AP2A2 mutation and defective endocytosis in a Malian family with hereditary spastic paraplegia.

Hereditary spastic paraplegia (HSP) comprises a large group of neurogenetic disorders characterized by progressive lower extremity spasticity. Neurological evaluation and genetic testing were completed in a Malian family with early-onset HSP. Three children with unaffected consanguineous parents presented with symptoms consistent with childhood-onset complicated HSP. Neurological evaluation found lower limb weakness, spasticity, dysarthria, seizures, and intellectual disability. Brain MRI showed corpus callosum thinning with cortical and spinal cord atrophy, and an EEG detected slow background in the index patient. Whole exome sequencing identified a homozygous missense variant in the adaptor protein (AP) complex 2 alpha-2 subunit (AP2A2) gene. Western blot analysis showed reduced levels of AP2A2 in patient-iPSC derived neuronal cells. Endocytosis of transferrin receptor (TfR) was decreased in patient-derived neurons. In addition, we observed increased axon initial segment length in patient-derived neurons. Xenopus tropicalis tadpoles with ap2a2 knockout showed cerebral edema and progressive seizures. Immunoprecipitation of the mutant human AP-2-appendage alpha-C construct showed defective binding to accessory proteins. We report AP2A2 as a novel genetic entity associated with HSP and provide functional data in patient-derived neuron cells and a frog model. These findings expand our understanding of the mechanism of HSP and improve the genetic diagnosis of this condition.

Deep phenotyping of post-infectious myalgic encephalomyelitis/chronic fatigue syndrome.

Post-infectious myalgic encephalomyelitis/chronic fatigue syndrome (PI-ME/CFS) is a disabling disorder, yet the clinical phenotype is poorly defined, the pathophysiology is unknown, and no disease-modifying treatments are available. We used rigorous criteria to recruit PI-ME/CFS participants with matched controls to conduct deep phenotyping. Among the many physical and cognitive complaints, one defining feature of PI-ME/CFS was an alteration of effort preference, rather than physical or central fatigue, due to dysfunction of integrative brain regions potentially associated with central catechol pathway dysregulation, with consequences on autonomic functioning and physical conditioning. Immune profiling suggested chronic antigenic stimulation with increase in naïve and decrease in switched memory B-cells. Alterations in gene expression profiles of peripheral blood mononuclear cells and metabolic pathways were consistent with cellular phenotypic studies and demonstrated differences according to sex. Together these clinical abnormalities and biomarker differences provide unique insight into the underlying pathophysiology of PI-ME/CFS, which may guide future intervention.

Direct interhemispheric cortical communication via thalamic commissures: a new white matter pathway in the primate brain.

Cortical neurons of eutherian mammals project to the contralateral hemisphere, crossing the midline primarily via the corpus callosum and the anterior, posterior, and hippocampal commissures. We recently reported and named the thalamic commissures (TCs) as an additional interhemispheric axonal fiber pathway connecting the cortex to the contralateral thalamus in the rodent brain. Here, we demonstrate that TCs also exist in primates and characterize the connectivity of these pathways with high-resolution diffusion-weighted MRI, viral axonal tracing, and fMRI. We present evidence of TCs in both New World (Callithrix jacchus and Cebus apella) and Old World primates (Macaca mulatta). Further, like rodents, we show that the TCs in primates develop during the embryonic period, forming anatomical and functionally active connections of the cortex with the contralateral thalamus. We also searched for TCs in the human brain, showing their presence in humans with brain malformations, although we could not identify TCs in healthy subjects. These results pose the TCs as a vital fiber pathway in the primate brain, allowing for more robust interhemispheric connectivity and synchrony and serving as an alternative commissural route in developmental brain malformations.

In vivo MRI is sensitive to remyelination in a nonhuman primate model of multiple sclerosis.

