Serum and cerebrospinal fluid neurofilament light chains measured by SIMOA™, Ella™, and Lumipulse™ in multiple sclerosis naïve patients.

BACKGROUND: Neurofilament light chains (NfL) are cytoskeletal biomarkers of axonal damage, about 40-fold higher in cerebrospinal fluid (CSF) compared to serum, and requiring ultrasensitive techniques to be measured in this latter fluid. OBJECTIVES: To compare CSF and serum NfL levels in multiple sclerosis (MS) patients using different platforms. METHODS: 60 newly diagnosed relapsing-remitting MS patients (38 females; median age: 36.5 years, range: 15-60) were enrolled before steroid or disease-modifying treatments. CSF and serum NfL were measured with: the commercial Ella™ microfluidic platform (Bio-Techne), the Lumipulse™ Chemiluminescent Enzyme ImmunoAssay (Fujirebio), and the SIMOA™ on the SR-X instrument using NF-light assays (Quanterix). RESULTS: CSF and serum NfL absolute levels strongly correlated between assays, although being more elevated with Ella™. Passing-Bablok regression showed high agreement in measuring CSF NfL between assays (with greater proportional difference using Ella™), and very high agreement for serum comparing SIMOA™ and Lumipulse™. Similarly, the Bland-Altman comparison evidenced lower biases for Lumipulse™ for both fluids. CONCLUSIONS: CSF and serum NfL in naïve MS patients are reliably measured with all assays. Although not interchangeable, SIMOA™ and Lumipulse™ showed high agreement for serum and CSF values.

Lack of association between IL-1ß, TNF-α, and IL-10 gene polymorphisms and sporadic Parkinson's disease in an Italian cohort.

OBJECTIVE: There is increasing evidence suggesting that neuroinflammation and microglia activation may play important roles in the pathway leading to neuronal cell death in Parkinson's disease (PD). Chronic activation of microglia may cause neuronal damage through the release of potentially cytotoxic molecules, such as pro-inflammatory cytokines. Different functional promoter polymorphisms within genes coding pro- or anti-inflammatory cytokines involved in the immune reactions in the brain might influence the risk of developing PD or the age of disease onset. AIM: To investigate the interleukin (IL)-1ß-511, tumor necrosis factor alpha (TNF-α)-308, and interleukin (IL)-10-1082 gene polymorphisms as susceptibility factors for PD. METHODS: We analyzed genotype and allele distributions of these polymorphisms in 146 Italian patients with PD and 156 healthy controls. RESULTS: None of the polymorphisms we investigated was found to be associated with PD or with age of disease onset. No significant differences between patients with PD and controls were found as regards the concomitant presence of variant alleles in the three polymorphisms studied. We found that only the combined genotype TNF-α-308GG/IL-1ß-511T+ is associated with a decreased risk of PD. CONCLUSION: Our results indicate that the cytokine gene polymorphisms we investigated are not related to the development of PD in the Italian population; further studies are warranted to clarify the role of the TNF-α-308GG/IL-1ß-511T+ combined genotype.

High level of TILs is an independent predictor of negative sentinel lymph node in women but not in men.

Factors that are most associated with positive lymph node status in melanoma are Breslow thickness and ulceration. However, there are other factors that have been little explored and could help in the identification of "at risk patients" harbouring occult metastasis. The objective of this study was to determine whether intensity of tumour-infiltrating lymphocytes (TILs) in a cohort study (N = 4133) is an independent predictor of sentinel lymph node (SLN) status in patients with primary cutaneous melanoma. Of the patients with cutaneous melanoma who resulted negative for nodal metastasis, 50.7% had moderate/marked TILs versus 27.7% among those patients who resulted positive for nodal metastasis. In the multivariate analysis, controlling for sex, age, mitotic rate, ulceration and Breslow, high levels of TILs in primary invasive melanoma was associated with a lower risk of developing SLN metastasis (OR 0.46; 95% CI 0.23-0.95, p = 0.037). When the analysis was stratified by sex, the protective effect of moderate/marked TIL remained only for women (OR 0.30; 95% CI 0.10-0.93, p = 0.037) but not for men (OR 0.51; 95% CI 0.19-1.34, p = 0.172). Other independent predictors of negative lymph node were low Breslow thickness (≤ 2.0 mm) and low mitotic rate. Besides predicting a negative lymph node response, TILs were also associated with a decreased risk of 10-year mortality among females with positive lymph node. Our findings suggest that high level of TILs is an independent predictor of negative SLN status among women. Further research is warranted to confirm our findings.

