The latency-associated nuclear antigen of Kaposi sarcoma-associated herpesvirus induces B cell hyperplasia and lymphoma

Kaposi sarcoma–associated herpesvirus (KSHV) is a human lymphotropic herpesvirus. It is implicated in B cell neoplasias such as primary effusion lymphoma and multicentric Castleman disease in AIDS patients. The KSHV latency-associated nuclear antigen (LANA) is consistently expressed in all KSHV-associated tumor cells and was shown to bind the tumor suppressor proteins p53 and pRb. To test LANA’s contribution to lymphomagenesis in vivo we generated transgenic mice expressing LANA under the control of its own promoter, which is B cell specific. All of the transgenic mice developed splenic follicular hyperplasia due to an expansion of IgM+IgD+ B cells and showed increased germinal center formation. We also observed lymphomas, implying that LANA can activate B cells and provide the first step toward lymphomagenesis

The immediate effects of dry needling for ankle and toe plantar flexors on spasticity and balance in patients with stroke

Background: Spasticity is one of the most important symptoms of stroke, which leads to movement constraints and disability. The presence of spasticity in the ankle and toe plantar flexor muscles disturbs the balance and gait of patients with stroke. Dry needling has been introduced as a new method for the treatment of spasticity. The aim of this study was to investigate the immediate effects of the ankle and toe plantar flexors dry needling on spasticity and balance in patients with stroke. Methods: This study was a clinical pretest-posttest study. Twenty patients with stroke (12 males and 8 females), the mean age of 56.5±13 years were included. The assessments were performed before dry needling, immediately after dry needling and 15 minutes after that. Dry needling was used to treat gastrocnemius (ankle plantar flexor) muscles, flexor digitorum longus, and flexor digitorum brevis of the affected lower limb of the patients, for one session. Each muscle was needled for one minute with fast in-fast out technique. The outcome measures of the study were modified modified Ashworth scale (MMAS) for the assessment of the severity of muscle spasticity, timed up and go test and one leg stance test, for balance evaluation. The study was conducted in neurological physical therapy, Clinic of Rehabilitation School, Tehran University of Medical Sciences in Iran, from April 2017 to April 2018. Results: The results showed a significant decrease in the ankle and toe plantar flexor muscles spasticity both immediately after dry needling and at 15 minutes follow-up (P=0.001). The duration of timed up and go test (P=0.001) and one leg stance test (P=0.001) improved significantly after dry needling and this improvement persisted for 15 minutes after dry needling. The effect size for timed up and go test and one leg stance test was small (Cohen'sd=0.33 and 0.32 respectively). Conclusion: This study suggests that dry needling is effective in improving spasticity of ankle and toe plantar flexor muscles and the balance of patients with stroke. Further research with larger sample size and control group is necessary. © 2020 Tehran University of Medical Sciences. All rights reserved

High-throughput imaging of ATG9A distribution as a diagnostic functional assay for adaptor protein complex 4-associated hereditary spastic paraplegia

Adaptor protein complex 4-associated hereditary spastic paraplegia is caused by biallelic loss-of-function variants in AP4B1, AP4M1, AP4E1 or AP4S1, which constitute the four subunits of this obligate complex. While the diagnosis of adaptor protein complex 4-associated hereditary spastic paraplegia relies on molecular testing, the interpretation of novel missense variants remains challenging. Here, we address this diagnostic gap by using patient-derived fibroblasts to establish a functional assay that measures the subcellular localization of ATG9A, a transmembrane protein that is sorted by adaptor protein complex 4. Using automated high-throughput microscopy, we determine the ratio of the ATG9A fluorescence in the trans-Golgi-network versus cytoplasm and ascertain that this metric meets standards for screening assays (Z'-factor robust >0.3, strictly standardized mean difference >3). The `ATG9A ratio' is increased in fibroblasts of 18 well-characterized adaptor protein complex 4-associated hereditary spastic paraplegia patients [mean: 1.54 +/- 0.13 versus 1.21 +/- 0.05 (standard deviation) in controls] and receiver-operating characteristic analysis demonstrates robust diagnostic power (area under the curve: 0.85, 95% confidence interval: 0.849-0.852). Using fibroblasts from two individuals with atypical clinical features and novel biallelic missense variants of unknown significance in AP4B1, we show that our assay can reliably detect adaptor protein complex 4 function. Our findings establish the 'ATG9A ratio' as a diagnostic marker of adaptor protein complex 4-associated hereditary spastic paraplegia

Homozygous missense WIPI2 variants cause a congenital disorder of autophagy with neurodevelopmental impairments of variable clinical severity and disease course.

