Giulia Maria Sancesario

Phase I Clinical Trial of Smad7 Knockdown Using Antisense Oligonucleotide in Patients With Active Crohn's Disease

In the gut of patients with Crohns disease (CD), high Smad7 blocks the immune-suppressive activity of transforming growth factor (TGF)-β1, thereby contributing to amplify inflammatory signals. In vivo in mice, knockdown of Smad7 with a Smad7 antisense oligonucleotide (GED0301) attenuates experimental colitis. Here, we provide results of a phase 1 clinical, open-label, dose-escalation study of GED0301 in patients with active, steroid-dependent/resistant CD, aimed at assessing the safety and tolerability of the drug. Patients were allocated to three treatment groups receiving oral GED0301 once daily for 7 days at doses of 40, 80, or 160 mg. A total of 15 patients were enrolled. No serious adverse event was registered. GED0301 was well tolerated and no patient dropped out during the study. Twenty-five adverse events were documented in 11 patients, the majority of whom were judged to be of mild intensity and unrelated to treatment. GED0301 treatment reduced the percentage of inflammatory cytokine-expressing CCR9-positive T cells in the blood. The study shows for the first time that GED0301 is safe and well tolerated in patients with active CD.

Orexinergic system dysregulation, sleep impairment, and cognitive decline in Alzheimer disease.

IMPORTANCE Nocturnal sleep disruption develops in Alzheimer disease (AD) owing to the derangement of the sleep-wake cycle regulation pathways. Orexin contributes to the regulation of the sleep-wake cycle by increasing arousal levels and maintaining wakefulness. OBJECTIVES To study cerebrospinal fluid levels of orexin in patients with AD, to evaluate the relationship of orexin cerebrospinal fluid levels with the degree of dementia and the cerebrospinal fluid AD biomarkers (tau proteins and β-amyloid 1-42), and to analyze potentially related sleep architecture changes measured by polysomnography. DESIGN, SETTING, AND PARTICIPANTS We conducted a case-control study from August 1, 2012, through May 31, 2013. We included 48 drug-naive AD patients referred to the Neurological Clinic of the University Hospital of Rome Tor Vergata. Based on the Mini-Mental State Examination score, 21 patients were included in mild AD group (score, ≥21), whereas 27 were included in the moderate to severe AD group (score, <21). The control group consisted of 29 nondemented participants of similar age and sex. EXPOSURE Laboratory assessment of cerebrospinal fluid levels of orexin, tau proteins, and β-amyloid 1-42 and polysomnographic assessment of sleep variables. MAIN OUTCOMES AND MEASURES Levels of orexin, tau proteins, and β-amyloid 1-42; macrostructural variables of nocturnal sleep (total sleep time, sleep efficiency, sleep onset and rapid eye movement [REM] sleep latencies, non-REM and REM sleep stages, and wakefulness after sleep onset); and Mini-Mental State Examination scores. RESULTS Patients with moderate to severe AD presented with higher mean (SD) orexin levels compared with controls (154.36 [28.16] vs 131.03 [26.55]; P < .01) and with more impaired nocturnal sleep with respect to controls and patients with mild AD. On the other hand, in the global AD group, orexin levels were positively correlated with total tau protein levels (r = 0.32; P = .03) and strictly related to sleep impairment. Finally, cognitive impairment, as measured by the Mini-Mental State Examination, was correlated with sleep structure deterioration. CONCLUSIONS AND RELEVANCE Our results demonstrate that, in AD, increased cerebrospinal fluid orexin levels are related to a parallel sleep deterioration, which appears to be associated with cognitive decline. Therefore, the orexinergic system seems to be dysregulated in AD, and its output and function appear to be overexpressed along the progression of the neurodegenerative process. This overexpression may result from an imbalance of the neurotransmitter networks regulating the wake-sleep cycle toward the orexinergic system promoting wakefulness.