Sentinel lymph node biopsy for high risk cutaneous squamous cell carcinoma: case series and review of the literature.

AIMS: Cutaneous squamous cell carcinoma (SCC) is the second most common skin cancer. The metastatic potential is generally low. However, there are subgroups of patients at higher risk, for whom sentinel lymph node biopsy (SLNB) might be useful. SLNB might allow the timely inclusion of high risk patients in more aggressive treatment protocols, sparing at the same time node-negative patients the morbidity of potentially unnecessary therapy. Our aim was to introduce the concept of SLNB for patients with high risk cutaneous SCC. PATIENTS AND METHODS: We examined a consecutive series of high risk cutaneous SCC patients undergoing SLNB at our large dermatological hospital, and performed a literature review and pooled analysis of all published cases of SLNB for cutaneous SCC. RESULTS: Among the 22 clinically node-negative patients undergoing SLNB at our hospital, one patient (4.5%) showed a histologically positive sentinel node and developed recurrences during follow-up. Sentinel node-negative patients showed no metastases at a median follow-up of 17 months (range: 6-64). The incidence of positive sentinel nodes in previous reports ranged between 12.5% and 44.4%. Pooling together patients from the present and previous studies (total 83 patients), we calculated an Odds Ratio of 2.76 (95% CI 1.2-6.5; p=0.02) of finding positive sentinel nodes for an increase in tumor size from <2 cm to 2.1-3 cm to >3 cm. CONCLUSIONS: Our case series and the pooled analysis support the concept that SLNB can be performed for high risk cutaneous SCC. Prospective multicenter studies are needed to examine the role, utility and cost-effectiveness of SLNB for this population.

Detection of anti-endomysial and anti-tissue transglutaminase autoantibodies in media following culture of oral biopsies from patients with untreated coeliac disease.

BACKGROUND AND AIMS: Coeliac disease is an autoimmune disorder characterised by high levels of anti-endomysial and anti-tissue transglutaminase autoantibodies in sera and media of cultured intestinal mucosa biopsies from affected patients. In this study, we wished to investigate whether anti-endomysial and anti-tissue transglutaminase antibodies can also be detected in culture media of oral mucosa specimens, and whether the mouth can be used as an area of immunological testing for coeliac disease. METHODS: Small intestine and cheek biopsy samples taken from 16 patients with active coeliac disease and from 11 controls were cultured in vitro for 48 h at 37 degrees C in presence of medium alone. Anti-endomysial and anti-tissue transglutaminase were detected in sera and in supernatants of these cultured biopsy samples by indirect immunofluorescence and enzyme immunoassay (EIA), respectively. RESULTS: Anti-endomysial and anti-tissue transglutaminase were positive in sera of 15/16 coeliac disease patients. Culture media of intestinal mucosa samples from 14/16 coeliac disease patients were anti-endomysial positive, while the same antibodies were positive in supernatants of cultured oral mucosa samples from 15/16 coeliac disease patients. Anti-tissue transglutaminase were positive in both intestinal and oral culture media of 15/16 coeliac disease patients. Neither anti-endomysial nor anti-tissue transglutaminase were found in sera or in culture supernatants of both intestinal and oral biopsy samples from 11 controls. CONCLUSIONS: Our study suggests a new immunological site to detect the pathognomonic autoantibodies of coeliac disease and confirms that the mouth is involved in this illness.

Cerebral venous hemodynamic abnormalities in episodic and chronic migraine.

