Roberta Mancuso

Microbes and Alzheimer's Disease

We are researchers and clinicians working on Alzheimer’s disease (AD) or related topics, and we write to express our concern that one particular aspect of the disease has been neglected, even thoug ...

Human endogenous retrovirus type W envelope expression in blood and brain cells provides new insights into multiple sclerosis disease

Background: The envelope protein from multiple sclerosis (MS) associated retroviral element (MSRV), a member of the Human Endogenous Retroviral family ‘W’ (HERV-W), induces dysimmunity and inflammation. Objective: The objective of this study was to confirm and specify the association between HERV-W/MSRV envelope (Env) expression and MS. Methods: 103 MS, 199 healthy controls (HC) and controls with other neurological diseases (28), chronic infections (30) or autoimmunity (30) were analysed with an immunoassay detecting Env in serum. Env RNA or DNA copy numbers in peripheral blood mononuclear cells (PBMC) were determined by a quantitative polymerase chain reaction (PCR). Env was detected by immunohistology in the brains of patients with MS with three specific monoclonals. Results: Env antigen was detected in a serum of 73% of patients with MS with similar prevalence in all clinical forms, and not in chronic infection, systemic lupus, most other neurological diseases and healthy donors (p<0.01). Cases with chronic inflammatory demyelinating polyneuropathy (5/8) and rare HC (4/103) were positive. RNA expression in PBMC and DNA copy numbers were significantly elevated in patients with MS versus HC (p<0.001). In patients with MS, DNA copy numbers were significantly increased in chronic progressive MS (secondary progressive MS vs relapsing–remitting MS (RRMS) p<0.001; primary progressive MS vs RRMS –<0.02). Env protein was evidenced in macrophages within MS brain lesions with particular concentrations around vascular elements. Conclusion: The association between MS disease and the MSRV-type HERV-W element now appears quite strong, as evidenced ex-vivo from serum and PBMC with post-mortem confirmation in brain lesions. Chronic progressive MS, RRMS and clinically isolated syndrome show different ELISA (Enzyme-Linked Immunosorbent Assay) and/or PCR profiles suggestive of an increase with disease evolution, and amplicon sequencing confirms the association with particular HERV-W elements.

The NLRP3 and NLRP1 inflammasomes are activated in Alzheimer's disease

BackgroundInterleukin-1 beta (IL-1β) and its key regulator, the inflammasome, are suspected to play a role in the neuroinflammation observed in Alzheimer’s disease (AD); no conclusive data are nevertheless available in AD patients.ResultsmRNA for inflammasome components (NLRP1, NLRP3, PYCARD, caspase 1, 5 and 8) and downstream effectors (IL-1β, IL-18) was up-regulated in severe and MILD AD. Monocytes co-expressing NLRP3 with caspase 1 or caspase 8 were significantly increased in severe AD alone, whereas those co-expressing NLRP1 and NLRP3 with PYCARD were augmented in both severe and MILD AD. Activation of the NLRP1 and NLRP3 inflammasomes in AD was confirmed by confocal microscopy proteins co-localization and by the significantly higher amounts of the pro-inflammatory cytokines IL-1β and IL-18 being produced by monocytes. In MCI, the expression of NLRP3, but not the one of PYCARD or caspase 1 was increased, indicating that functional inflammasomes are not assembled in these individuals: this was confirmed by lack of co-localization and of proinflammatory cytokines production.ConclusionsThe activation of at least two different inflammasome complexes explains AD-associated neuroinflammation. Strategies targeting inflammasome activation could be useful in the therapy of AD.

