endogenous inhibitor of TNF production, or the induction of IL-6. These results indicate that LPS-induced IL-10 can act as an important endogenous inhibitor of brain TNF production and suggest an anti-inflammatory role for IL-10 in the central nervous system.