Alterations of cerebral venous drainage have been demonstrated in chronic migraine (CM), suggesting that cerebral venous hemodynamic abnormalities (CVHAs) play a role in this condition. The aim of the present study was to look for a correlation between CM and CVHAs. We recruited 33 subjects suffering from CM with or without analgesic overuse, 29 episodic migraine (EM) patients with or without aura, and 21 healthy subjects as controls (HCs). CVHAs were evaluated by transcranial and extracranial echo-color Doppler evaluation of five venous hemodynamic parameters. CVHAs were significantly more frequent in the CM and EM patients than in the HCs. In the migraine patients, CVHAs were not correlated with clinical features. The significantly greater frequency of CVHAs observed in the migraineurs may reflect a possible relationship between migraine and these abnormalities. Prospective longitudinal studies are needed to investigate whether CVHAs have a role in the processes of migraine chronification.

Functional correlates of nicotine administration: similarity with drugs of abuse.

Changes in the local utilization of cerebral glucose resulting from administered drugs acting on the central nervous system can be evaluated quantitatively by the [14C]2-deoxyglucose method. We report the findings obtained by the [14C]2-deoxyglucose method that contribute to understanding the cerebral functional effects of drugs of abuse and discuss in particular the similarities between nicotine and other addictive drugs. A common consequence of the intravenous administration of psychomotor stimulants and opioids in the rat is the increase in glucose utilization in the shell of nucleus accumbens. This functional change is accompanied by increased local extracellular concentrations of dopamine. Altered functional activity and dopamine neurotransmission in the shell of the nucleus accumbens thus represent distinctive neurobiological markers of the addictive properties of several drugs, independently of the specific neurochemical mechanisms of action. It has recently been shown that the intravenous administration of a pulse of nicotine, at single-unit doses corresponding to those that maintain self-administration in the rat, produces neurochemical and metabolic changes in the shell of the nucleus accumbens that closely resemble those of psychomotor stimulants and opioids. The latter results demonstrate that nicotine shares with highly addictive drugs a distinct neurochemical and functional consequence. They therefore contribute to the neurochemical definition of the addictive nature of nicotine. These neurochemical and functional changes may contribute to the changes in expression of intracellular second messengers and neurotransmitter/receptor systems observed particularly in the shell following the administration of drugs of abuse.

A two-step mechanism by which corticotropin-releasing hormone releases hypothalamic beta-endorphin: the role of vasopressin and G-proteins.

It is well established that in the pituitary gland corticotropin-releasing hormone (CRH) stimulates the release of beta-endorphin (beta-E) via a cAMP-linked mechanism. Studies of the mechanisms underlying the CRH stimulation of beta-E release from rat hypothalamic slices perifused in vitro are reported in this paper. The data indicate that both a cAMP-dependent and non-cAMP-dependent mechanism mediate the action of CRH in the hypothalamus. The presence of a cAMP-linked mechanism was suggested by the finding that cholera toxin (0.1-10 nM) and forskolin (2.5 x 10(-6) M), both of which act to raise intracellular cAMP levels, stimulated the release of beta-E. In both cases, no further stimulation was seen upon addition of CRH (10(-8)M). However, it was also found that preincubation of the tissue with pertussis toxin (PTX; 100 ng/ml) prevented both the CRH- and forskolin-stimulated release of beta-E. This indicated that, in addition to the cAMP-linked mechanism, a further messenger system which is connected to a PTX-sensitive G-protein may also play a role. The latter observation also implied that a further substance, which utilizes a separate second messenger system, might be involved in the CRH stimulation of beta-E release. In this regard the role of arginine vasopressin (AVP) was investigated due to the known interaction between CRH and AVP in the pituitary gland. AVP (10(-12) to 10(-6)M) itself potently and dose-dependently stimulated beta-E release, producing a maximal increase of 220% above basal levels. The AVP-induced release of beta-E was abolished in PTX-pretreated hypothalami. The apparently obligatory requirement of AVP for the CRH-stimulation of beta-E release was illustrated by the finding that blockade of AVP receptors using the AVP antagonist d(CH2)5 [Tyr(OEt)2,Val4]-AVP almost completely attenuated the CRH-stimulated release of beta-E. Furthermore, in the presence of a high concentration of AVP (10(-6)M) no further stimulation of release was seen with CRH (10(-8)M). These data therefore strongly indicate that CRH acts via the intermediacy of AVP to release beta-E from hypothalamic slices in vitro and that two separate second messenger systems are involved: a cAMP-linked mechanism connected to a cholera toxin-sensitive G-protein (CRH) and a second system linked to a PTX-sensitive G-protein (AVP).