Remyelination is crucial to recover from inflammatory demyelination in multiple sclerosis (MS). Investigating remyelination in vivo using magnetic resonance imaging (MRI) is difficult in MS, where collecting serial short-interval scans is challenging. Using experimental autoimmune encephalomyelitis (EAE) in common marmosets, a model of MS that recapitulates focal cerebral inflammatory demyelinating lesions, we investigated whether MRI is sensitive to, and can characterize, remyelination. In six animals followed with multisequence 7 T MRI, 31 focal lesions, predicted to be demyelinated or remyelinated based on signal intensity on proton density-weighted images, were subsequently assessed with histopathology. Remyelination occurred in four of six marmosets and 45% of lesions. Radiological-pathological comparison showed that MRI had high statistical sensitivity (100%) and specificity (90%) for detecting remyelination. This study demonstrates the prevalence of spontaneous remyelination in marmoset EAE and the ability of in vivo MRI to detect it, with implications for preclinical testing of pro-remyelinating agents.

Unstable EBV latency drives inflammation in multiple sclerosis patient derived spontaneous B cells.

Epidemiological studies have demonstrated that Epstein-Barr virus (EBV) is a known etiologic risk factor, and perhaps prerequisite, for the development of MS. EBV establishes life-long latent infection in a subpopulation of memory B cells. Although the role of memory B cells in the pathobiology of MS is well established, studies characterizing EBV-associated mechanisms of B cell inflammation and disease pathogenesis in EBV (+) B cells from MS patients are limited. Accordingly, we analyzed spontaneous lymphoblastoid cell lines (SLCLs) from multiple sclerosis patients and healthy controls to study host-virus interactions in B cells, in the context of an individual's endogenous EBV. We identify differences in EBV gene expression and regulation of both viral and cellular genes in SLCLs. Our data suggest that EBV latency is dysregulated in MS SLCLs with increased lytic gene expression observed in MS patient B cells, especially those generated from samples obtained during "active" disease. Moreover, we show increased inflammatory gene expression and cytokine production in MS patient SLCLs and demonstrate that tenofovir alafenamide, an antiviral that targets EBV replication, decreases EBV viral loads, EBV lytic gene expression, and EBV-mediated inflammation in both SLCLs and in a mixed lymphocyte assay. Collectively, these data suggest that dysregulation of EBV latency in MS drives a pro-inflammatory, pathogenic phenotype in memory B cells and that this response can be attenuated by suppressing EBV lytic activation. This study provides further support for the development of antiviral agents that target EBV-infection for use in MS.

Hereditary spastic paraplegia in Mali: epidemiological and clinical features.

BACKGROUND AND PURPOSE: Hereditary spastic paraplegia (HSP) is a group of neurodegenerative diseases divided into pure and complex forms, with spasticity in lower limbs only, or associated with other neurologic and non-neurologic manifestations, respectively. Although widely reported in other populations, very little data exist in sub-Saharan Africa. METHODS: Patients with neurodegenerative features were evaluated over a 19-month period at the Department of Neurology, Teaching Hospital of Point "G", Bamako, Mali. The diagnosis of HSP was considered based on family history and the absence of other known non-genetic causes. Genetic analysis including candidate gene and whole exome sequencing was performed and variant pathogenicity was tested using prediction tools and ACMG guidelines. RESULTS: Of the 170 families with hereditary neurological disorders enrolled, 16 had features consistent with HSP, a frequency of 9%. The average age of onset was 14.7 years with 46% starting before age 6. The male/female ratio was 2.6:1. Complex forms were seen in 75% of cases, and pure forms in 25%. Pyramidal findings were present in all patients. Associated features included mental retardation, peripheral neuropathy, epilepsy, oculomotor impairment and urinary urgency. Most patients were treated with a muscle relaxant and physical therapy, and restorative surgery was done in one. Genetic testing identified novel variants in three families (19%). CONCLUSION: This study confirms the clinical variability of HSPs and adds African data to the current literature.

MPAPASS software enables stitched multiplex, multidimensional EV repertoire analysis and a standard framework for reporting bead-based assays.