Tumor necrosis factor is elevated in progressive multiple sclerosis and causes excitotoxic neurodegeneration

Background: Chronic inflammation leads to gray matter damage in progressive multiple sclerosis (MS), but the mechanism linking inflammation and neurodegeneration is unclear. Objective: The objective of this paper is to investigate the synaptic mechanism of inflammatory neurodegeneration in progressive forms of MS. Methods: Cytokine and neurofilament-light were determined in cerebrospinal fluid (CSF) of MS patients. In vitro electrophysiology and cell swelling experiments were performed to measure the effects of inflammatory cytokines in the CSF of MS patients on synaptic transmission and neuronal integrity. Results: Tumor necrosis factor-α (TNF) was higher in CSF of progressive MS subjects, and caused excitotoxic neuronal death in vitro. In murine brain slices incubated in the presence of CSF from progressive MS, in fact, we observed increased spontaneous excitatory postsynaptic currents (sEPSCs) and glutamate-mediated neuronal swelling through a mechanism dependent on enhanced TNF signaling. We also suggested a pathogenic role of B cells in TNF CSF increase, exacerbation of glutamatergic transmission and neuronal damage, since CNS depletion of B cells with intrathecal rituximab caused a dramatic reduction of TNF levels, of TNF-induced sEPSC alterations, and of neurofilament CSF concentrations in a patient with progressive MS. Conclusion: Our results point to TNF as a primary neurotoxic molecule in progressive forms of MS.

Cognitive and cortical plasticity deficits correlate with altered amyloid-β CSF levels in multiple sclerosis.

Cognitive dysfunction is of frequent observation in multiple sclerosis (MS). It is associated with gray matter pathology, brain atrophy, and altered connectivity, and recent evidence showed that acute inflammation can exacerbate mental deficits independently of the primary functional system involved. In this study, we measured cerebrospinal fluid (CSF) levels of amyloid-β1−42 and τ protein in MS and in clinically isolated syndrome patients, as both proteins have been associated with cognitive decline in Alzheimers disease (AD). In AD, amyloid-β1–42 accumulates in the brain as insoluble extracellular plaques, possibly explaining why soluble amyloid-β1–42 is reduced in the CSF of these patients. In our sample of MS patients, amyloid-β1–42 levels were significantly lower in patients cognitively impaired (CI) and were inversely correlated with the number of Gadolinium-enhancing (Gd+) lesions at the magnetic resonance imaging (MRI). Positive correlations between amyloid-β1–42 levels and measures of attention and concentration were also found. Furthermore, abnormal neuroplasticity of the cerebral cortex, explored with θ burst stimulation (TBS), was observed in CI patients, and a positive correlation was found between amyloid-β1–42 CSF contents and the magnitude of long-term potentiation-like effects induced by TBS. No correlation was conversely found between τ protein concentrations and MRI findings, cognitive parameters, and TBS effects in these patients. Together, our results indicate that in MS, central inflammation is able to alter amyloid-β metabolism by reducing its concentration in the CSF and leading to impairment of synaptic plasticity and cognitive function.

Aβ1–42 Detection in CSF of Alzheimer's disease is influenced by temperature: Indication of reversible Aβ1–42 aggregation?

Amyloid-beta 1-42 (Abeta1-42), peptide detectable in cerebrospinal fluid (CSF), has been extensively studied as diagnostic marker for Alzheimers disease; however, results are variable. We investigated whether Abeta1-42 detection in CSF may be affected by handling temperature after lumbar puncture. CSF was collected from patients affected by probable AD (n=27), other dementias (OD) (n=24), or other neurological disorders without cognitive impairment (OND) (n=23). After lumbar puncture, CSF samples were either maintained at 37 degrees C, or handled according to standard procedures and centrifuged at 4 degrees C for 10 min; thereafter, one aliquot was further stored at 4 degrees C and another at 37 degrees C, before freezing all samples 90 min later at -80 degrees C, pending analysis. Abeta1-42 and total tau were determined using a commercially available sandwich enzyme-linked immunosorbent assay ELISA. Reduced Abeta1-42 and increased total tau CSF levels were confirmed as characteristic hallmarks of the OD and AD groups, providing standard measurement in samples stored at 4 degrees C before freezing. However, avoiding cooling or reheating CSF from 4 to 37 degrees C before freezing strikingly increased the Abeta1-42 concentration detectable in the AD group (P<0.01), but not in control groups. The results indicate that a pool of Abeta1-42 cannot be detectable in the CSF of AD patients, because standard preanalytical cooling masks in some ways the epitope recognized by Abeta1-42 specific antibodies. Moreover, our study suggests that low temperature could induce Abeta1-42 conformational changes and multimeric aggregates in probable AD, but, more importantly, Abeta1-42 aggregation could be reversible.