HHV8 a subtype is associated with rapidly evolving classic Kaposi's sarcoma

The link between human herpesvirus 8 (KSHV) and Kaposis sarcoma (KS) has been proven, but many important aspects including risk factors, genetic predisposition to tumor development, transmission of KSHV, and the pathogenic potential of different genotypes remain to be elucidated. Possible associations between clinical parameters and antibody levels, viral load fluctuations, and viral genotype were analyzed by quantitative real‐time PCR, an in‐house developed IFA assay, and sequence analysis of ORF K1‐VR1 in blood, serum and saliva of 38 subjects with classic KS (cKS). KSHV lytic antibodies were significantly increased in stage IV compared to stage I and II patients (p = 0.006 and p = 0.041, respectively). KSHV blood, serum, and saliva viral load was comparable in all stages. The highest viral loads were detected in saliva, and they decreased in stages III–IV compared to stages I–II patients. Higher concentrations of lytic antibodies and higher viral loads were observed in fast progressing cKS patients, in whom KSHV detection from blood was also more frequent. Type A KSHV strain was almost exclusively present in rapid progressors (12/17 cases), while C type was mainly present in slow progressing patients (6/7 cases). Finally, detection of type A KSHV strain associated with higher blood viral loads. KSHV lytic antibody levels and viral load can be used to monitor clinical evolution of cKS. Infection supported by KSHV A subtype is associated with more rapid progressive disease. Careful monitoring and aggressive therapeutic protocols should be considered in patients with KSHV A‐supported infection. J. Med. Virol. 80:2153–2160, 2008.

Molecular Analysis of JC Virus Genotypes Circulating Among the Italian Healthy Population

JC virus (JCV) is an unique virus, but eight different JCV genotypes and various subtypes have been individuated, with a geographic distribution that has been described in general but still needs to be detailed. To define JCV genotype epidemiology in Italy, the authors collected urine from 211 healthy individuals living in Northern, Central, and Southern Continental Italy, and in the two main Italian islands. As screening, JCV DNA was searched using a polymerase chain reaction (PCR) designed to amplify the highly conserved large T (LT) antigen-coding region. Then, to define JCV genotype and trascriptional control region (TCR) organization, fragments of major capsid protein (VP1) and TCR region were amplified and subjected to nucleotide sequencing. The mean frequency of JCV viruria was of 46%, without differences among the four geographically divided groups and between females and males. JCV types 1 and 4 were the most frequently detected, whereas JCV type 2 was rare, and type 3 was found only in one subject. The low frequency of JCV type 2 contrasts with the reported high frequency of this subtype in progressive multifocal leukoen-cephalopathy (PML), and supports its specific role in PML. The data indicate that JCV genotype epidemiology in Italy is quite different from that of other European countries; moreover, differences between the various Italian regions have been observed. An unexpected high frequency of a new variant of JCV type 4 with a stable point mutation (C → G) at nucleotide 1851 was found. Furthermore, all the urinary strains had a TCR showing an archetypal organization.

A case of a progressive multifocal leukoencephalopathy patient with four different JC virus transcriptional control region rearrangements in cerebrospinal fluid, blood, serum, and urine

JC virus (JCV) is the etiological agent of progressive multifocal leukoencephalopathy (PML), a fatal demyelinating disease of the central nervous system (CNS). During the acquired immunodeficiency syndrome (AIDS) epidemic, it was the cause of the death in up to 8% of AIDS patients. The genomic organization of JCV and, in particular, the hypervariability of the transcriptional control region (TCR), a regulatory noncoding region, are well known. Given that the TCR plays a central role in the viral replication of JCV, a crucial role in the determination of the neurotropism and in the pathogenic capabilities of the virus is also suspected. Here the authors describe a case of PML that did not respond to highly active antiretroviral therapy (HAART) therapy. There was a simultaneous presence of JCV strains with four different TCR structures in urine, peripheral blood cells, serum, and cerebrospinal fluid (CSF) samples. These data confirmed that the presence of the archetype TCR is restricted to urine, while also suggesting that the degree of the rearrangement varies and increases from the peripheral blood to CSF.

Detection of viral DNA sequences in the cerebrospinal fluid of patients with multiple sclerosis.

The role of viruses in the pathogenesis of multiple sclerosis (MS) is a subject of heated debate. The presence of six different neurotropic viruses was sought, including JC virus (JCV), varicella zoster virus (VZV), human herpesvirus 6 (HHV‐6), and Epstein‐Barr virus (EBV), in cerebrospinal fluid (CSF) samples collected from 51 patients with MS and 30 patients with other neurological diseases. Cell‐free or cell‐associated viral DNA in CSF samples was detected by real‐time PCR, and viral loads were determined. Magnetic resonance imaging (MRI) examinations were also performed to look for active lesions. Cell‐associated JCV DNA was detected in 3 of the 51 patients with MS and in 2 of the 30 patients with other neurological disease. Cell‐free JCV DNA was detected in one additional patient with MS. Cell‐free VZV DNA was detected in one patient without MS, cell‐free HHV‐6 was detected in one patient with MS, and cell‐free EBV was detected in one patient with MS. All other study patients had no detectable viral DNA in CSF samples and no double infections were found. The small percentage of patients with detectable viral DNA in CSF samples was comparable between patients with MS and those with other neurological disease, and presence of viral DNA was not a predictor of brain lesions. Additional observations suggest that cell trafficking from the periphery, rather than leakage through the blood–brain barrier, results in the transport of viruses to the CNS, where local immunosurveillance can control viral replication in immunocompetent individuals. J. Med. Virol. 82:1051–1057, 2010.