Extracellular vesicles (EVs) of various types are released or shed from all cells. EVs carry proteins and contain additional protein and nucleic acid cargo that relates to their biogenesis and cell of origin. EV cargo in liquid biopsies is of widespread interest owing to its ability to provide a retrospective snapshot of cell state at the time of EV release. For the purposes of EV cargo analysis and repertoire profiling, multiplex assays are an essential tool in multiparametric analyte studies but are still being developed for high-parameter EV protein detection. Although bead-based EV multiplex analyses offer EV profiling capabilities with conventional flow cytometers, the utilization of EV multiplex assays has been limited by the lack of software analysis tools for such assays. To facilitate robust EV repertoire studies, we developed multiplex analysis post-acquisition analysis (MPAPASS) open-source software for stitched multiplex analysis, EV database-compatible reporting, and visualization of EV repertoires.

Cortical lesion hotspots and association of subpial lesions with disability in multiple sclerosis.

BACKGROUND: Dramatic improvements in visualization of cortical (especially subpial) multiple sclerosis (MS) lesions allow assessment of impact on clinical course. OBJECTIVE: Characterize cortical lesions by 7 tesla (T) T2*-/T1-weighted magnetic resonance imaging (MRI); determine relationship with other MS pathology and contribution to disability. METHODS: Sixty-four adults with MS (45 relapsing-remitting/19 progressive) underwent 3 T brain/spine MRI, 7 T brain MRI, and clinical testing. RESULTS: Cortical lesions were found in 94% (progressive: median 56/range 2-203; relapsing-remitting: 15/0-168; p = 0.004). Lesion distribution across 50 cortical regions was nonuniform (p = 0.006), with highest lesion burden in supplementary motor cortex and highest prevalence in superior frontal gyrus. Leukocortical and white matter lesion volumes were strongly correlated (r = 0.58, p < 0.0001), while subpial and white matter lesion volumes were moderately correlated (r = 0.30, p = 0.002). Leukocortical (p = 0.02) but not subpial lesions (p = 0.40) were correlated with paramagnetic rim lesions; both were correlated with spinal cord lesions (p = 0.01). Cortical lesion volumes (total and subtypes) were correlated with expanded disability status scale, 25-foot timed walk, nine-hole peg test, and symbol digit modality test scores. CONCLUSION: Cortical lesions are highly prevalent and are associated with disability and progressive disease. Subpial lesion burden is not strongly correlated with white matter lesions, suggesting differences in inflammation and repair mechanisms.

Management of HAM/TSP: Systematic Review and Consensus-based Recommendations 2019.

PURPOSE OF REVIEW: To provide an evidence-based approach to the use of therapies that are prescribed to improve the natural history of HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP)-a rare disease. RECENT FINDINGS: All 41 articles on the clinical outcome of disease-modifying therapy for HAM/TSP were included in a systematic review by members of the International Retrovirology Association; we report here the consensus assessment and recommendations. The quality of available evidence is low, based for the most part on observational studies, with only 1 double-masked placebo-controlled randomized trial. SUMMARY: There is evidence to support the use of both high-dose pulsed methyl prednisolone for induction and low-dose (5 mg) oral prednisolone as maintenance therapy for progressive disease. There is no evidence to support the use of antiretroviral therapy. There is insufficient evidence to support the use of interferon-α as a first-line therapy.

Animal Models of Alzheimer's Disease Should Be Controlled for Roseolovirus.

Animal models to study Alzheimer's disease (AD) pathogenesis are under development. Since herpesviruses have been postulated to be capable of triggering the pathogenic process, AD animal models (mouse, pig, and non-human primates) should be controlled for the presence of these viruses. Only virus-free models allow studying the genetic factors and the effect of adding viruses. Roseoloviruses such as human herpesvirus 6 and the related viruses in the animals are the main topic of this commentary.

Atomic structure of the human herpesvirus 6B capsid and capsid-associated tegument complexes.