Potential role of IL-13 in neuroprotection and cortical excitability regulation in multiple sclerosis

Background: Inflammation triggers secondary neurodegeneration in multiple sclerosis (MS). Objectives: It is unclear whether classical anti-inflammatory cytokines have the potential to interfere with synaptic transmission and neuronal survival in MS. Methods: Correlation analyses between cerebrospinal fluid (CSF) contents of anti-inflammatory cytokines and molecular, imaging, clinical, and neurophysiological measures of neuronal alterations were performed. Results: Our data suggest that interleukin-13 (IL-13) plays a neuroprotective role in MS brains. We found, in fact, that the levels of IL-13 in the CSF of MS patients were correlated with the contents of amyloid-β1-42. Correlations were also found between IL-13 and imaging indexes of axonal and neuronal integrity, such as the retinal nerve fibre layer thickness and the macular volume evaluated by optical coherence tomography. Furthermore, the levels of IL-13 were related to better performance in the low-contrast acuity test and Multiple Sclerosis Functional Composite scoring. Finally, by means of transcranial magnetic stimulation, we have shown that GABAA-mediated cortical inhibition was more pronounced in patients with high IL-13 levels in the CSF, as expected for a neuroprotective, anti-excitotoxic effect. Conclusions: The present correlation study provides some evidence for the involvement of IL-13 in the modulation of neuronal integrity and synaptic function in patients with MS.

CSF lactate levels, τ proteins, cognitive decline: a dynamic relationship in Alzheimer's disease

Objectives To investigate, in patients with Alzheimers Disease (AD), the possible interplay linking alteration of neuronal energy metabolism, as measured via cerebrospinal fluid (CSF) lactate concentration, to severity of AD neurodegenerative processes and impairment of cognitive abilities. Methods In this study we measured and correlated CSF lactate concentrations, AD biomarker levels (τ-proteins and β-amyloid) and Mini-Mental State Examination (MMSE) score in a population of drug-naïve patients with AD ranging from mild (MMSE≥21/30) to moderate-severe (MMSE<21/30) cognitive decline. They were compared to healthy controls and patients with vascular dementia (VaD). Results Patients with AD (n=145) showed a significant increase of CSF lactate concentration compared to controls (n=80) and patients with VaD (n=44), which was higher in mild (n=67) than in patients with moderate–severe AD (n=78). Moreover, we found, in either the whole AD population or both subgroups, a CSF profile in which higher CSF levels of t-τ and p-τ proteins corresponded to lower concentrations of lactate. Conclusions We verified the occurrence of high CSF lactate levels in patients with AD, which may be ascribed to mitochondria impairment. Hypothesising that τ proteins may exert a detrimental effect on the entire cellular energy metabolism, the negative correlation found between lactate and τ-protein levels may allow speculation that τ toxicity, already demonstrated to have affected mitochondria, could also impair glycolytic metabolism with a less evident increase of lactate levels in more severe AD. Thus, we suggest a dynamic relationship between neuronal energy metabolism, τ proteins and cognitive decline in AD and propose the clinical potential of assessing CSF lactate levels in patients with AD to better define the neuronal brain metabolism damage.

Cerebrospinal Fluid Levels of a 20–22 kDa NH2 Fragment of Human Tau Provide a Novel Neuronal Injury Biomarker in Alzheimer's Disease and Other Dementias

Truncation at N-terminal domain of tau protein is early associated with neurofibrillary pathology in several human tauopathies, including Alzheimers disease (AD). In affected subjects, the monitoring of total (t-tau) and/or phosphorylated tau (p-tau) levels in cerebrospinal fluid (CSF) provides a reliable, indirect evaluation of cellular changes occurring in vivo and the identification of additional CSF biomarkers would better assist with the clinical practice, allowing a broader profile of underlying ongoing neurodegeneration. Here we show that a 20-22 kDa NH2-truncated form of human tau (i.e., NH2htau), a neurotoxic fragment of the full length protein (htau40) that we previously found in synapses from subjects affected by different tauopathies: (i) is not a normal constituent of CSF, unlike t-tau and p-tau, being exceptionally detected in patients without cognitive impairment; (ii) discriminates, with a weak specificity of 65% but a high sensitivity of 85%, patients carrying a large spectrum of neurodegenerative diseases associated with cognitive deterioration (i.e., AD, frontotemporal lobar degeneration, Parkinsons disease with dementia, vascular dementia, mixed dementia, etc.) from subjects affected by other neurological disorders without mnesic disability; and (iii) is a neuronal injury biomarker as its levels in CSF are not related to the severity and progression of cognitive decline. The dynamic evaluation of NH2htau in CSF might add some useful hints in the ordinary clinical practice as it provides a novel, general biomarker for human tauopathies and other neurodegenerative diseases associated with dementia.