JC virus viremia in interferon-β –treated and untreated Italian multiple sclerosis patients and healthy controls

Following the development of progressive multifocal leukoencephalopathy (PML) in two multiple sclerosis (MS) patients treated with natalizumab and interferon-β (IFNβ), a possible correlation between JC virus (JCV), the etiological agent of PML, and MS has received heightened interest. In particular, attention has focused on assessing whether IFNβ treatment could affect the replication of JCV and thus its frequency in the peripheral blood of MS patients and whether the presence of JCV DNA in peripheral blood could be a predictive marker of the risk of developing PML. In order to answer to these questions, peripheral blood samples were collected from 59 INFβ-treated, 39 untreated relapsing-remitting MS patients, and 98 healthy controls (HCs) and JCV DNA levels were determined and quantified by means of a real-time polymerase chain reaction (Q-PCR) assay. Overall, no differences were found in the presence or viral load of JCV DNA of MS patients and the HCs, but JCV DNA was significantly less frequent in the peripheral blood of IFNβ-treated patients (13.6%) compared to the untreated MS patients (46.1%) and the healthy controls (28.6%). These results suggest that the presence of JCV in the blood of MS patients cannot be considered as a marker or a risk factor for PML development. In addition, they indicate that treatment with INFβ can lead to the reduction of presence of the JCV genome in the peripheral blood of MS patients and, thus, that this drug probably does not increase the risk of PML in MS patients treated with IFNβ.

Nucleotide sequence and restriction fragment-length polymorphism analysis of human T-cell lymphotropic virus type II (HTLV-II) in Southern Europe : Evidence for the HTLV-IIa and HTLV-IIb subtypes

Human T-cell lymphotropic virus type II (HTLV-II) has been subtyped into two major groups, IIa and IIb, according to molecular studies involving env gene sequencing. Subsequently, this retrovirus was further subclassified by examining the long terminal repeat (LTR), the most divergent genomic region. Sequence analysis and restriction fragment-length polymorphism (RFLP) applied to the LTR region identified either four or five groups within the IIa subtype (depending on the restriction enzyme sets used) and six within the IIb subtype. In this study, we analyzed the LTR sequences of 29 samples obtained from HTLV-II-infected individuals living in Spain and Italy, which included 24 injecting drug users (IDUs), three blood donors, and two subjects at risk for HIV/HTLV infection. Sequence analysis and phylogenetic analysis of 720 base pairs of the LTR performed in 10 Spanish samples showed that all of these samples belonged to IIb subtype, with a divergence of 7.5% and 1.66% compared with MoT (IIa) and NRA/G12 (IIb) isolates, respectively. RFLP analysis demonstrated the presence of the IIb 4-subtype restriction pattern in 26 samples, a IIb5-subtype pattern in one Italian IDU, and a IIa0-subtype pattern in two Italian samples (blood donors), according to W.M. Switzers nomenclature. This is the first report of the presence of IIb5 in Southern Europe and IIa0 among Italian blood donors. RFLP correlated with nucleotide sequence and phylogenetic data obtained in this study, demonstrating the ability of the RFLP method to predict the phylogroup of HTLV-II-infected samples.

Titers of Herpes Simplex Virus Type 1 Antibodies Positively Correlate with Grey Matter Volumes in Alzheimer's Disease

HSV-1 infection of the central nervous system targets the same brain regions most affected in Alzheimers disease (AD) and could play a pathogenic role in AD. HSV-1 serum IgG titers were analyzed in patients with mild AD (n = 83) and healthy controls (HC, n = 51); results were correlated with cortical grey matter (GM) volumes as analyzed by MRI. Seroprevalence and antibody (Ab) titers were comparable between AD and HC; elevated Ab titers (>75th percentile) were nevertheless significantly more frequent in AD and were positively correlated with cortical bilateral temporal and orbitofrontal GM volumes. HSV-1-specific-Ab could possibly play a protective role in the early stages of AD.