WIPI2 is a member of the human WIPI protein family (seven-bladed b-propeller proteins binding phosphatidylinositols, PROPPINs), which play a pivotal role in autophagy and has been implicated in the pathogenesis of several neurological conditions. The homozygous WIPI2 variant c.745G>A; p.(Val249Met) (NM_015610.4) has recently been associated with a neurodevelopmental disorder in a single family. Using exome sequencing and Sanger segregation analysis, here, two novel homozygous WIPI2 variants [c.551T>G; p.(Val184Gly) and c.724C>T; p.(Arg242Trp) (NM_015610.4)] were identified in four individuals of two consanguineous families. Additionally, follow-up clinical data were sought from the previously reported family. Three non-ambulant affected siblings of the first family harbouring the p.(Val184Gly) missense variant presented with microcephaly, profound global developmental delay/intellectual disability, refractory infantile/childhood-onset epilepsy, progressive tetraplegia with joint contractures and dyskinesia. In contrast, the proband of the second family carrying the p.(Arg242Trp) missense variant, similar to the initially reported WIPI2 cases, presented with a milder phenotype, encompassing moderate intellectual disability, speech and visual impairment, autistic features, and an ataxic gait. Brain MR imaging in five patients showed prominent white matter involvement with a global reduction in volume, posterior corpus callosum hypoplasia, abnormal dentate nuclei and hypoplasia of the inferior cerebellar vermis. To investigate the functional impact of these novel WIPI2 variants, we overexpressed both in WIPI2-knockout HEK293A cells. In comparison to wildtype, expression of the Val166Gly WIPI2b mutant resulted in a deficient rescue of LC3 lipidation whereas Arg224Trp mutant increased LC3 lipidation, in line with the previously reported Val231Met variant. These findings support a dysregulation of the early steps of the autophagy pathway. Collectively, our findings provide evidence that biallelic WIPI2 variants cause a neurodevelopmental disorder of variable severity and disease course. Our report expands the clinical spectrum and establishes WIPI2-related disorder as a congenital disorders of autophagy

Murine Gamma-herpesvirus Immortalization of Fetal Liver-Derived B Cells Requires both the Viral Cyclin D Homolog and Latency-Associated Nuclear Antigen

Human gammaherpesviruses are associated with the development of lymphoproliferative diseases and B cell lymphomas, particularly in immunosuppressed hosts. Understanding the molecular mechanisms by which human gammaherpesviruses cause disease is hampered by the lack of convenient small animal models to study them. However, infection of laboratory strains of mice with the rodent virus murine gammaherpesvirus 68 (MHV68) has been useful in gaining insights into how gammaherpesviruses contribute to the genesis and progression of lymphoproliferative lesions. In this report we make the novel observation that MHV68 infection of murine day 15 fetal liver cells results in their immortalization and differentiation into B plasmablasts that can be propagated indefinitely in vitro, and can establish metastasizing lymphomas in mice lacking normal immune competence. The phenotype of the MHV68 immortalized B cell lines is similar to that observed in lymphomas caused by KSHV and resembles the favored phenotype observed during MHV68 infection in vivo. All established cell lines maintained the MHV68 genome, with limited viral gene expression and little or no detectable virus production - although virus reactivation could be induced upon crosslinking surface Ig. Notably, transcription of the genes encoding the MHV68 viral cyclin D homolog (v-cyclin) and the homolog of the KSHV latency-associated nuclear antigen (LANA), both of which are conserved among characterized γ2-herpesviruses, could consistently be detected in the established B cell lines. Furthermore, we show that the v-cyclin and LANA homologs are required for MHV68 immortalization of murine B cells. In contrast the M2 gene, which is unique to MHV68 and plays a role in latency and virus reactivation in vivo, was dispensable for B cell immortalization. This new model of gammaherpesvirus-driven B cell immortalization and differentiation in a small animal model establishes an experimental system for detailed investigation of the role of gammaherpesvirus gene products and host responses in the genesis and progression of gammaherpesvirus-associated lymphomas, and presents a convenient system to evaluate therapeutic modalities

KSHV Reactivation from Latency Requires Pim-1 and Pim-3 Kinases to Inactivate the Latency-Associated Nuclear Antigen LANA

Host signal-transduction pathways are intimately involved in the switch between latency and productive infection of herpes viruses. As with other herpes viruses, infection by Kaposi's sarcoma herpesvirus (KSHV) displays these two phases. During latency only few viral genes are expressed, while in the productive infection the virus is reactivated with initiation of extensive viral DNA replication and gene expression, resulting in production of new viral particles. Viral reactivation is crucial for KSHV pathogenesis and contributes to the progression of KS. We have recently identified Pim-1 as a kinase reactivating KSHV upon over-expression. Here we show that another Pim family kinase, Pim-3, also induces viral reactivation. We demonstrate that expression of both Pim-1 and Pim-3 is induced in response to physiological and chemical reactivation in naturally KSHV-infected cells, and we show that they are required for KSHV reactivation under these conditions. Furthermore, our data indicate that Pim-1 and Pim-3 contribute to viral reactivation by phosphorylating the KSHV latency-associated nuclear antigen (LANA) on serine residues 205 and 206. This counteracts the LANA–mediated repression of the KSHV lytic gene transcription. The identification of Pim family kinases as novel cellular regulators of the gammaherpesvirus life cycle facilitates a deeper understanding of virus–host interactions during reactivation and may represent potential novel targets for therapeutic intervention