Cerebrospinal fluid lactate levels and brain [18F]FDG PET hypometabolism within the default mode network in Alzheimer’s disease

PurposeIt has been suggested that neuronal energy metabolism may be involved in Alzheimer’s disease (AD). In this view, the finding of increased cerebrospinal fluid (CSF) lactate levels in AD patients has been considered the result of energetic metabolism dysfunction. Here, we investigated the relationship between neuronal energy metabolism, as measured via CSF lactate levels, and cerebral glucose metabolism, as stated at the 2-deoxy-2-(18F)fluoro-D-glucose positron emission tomography ([18F]FDG PET) in AD patients.MethodsAD patients underwent lumbar puncture to measure CSF lactate levels and [18F]FDG PET to assess brain glucose metabolism. CSF and PET data were compared to controls. Since patients were studied at rest, we specifically investigated brain areas active in rest-condition owing to the Default Mode Network (DMN). We correlated the CSF lactate concentrations with the [18F]FDG PET data in brain areas owing to the DMN, using sex, age, disease duration, Mini Mental State Examination, and CSF levels of tau proteins and beta-amyloid as covariates.ResultsAD patients (n = 32) showed a significant increase of CSF lactate levels compared to Control 1 group (n = 28). They also showed brain glucose hypometabolism in the DMN areas compared to Control 2 group (n = 30). Within the AD group we found the significant correlation between increased CSF lactate levels and glucose hypometabolism in Broadman areas (BA) owing to left medial prefrontal cortex (BA10, mPFC), left orbitofrontal cortex (BA11, OFC), and left parahippocampal gyrus (BA 35, PHG).ConclusionWe found high CSF levels of lactate and glucose hypometabolism within the DMN in AD patients. Moreover, we found a relationship linking the increased CSF lactate and the reduced glucose consumption in the left mPFC, OFC and PHG, owing to the anterior hub of DMN. These findings could suggest that neural glucose hypometabolism may affect the DMN efficiency in AD, also proposing the possible role of damaged brain energetic machine in impairing DMN.

The Load of Amyloid-β Oligomers is Decreased in the Cerebrospinal Fluid of Alzheimer's Disease Patients

Amyloid-β (Aβ) oligomers are heterogeneous and instable compounds of variable molecular weight. Flow cytometry and fluorescence resonance energy transfer (FRET)-based methods allow the simultaneous detection of Aβ oligomers with low and high molecular weight in their native form. We evaluated whether an estimate of different species of Aβ oligomers in the cerebrospinal fluid (CSF) with or without dilution with RIPA buffer could be more useful in the diagnosis of Alzheimers disease (AD) than the measurement of Aβ42 monomers, total tau (t-tau), and phosphorylated tau (p-tau). Increased t-tau (p < 0.01) and p-tau (p < 0.01), and decreased Aβ42 (p < 0.01), were detected in the CSF of patients with AD (n = 46), compared to patients with other dementia (OD) (n = 35) or with other neurological disorders (OND) (n = 56). In native CSF (n = 137), the levels of Aβ oligomers were lower (p < 0.05) in AD than in OD and OND patients; in addition, the ratio Aβ oligomers/p-tau was lower in AD than in OD (p < 0.01) and OND (p < 0.05) patients, yielding a sensitivity of 75% and a specificity of 64%. However, in CSF diluted with RIPA (n = 30), Aβ oligomers appeared higher (p < 0.05) in AD than in OND patients, suggesting they become partially disaggregated and more easily detectable after RIPA. In conclusion, FRET analysis in native CSF is essential to correctly determine the composition of Aβ oligomers. In this experimental setting, the simultaneous estimate of low and high molecular weight Aβ oligomers is as useful as the other biomarkers in the diagnosis of AD. The low amount of Aβ oligomers detected in native CSF of AD may be inversely related to their levels in the brain, as occurs for Aβ monomers, representing a biomarker for the amyloid pathogenic cascade.