Effect of collateral blood flow and cerebral vasomotor reactivity on the outcome of carotid artery occlusion.

BACKGROUND AND PURPOSE: Evidence suggests that an alteration in cerebral hemodynamics plays a relevant role in the occurrence of stroke in patients with carotid occlusion. The purpose of the present study was to evaluate the relationships among baseline characteristics, type and number of collateral pathways, cerebral vasomotor reactivity (VMR), and outcome of patients with carotid occlusion. METHODS: One hundred four patients with symptomatic or asymptomatic internal carotid artery occlusion were followed up prospectively for a median period of 24 months. Cerebral VMR to apnea was calculated with transcranial Doppler ultrasonography by means of the breath-holding index (BHI) in the middle cerebral arteries. The patency of the 3 major intracranial collateral vessels was also evaluated. RESULTS: During the follow-up period, 18 patients experienced an ischemic stroke ipsilateral to internal carotid artery occlusion. Among factors considered, only older age, number of collateral pathways, and BHI values in the middle cerebral artery ipsilateral to the occluded side were significantly associated with the risk of ipsilateral stroke (P<0.001, P=0.008, and P<0.001, respectively; multiple Cox regression analysis). A normal VMR and favorable prognosis characterized patients with full collateral development; in this group, no patient experienced an ischemic event. On the other hand, an impaired VMR and increased probability of experiencing a stroke were found in patients without collateral pathways; the annual risk of ipsilateral stroke in this group was 32.7%. Patients with 1 or 2 collateral pathways showed a different VMR ranging from normal to strongly reduced BHI values. The ipsilateral stroke event risk was 17.5% in patients with 1 collateral vessel and 2.7% in patients with 2 collateral pathways. In this case, the risk of cerebrovascular events occurring during the follow-up period was significantly related to VMR. CONCLUSIONS: These data suggest that cerebral hemodynamic status in patients with carotid occlusive disease is influenced by both individual anatomic and functional characteristics. The planning of strategies to define the risk profile and any attempt to influence patients' outcome should be based on the evaluation of the intracranial hemodynamic adaptive status, with particular attention to the number of collateral vessels and the related VMR.

Parkinson's disease and somatosensory evoked potentials: apomorphine-induced transient potentiation of frontal components.

We recorded somatosensory evoked potentials (SEPs) to median nerve stimulation from parietal and frontal districts in 32 patients with Parkinson's disease by evaluating latency/amplitude characteristics of the parietal P14-N20-P25 and of the frontal P20-N30-P40 wave complexes before and 10, 20, 30, and 60 minutes after subcutaneous administration of apomorphine chloride. The frontal complex N30-P40 was smaller than normal in 17 patients in baseline recordings. Following apomorphine, the parietal responses did not significantly vary in amplitude, but the frontal complex showed a remarkable amplitude potentiation in 22 of 32 patients (68.7%, p < 0.001), 19 of whom were also improving clinically. Amplitude potentiation was evident 10 minutes after apomorphine and faded away nearly in parallel with the end of its clinical efficacy. There were no SEP changes in three healthy controls after apomorphine.

Effects of chronic treatment with imipramine, trazodone and electroshock on the behavioural and electroencephalographic modifications induced by clonidine in the rat.

The influence of some typical and atypical antidepressant drugs on the EEG and behavioural effects of clonidine has been studied. The results indicate that chronic administration of imipramine or of electroshock led to the abolition of the behavioural depression and of the EEG synchronisation induced by clonidine. Chronic administration of trazodone was without effect. These results support the hypothesis that chronic treatment with imipramine and electroshock elicits a down-regulation of noradrenergic presynaptic receptors. These structures seem to be largely unaffected by trazodone, which could have a favourable effect on depression through its influence on beta-adrenergic receptors and on the 5-HT system.