Abortive Lytic Reactivation of KSHV in CBF1/CSL Deficient Human B Cell Lines

Since Kaposi's sarcoma associated herpesvirus (KSHV) establishes a persistent infection in human B cells, B cells are a critical compartment for viral pathogenesis. RTA, the replication and transcription activator of KSHV, can either directly bind to DNA or use cellular DNA binding factors including CBF1/CSL as DNA adaptors. In addition, the viral factors LANA1 and vIRF4 are known to bind to CBF1/CSL and modulate RTA activity. To analyze the contribution of CBF1/CSL to reactivation in human B cells, we have successfully infected DG75 and DG75 CBF1/CSL knock-out cell lines with recombinant KSHV.219 and selected for viral maintenance by selective medium. Both lines maintained the virus irrespective of their CBF1/CSL status. Viral reactivation could be initiated in both B cell lines but viral genome replication was attenuated in CBF1/CSL deficient lines, which also failed to produce detectable levels of infectious virus. Induction of immediate early, early and late viral genes was impaired in CBF1/CSL deficient cells at multiple stages of the reactivation process but could be restored to wild-type levels by reintroduction of CBF1/CSL. To identify additional viral RTA target genes, which are directly controlled by CBF1/CSL, we analyzed promoters of a selected subset of viral genes. We show that the induction of the late viral genes ORF29a and ORF65 by RTA is strongly enhanced by CBF1/CSL. Orthologs of ORF29a in other herpesviruses are part of the terminase complex required for viral packaging. ORF65 encodes the small capsid protein essential for capsid shell assembly. Our study demonstrates for the first time that in human B cells viral replication can be initiated in the absence of CBF1/CSL but the reactivation process is severely attenuated at all stages and does not lead to virion production. Thus, CBF1/CSL acts as a global hub which is used by the virus to coordinate the lytic cascade

EPG5-related Vici syndrome: a paradigm of neurodevelopmental disorders with defective autophagy

Vici syndrome is a progressive neurodevelopmental multisystem disorder due to recessive mutations in the key autophagy gene EPG5. We report genetic, clinical, neuroradiological, and neuropathological features of 50 children from 30 families, as well as the neuronal phenotype of EPG5 knock-down in Drosophila melanogaster. We identified 39 different EPG5 mutations, most of them truncating and predicted to result in reduced EPG5 protein. Most mutations were private, but three recurrent mutations (p.Met2242Cysfs*5, p.Arg417*, and p.Gln336Arg) indicated possible founder effects. Presentation was mainly neonatal, with marked hypotonia and feeding difficulties. In addition to the five principal features (callosal agenesis, cataracts, hypopigmentation, cardiomyopathy, and immune dysfunction), we identified three equally consistent features (profound developmental delay, progressive microcephaly, and failure to thrive). The manifestation of all eight of these features has a specificity of 97%, and a sensitivity of 89% for the presence of an EPG5 mutation and will allow informed decisions about genetic testing. Clinical progression was relentless and many children died in infancy. Survival analysis demonstrated a median survival time of 24 months (95% confidence interval 0–49 months), with only a 10th of patients surviving to 5 years of age. Survival outcomes were significantly better in patients with compound heterozygous mutations (P = 0.046), as well as in patients with the recurrent p.Gln336Arg mutation. Acquired microcephaly and regression of skills in long-term survivors suggests a neurodegenerative component superimposed on the principal neurodevelopmental defect. Two-thirds of patients had a severe seizure disorder, placing EPG5 within the rapidly expanding group of genes associated with early-onset epileptic encephalopathies. Consistent neuroradiological features comprised structural abnormalities, in particular callosal agenesis and pontine hypoplasia, delayed myelination and, less frequently, thalamic signal intensity changes evolving over time. Typical muscle biopsy features included fibre size variability, central/internal nuclei, abnormal glycogen storage, presence of autophagic vacuoles and secondary mitochondrial abnormalities. Nerve biopsy performed in one case revealed subtotal absence of myelinated axons. Post-mortem examinations in three patients confirmed neurodevelopmental and neurodegenerative features and multisystem involvement. Finally, downregulation of epg5 (CG14299) in Drosophila resulted in autophagic abnormalities and progressive neurodegeneration. We conclude that EPG5-related Vici syndrome defines a novel group of neurodevelopmental disorders that should be considered in patients with suggestive features in whom mitochondrial, glycogen, or lysosomal storage disorders have been excluded. Neurological progression over time indicates an intriguing link between neurodevelopment and neurodegeneration, also supported by neurodegenerative features in epg5-deficient Drosophila, and recent implication of other autophagy regulators in late-onset neurodegenerative disease