Human herpesvirus 6B (HHV-6B) belongs to the ß-herpesvirus subfamily of the Herpesviridae. To understand capsid assembly and capsid-tegument interactions, here we report atomic structures of HHV-6B capsid and capsid-associated tegument complex (CATC) obtained by cryoEM and sub-particle reconstruction. Compared to other ß-herpesviruses, HHV-6B exhibits high similarity in capsid structure but organizational differences in its CATC (pU11 tetramer). 180 "VΛ"-shaped CATCs are observed in HHV-6B, distinguishing from the 255 "Λ"-shaped dimeric CATCs observed in murine cytomegalovirus and the 310 "Δ"-shaped CATCs in human cytomegalovirus. This trend in CATC quantity correlates with the increasing genomes sizes of these ß-herpesviruses. Incompatible distances revealed by the atomic structures rationalize the lack of CATC's binding to triplexes Ta, Tc, and Tf in HHV-6B. Our results offer insights into HHV-6B capsid assembly and the roles of its tegument proteins, including not only the ß-herpesvirus-specific pU11 and pU14, but also those conserved across all subfamilies of Herpesviridae.

Essential Role of Human T Cell Leukemia Virus Type 1 orf-I in Lethal Proliferation of CD4+ Cells in Humanized Mice.

Human T cell leukemia virus type 1 (HTLV-1) is the ethological agent of adult T cell leukemia/lymphoma (ATLL) and a number of lymphocyte-mediated inflammatory conditions, including HTLV-1-associated myelopathy/tropical spastic paraparesis. HTLV-1 orf-I encodes two proteins, p8 and p12, whose functions in humans are to counteract innate and adaptive responses and to support viral transmission. However, the in vivo requirements for orf-I expression vary in different animal models. In macaques, the ablation of orf-I expression by mutation of its ATG initiation codon abolishes the infectivity of the molecular clone HTLV-1p12KO In rabbits, HTLV-1p12KO is infective and persists efficiently. We used humanized mouse models to assess the infectivity of both wild-type HTLV-1 (HTLV-1WT) and HTLV-1p12KO We found that NOD/SCID/γC-/- c-kit+ mice engrafted with human tissues 1 day after birth (designated NSG-1d mice) were highly susceptible to infection by HTLV-1WT, with a syndrome characterized by the rapid polyclonal proliferation and infiltration of CD4+ CD25+ T cells into vital organs, weight loss, and death. HTLV-1 clonality studies revealed the presence of multiple clones of low abundance, confirming the polyclonal expansion of HTLV-1-infected cells in vivo HTLV-1p12KO infection in a bone marrow-liver-thymus (BLT) mouse model prone to graft-versus-host disease occurred only following reversion of the orf-I initiation codon mutation within weeks after exposure and was associated with high levels of HTLV-1 DNA in blood and the expansion of CD4+ CD25+ T cells. Thus, the incomplete reconstitution of the human immune system in BLT mice may provide a window of opportunity for HTLV-1 replication and the selection of viral variants with greater fitness.IMPORTANCE Humanized mice constitute a useful model for studying the HTLV-1-associated polyclonal proliferation of CD4+ T cells and viral integration sites in the human genome. The rapid death of infected animals, however, appears to preclude the clonal selection typically observed in human ATLL, which normally develops in 2 to 5% of individuals infected with HTLV-1. Nevertheless, the expansion of multiple clones of low abundance in these humanized mice mirrors the early phase of HTLV-1 infection in humans, providing a useful model to investigate approaches to inhibit virus-induced CD4+ T cell proliferation.

HTLV-1 HBZ Protein Resides Exclusively in the Cytoplasm of Infected Cells in Asymptomatic Carriers and HAM/TSP Patients.