Effects of chronic treatment with imipramine on the behavioural and electroencephalographic modifications induced by clonidine in the rat.

The influence of repeated administration of imipramine on the EEG and behavioural effects of clonidine has been studied in the rat bearing chronic electrodes. Clonidine induced behavioural depression and EEG synchronization in control rats. Mydriasis, hyperirritability, stereotyped behaviour and EEG desynchronization were elicited by clonidine on the first and second days after discontinuation of the treatment with imipramine (15 mg/kg, i.p., daily for 21 days). On the 9th. day the animals responded to clonidine with sedation and EEG synchronization. These results support the hypothesis that chronic treatment with tricyclic antidepressant drugs changes the sensitivity of central noradrenergic neurones.

An increased expression of the mGlu5 receptor protein following LTP induction at the perforant path-dentate gyrus synapse in freely moving rats.

The involvement of metabotropic glutamate (mGlu) receptors in the induction of long-term potentiation (LTP) in vivo has been consistently documented. We have investigated whether LTP induction in the dentate gyrus of rats leads to changes in expression of mGlu2/3 or -5 receptor subtypes in the hippocampus. LTP was induced at the medial perforant path-dentate gyrus synapses, and mGlu receptor expression was examined by Western blot or in situ hybridization. An up-regulation of mGlu5 receptors was observed in the hippocampus both 24 and 48 h following LTP induction. This effect was restricted to the dentate gyrus and CA1 region, whereas no changes in mGlu5 receptor protein (but an increase in mRNA levels) were observed in the CA3 region. The increased expression of mGlu5 receptors was directly related to the induction of LTP, because it was not observed when tetanic stimulation was carried out in animals treated with the NMDA receptor antagonist, 2-amino-5-phosphonopentanoate (AP5). Western blot analysis also showed a reduced expression of mGlu2/3 receptors in the whole hippocampus 24 h after LTP induction, indicating that the increased expression of mGlu5 receptors was specific. These data suggest that an up-regulation of mGlu5 receptors is a component of the plastic changes that follow the induction of LTP at the perforant path-dentate gyrus synapse.

Effect of electroconvulsive shock treatment on the opioid-mediated inhibition of serotonin release in rat hippocampal slices.

We investigated the influence of electroconvulsive shock treatment on the opioid-mediated inhibition of serotonin (5-HT) release. The overflow of [3H]serotonin elicited by high potassium (30 mM) in superfused slices of rat hippocampus was significantly inhibited in the presence of two agonists, mu-selective [D-Ala2,N-methyl-Phe4,Gly5ol]enkephalin (DAGO) and delta-selective [D-Pen2,D-Pen5]enkephalin (DPDPE) in control animals. The repeated (once daily for 7 days) electroconvulsive shock treatment significantly reversed the inhibitory effect of both DAGO and DPDPE. Single electroconvulsive shock (1 day) had no effect on DAGO inhibition, on the contrary a trend to antagonism of the DPDPE effect was reported. These results suggested that the opioids are associated with the experimental induced seizures and that the ECS treatment might regulate the serotonin release throughout the opioid receptor system.

Psychostimulant drugs increase glucose utilization in the shell of the rat nucleus accumbens.

The nucleus accumbens (NAc) is a heterogeneous area, divided into a 'shell' and a 'core' on the basis of morphological and histochemical criteria. Moreover, the two portions have different anatomical connections. In order to test whether the two portions of NAc are functionally distinct, we measured local cerebral glucose utilization in rats administered intravenously with cocaine (1 mg kg-1) or amphetamine (0.5 mg kg-1). The results of the study show that, at these dosages, both drugs increase glucose utilization in the shell, but not in the core of NAc. This differential effect might reflect functional differences between the two portions of NAc, probably relevant to the abuse liability of psychostimulants.

Effects of single and repeated electroconvulsive shock on local cerebral glucose utilization in the conscious rat.

Local rates of cerebral glucose utilization (LCGU) were determined in adult rats submitted to electroconvulsive shock (ECS) treatment. One group of rats (n = 5) received a single ECS, a second group (n = 5) was treated once daily for 7 days. A third group (n = 5) of unshocked rats served as control. LCGU measurements were performed one day after either the single or the last ECS. Following a single ECS, most of the 45 brain regions examined exhibited lower rates of LCGU than controls, in contrast after repeated ECS treatment the metabolic activity results increased in segments of the hippocampus. The results indicate that repeated ECS treatment selectively increases metabolic rates within the hippocampus, a structure of the limbic system thought to be related to affective disorders and memory.

Dopamine release in striatal slices of rats previously submitted to electroconvulsive shock.

The effects of single and repeated electroconvulsive shock (ECS) on the release of dopamine from rat striatal slices were investigated using the fractional release technique. Experiments were performed 24 h after the single or the last of seven ECS sessions. Repeated, but not single, ECS was associated with reduced dopamine release in response to chemical stimulation. These results suggest that repeated ECS affects the regulation of striatal dopamine presynaptic receptors.

Differential effects of acute administration of clozapine or haloperidol on local cerebral glucose utilization in the rat.

We employed the [14C]2-deoxyglucose method in order to map local brain metabolic activity of rats administered 1, 5, or 20 mg/kg of clozapine, or 0.5 mg/kg of haloperidol, as compared to saline. Clozapine produced a dose-dependent reduction of glucose utilization. At the dose of 1 mg/kg, the effects were limited to limbic areas. An additional number of structures were significantly affected following administration of 5 mg/kg (the whole hippocampal formation and septal area, and cortical limbic areas). The dose of 20 mg/kg markedly reduced glucose utilization in most of the areas examined. Haloperidol (0.5 mg/kg) reduced glucose utilization of the orbital cortex, hippocampal formation and septal area, globus pallidus, amygdala, ventral thalamus, and substantia nigra reticulata. The results show that acute administration of clozapine or haloperidol are associated with different distribution patterns of altered cerebral energy metabolism. Clozapine differently from haloperidol, reduces energy metabolism of the nucleus accumbens and other limbic areas. Haloperidol, but not clozapine (1 or 5 mg/kg), affects the substantia nigra reticulata.

Repeated electroconvulsive shock increases glial fibrillary acidic protein, ornithine decarboxylase, somatostatin and cholecystokinin immunoreactivities in the hippocampal formation of the rat.

Rats were submitted to single or repeated (7 days, one session for each day) sessions of electroconvulsive shock. A computer-assisted morphometric and microdensitometric analysis of glial fibrillary acidic protein-, ornithine decarboxylase-, somatostatin- and cholecystokinin-like immunoreactivities was performed in the hippocampal formation and other brain areas. The results of the study showed a significant increase of the intensity of the immunostaining for glial fibrillary acidic protein, ornithine decarboxylase, somatostatin and cholecystokinin in the hippocampal formation and distinctively in the dentate gyrus following repeated, but not single, electroconvulsive shock. No significant change was found in the number of somatostatin- and cholecystokinin-like immunoreactive cell bodies in any hippocampal subregion and in the number of glial cells in the hilus of dentate gyrus in rats treated with single or repeated electroconvulsive shock. It is a distinct possibility that the observed increase in the content of the neuropeptides in the hippocampal formation reflects a compensatory response of the brain to seizure-inducing stimuli and that such an increase may play a role in the therapeutic effect of electroconvulsive shock.

Pertussis toxin inactivates the presynaptic serotonin autoreceptor in the hippocampus.

The serotonin (5-HT) agonists, D-lysergic acid diethylamide (LSD) and 5-methoxytryptamine (5-Me-O-T), dose dependently inhibited the K+-evoked outflow of [3H]5-HT from preloaded rat hippocampal slices in vitro, indicating activation of the 5-HT autoreceptor. However, this effect was abolished in slices pretreated with pertussis toxin. It is thus concluded that the 5-HT autoreceptor in the hippocampus is coupled to G proteins.