Human T cell lymphotropic virus type 1 (HTLV-1) is the causative agent of adult T cell leukemia/lymphoma (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) in a subset of infected subjects. Two viral proteins, Tax-1 and HTLV-1 basic leucine zipper factor (HBZ), play important roles in the pathogenesis of both diseases. We recently demonstrated that HBZ, previously considered a nuclear protein, is exclusively localized in the cytoplasm of peripheral blood mononuclear cells (PBMCs) of HAM/TSP patients. Here, the analysis of a larger panel of HAM/TSP cases confirmed that HBZ is a cytoplasmic protein, while Tax-1 preferentially localized in the cytoplasm with fewer speckle-like dots in the nucleus. More importantly, here we report for the first time that HBZ, when expressed in asymptomatic carriers (AC), is also confined in the cytoplasm. Similarly, Tax-1 was preferentially expressed in the cytoplasm in a significant proportion of AC. Interestingly, in both HAM/TSP and AC patients, the expression of HBZ and Tax-1 was rarely found in the same cell. We observed only few cases coexpressing the two oncoprotein in a very limited number of cells. In representative AC and HAM/TSP patients, cells expressing cytoplasmic HBZ were almost exclusively found in the CD4+ T cell compartment and very rarely in CD8+ T cells. Interestingly, at least in the cases analyzed, the expression of thymocite-expressed molecule involved in selection (THEMIS) is dispensable for the cytoplasmic localization of HBZ in both AC and HAM/TSP. The study of an HTLV-1-immortalized cell line established from an HAM/TSP patient confirmed HBZ as a resident cytoplasmic protein not shuttling between the cytoplasm and nucleus. These results extend our previous observation on the dichotomy of HBZ localization between HAM/TSP and ATL, pointing to the exclusive either cytoplasmic or nuclear localization in the two diseased states, respectively. Moreover, they show a rather selective expression in distinct cells of either HBZ or Tax-1. The unprecedented observation that HBZ is expressed only in the cytoplasm in AC strongly suggests a progressive modification of HBZ localization during the disease states associated to HTLV-1 infection. Future studies will clarify whether the distinct HBZ intracellular localization is a marker or a causative event of disease evolution.

Pembrolizumab Treatment for Progressive Multifocal Leukoencephalopathy.

BACKGROUND: Progressive multifocal leukoencephalopathy (PML) is an opportunistic brain infection that is caused by the JC virus and is typically fatal unless immune function can be restored. Programmed cell death protein 1 (PD-1) is a negative regulator of the immune response that may contribute to impaired viral clearance. Whether PD-1 blockade with pembrolizumab could reinvigorate anti-JC virus immune activity in patients with PML was unknown. METHODS: We administered pembrolizumab at a dose of 2 mg per kilogram of body weight every 4 to 6 weeks to eight adults with PML, each with a different underlying predisposing condition. Each patient received at least one dose but no more than three doses. RESULTS: Pembrolizumab induced down-regulation of PD-1 expression on lymphocytes in peripheral blood and in cerebrospinal fluid (CSF) in all eight patients. Five patients had clinical improvement or stabilization of PML accompanied by a reduction in the JC viral load in the CSF and an increase in in vitro CD4+ and CD8+ anti-JC virus activity. In the other three patients, no meaningful change was observed in the viral load or in the magnitude of antiviral cellular immune response, and there was no clinical improvement. CONCLUSIONS: Our findings are consistent with the hypothesis that in some patients with PML, pembrolizumab reduces JC viral load and increases CD4+ and CD8+ activity against the JC virus; clinical improvement or stabilization occurred in five of the eight patients who received pembrolizumab. Further study of immune checkpoint inhibitors in the treatment of PML is warranted. (Funded by the National Institutes of Health.).

Viruses and multiple sclerosis.

Multiple sclerosis (MS) is a heterogeneous disease that develops as an interplay between the immune system and environmental stimuli in genetically susceptible individuals. There is increasing evidence that viruses may play a role in MS pathogenesis acting as these environmental triggers. However, it is not known if any single virus is causal, or rather several viruses can act as triggers in disease development. Here, we review the association of different viruses to MS with an emphasis on two herpesviruses, Epstein-Barr virus (EBV) and human herpesvirus 6 (HHV-6). These two agents have generated the most impact during recent years as possible co-factors in MS disease development. The strongest argument for association of EBV with MS comes from the link between symptomatic infectious mononucleosis and MS and from seroepidemiological studies. In contrast to EBV, HHV-6 has been found significantly more often in MS plaques than in MS normal appearing white matter or non-MS brains and HHV-6 re-activation has been reported during MS clinical relapses. In this review we also suggest new strategies, including the development of new infectious animal models of MS and antiviral MS clinical trials, to elucidate roles of different viruses in the pathogenesis of this disease. Furthermore, we introduce the idea of using unbiased sequence-independent pathogen discovery methodologies, such as next generation sequencing, to study MS brain tissue or body fluids for detection of known viral sequences or potential novel viral agents.

The fate of porous hydroxyapatite granules used in facial skeletal augmentation.

Facial appearance is largely determined by the morphology of the underlying skeleton. Hydroxyapatite is one of several materials available to enhance projection of the facial skeleton. This study evaluated the long-term maintenance of augmented bony projection when porous hydroxyapatite granules are used on the facial skeleton. Ten female patients aged 28-58 years were studied following aesthetic augmentation of the facial skeleton at 24 sites using porous hydroxyapatite granules. Postoperative CT scans at 3 months served as the baseline measurement and compared with scans taken at 1 and 2 years, with the thickness of the hydroxyapatite measured in axial and coronal planes. Thickness of original bone plus overlay of hydroxyapatite, thickness of the overlying soft tissue, and the overall projection (bone plus soft tissue) were recorded. It was found that 99.7% of the hydroxyapatite was maintained at 2 years, with no statistical difference (t test) from the baseline measurement. The overall projection (bony and soft tissue) was maintained as there was no evidence of native bone resorption or soft tissue atrophy. Radiographic results confirmed that the use of porous hydroxyapatite granules for enhancement of the facial skeleton is not only a predictable procedure, but maintains full bony projection at 2 years.

Human T cell leukemia virus reactivation with progression of adult T-cell leukemia-lymphoma.

BACKGROUND: Human T-cell leukemia virus-associated adult T-cell leukemia-lymphoma (ATLL) has a very poor prognosis, despite trials of a variety of different treatment regimens. Virus expression has been reported to be limited or absent when ATLL is diagnosed, and this has suggested that secondary genetic or epigenetic changes are important in disease pathogenesis. METHODS AND FINDINGS: We prospectively investigated combination chemotherapy followed by antiretroviral therapy for this disorder. Nineteen patients were prospectively enrolled between 2002 and 2006 at five medical centers in a phase II clinical trial of infusional chemotherapy with etoposide, doxorubicin, and vincristine, daily prednisone, and bolus cyclophosphamide (EPOCH) given for two to six cycles until maximal clinical response, and followed by antiviral therapy with daily zidovudine, lamivudine, and alpha interferon-2a for up to one year. Seven patients were on study for less than one month due to progressive disease or chemotherapy toxicity. Eleven patients achieved an objective response with median duration of response of thirteen months, and two complete remissions. During chemotherapy induction, viral RNA expression increased (median 190-fold), and virus replication occurred, coincident with development of disease progression. CONCLUSIONS: EPOCH chemotherapy followed by antiretroviral therapy is an active therapeutic regimen for adult T-cell leukemia-lymphoma, but viral reactivation during induction chemotherapy may contribute to treatment failure. Alternative therapies are sorely needed in this disease that simultaneously prevent virus expression, and are cytocidal for malignant cells.

Multicenter comparison of PCR assays for detection of human herpesvirus 6 DNA in serum.

Human herpesvirus 6 (HHV-6) is a ubiquitous virus with which infections have been associated with pathologies ranging from delayed bone marrow engraftment to a variety of neurological diseases. The lack of a standardized assay that can be used to detect and estimate HHV-6 DNA contents in various clinical specimens can lead and has led to discordant results among investigators and on the potential association of HHV-6 to diseases. To identify the most reliable and sensitive assays, an identical set of 11 coded serum samples spiked with various quantities of the HHV-6A variant (range, 4 to 400,000 genome copies/ml) was sent to eight independent laboratories around the world. Each laboratory was asked to estimate the HHV-6 DNA content by use of its own protocols and assays. Among the various assays, three TaqMan-based real-time PCR assays yielded quantities that were closest to the quantity of HHV-6 that had been spiked. To provide better homogeneity between the results from the different laboratories working on HHV-6, we propose that investigators interested in quantifying HHV-6 in clinical samples adopt one of these assays.