Human herpesvirus multiplex ddPCR detection in brain tissue from low- and high-grade astrocytoma cases and controls.

BACKGROUND: Glioblastoma (GBM) is a fatal CNS malignancy, representing 50 % of all gliomas with approximately 12-18 months survival time after initial diagnosis. Recently, the human herpesvirus cytomegalovirus (CMV) has been suggested to have an oncogenic role, yet this association remains controversial. In addition, human herpesvirus 6 (HHV-6) and Epstein-Barr virus (EBV) have also been associated with low-grade gliomas, but few studies have examined HHV-6 and EBV in glioblastomas. Droplet digital PCR (ddPCR) is a highly precise diagnostic tool that enables the absolute quantification of target DNA. This study examines the association between multiple human herpesviruses and astrocytomas. METHODS: This study analyzed 112 brain tissue specimens, including 45 glioblastoma, 12 astrocytoma grade III, 2 astrocytoma grade II, 4 astrocytoma grade I, and 49 controls. All brain tissue samples were de-identified and pathologically confirmed. Each tissue block was sectioned for DNA extraction and CMV, EBV, HHV-6A and HHV-6B, and a cellular housekeeping gene were amplified by ddPCR. RESULTS: Neither CMV nor HHV-6A were detected in any of the astrocytoma samples. However, HHV-6B (p = 0.147) and EBV (p = 0.049) had a higher positivity frequency in the GBM compared to the controls. CONCLUSION: The undetectable CMV DNA in the astrocytoma cohort does not support the observation of an increased prevalence of CMV DNA in GBM, as reported in other studies. EBV has a significantly higher positivity in the GBM cohort compared to the controls, while HHV-6B has a higher but not statistically significant positivity in the case cohort. Whether these viruses play an oncogenic role in GBM remains to be further investigated

Novel Marmoset (Callithrix jacchus) Model of Human Herpesvirus 6A and 6B Infections: Immunologic, Virologic and Radiologic Characterization

Human Herpesvirus 6 (HHV-6) is a ubiquitous virus with an estimated seroprevalence of 95% in the adult population. HHV-6 is associated with several neurologic disorders, including multiple sclerosis, an inflammatory demyelinating disease affecting the CNS. Animal models of HHV-6 infection would help clarify its role in human disease but have been slow to develop because rodents lack CD46, the receptor for cellular entry. Therefore, we investigated the effects of HHV-6 infections in a non-human primate, the common marmoset Callithrix jacchus. We inoculated a total of 12 marmosets with HHV-6A and HHV-6B intravenously and HHV-6A intranasally. Animals were monitored for 25 weeks post-inoculation clinically, immunologically and by MRI. Marmosets inoculated with HHV-6A intravenously exhibited neurologic symptoms and generated virus-specific antibody responses, while those inoculated intravenously with HHV-6B were asymptomatic and generated comparatively lower antibody responses. Viral DNA was detected at a low frequency in paraffin-embedded CNS tissue of a subset of marmosets inoculated with HHV-6A and HHV-6B intravenously. When different routes of HHV-6A inoculation were compared, intravenous inoculation resulted in virus-specific antibody responses and infrequent detection of viral DNA in the periphery, while intranasal inoculation resulted in negligible virus-specific antibody responses and frequent detection of viral DNA in the periphery. Moreover, marmosets inoculated with HHV-6A intravenously exhibited neurologic symptoms, while marmosets inoculated with HHV-6A intranasally were asymptomatic. We demonstrate that a marmoset model of HHV-6 infection can serve to further define the contribution of this ubiquitous virus to human neurologic disorders

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

Targeting the SIN3A-PF1 interaction inhibits epithelial to mesenchymal transition and maintenance of a stem cell phenotype in triple negative breast cancer

Triple negative breast cancer (TNBC) is characterized by a poorly differentiated phenotype and limited treatment options. Aberrant epigenetics in this subtype represent a potential therapeutic opportunity, but a better understanding of the mechanisms contributing to the TNBC pathogenesis is required. The SIN3 molecular scaffold performs a critical role in multiple cellular processes, including epigenetic regulation, and has been identified as a potential therapeutic target. Using a competitive peptide corresponding to the SIN3 interaction domain of MAD (Tat-SID), we investigated the functional consequences of selectively blocking the paired amphipathic α-helix (PAH2) domain of SIN3. Here, we report the identification of the SID-containing adaptor PF1 as a factor required for maintenance of the TNBC stem cell phenotype and epithelial-to-mesenchymal transition (EMT). Tat-SID peptide blocked the interaction between SIN3A and PF1, leading to epigenetic modulation and transcriptional downregulation of TNBC stem cell and EMT markers. Importantly, Tat-SID treatment also led to a reduction in primary tumor growth and disseminated metastatic diseasein vivo. In support of these findings, knockdown ofPF1expression phenocopied treatment with Tat-SID bothin vitroandin vivo. These results demonstrate a critical role for a complex containing SIN3A and PF1 in TNBC and provide a rational for its therapeutic targeting

Detection of HHV-6 and EBV and Cytokine Levels in Saliva From Children With Seizures: Results of a Multi-Center Cross-Sectional Study

Background and Objective: One third of children with epilepsy are refractory to medications. Growing data support a role of common childhood infections with neurotropic viruses and inflammation in epileptogenesis. Our objective was to determine the frequency of Human Herpesvirus-6 (HHV-6) and Epstein-Barr Virus (EBV) infection and cytokine levels in saliva from children with seizures compared to healthy controls and to controls with a febrile illness without seizures.Methods: In this cross-sectional multi-center study, we collected saliva from 115 consecutive children with acute seizures (cases), 51 children with a fever and no seizures or underlying neurological disease (fever controls) and 46 healthy children (healthy controls). Specimens were analyzed by a novel droplet digital PCR for HHV-6 and EBV viral DNA and a bead-based immunoassay for neuroinflammatory cytokines.Results: Cases included febrile seizures (n = 30), acute seizures without (n = 53) and with fever (n = 4) in chronic epilepsy, new onset epilepsy (n = 13), febrile status epilepticus (n = 3), and first lifetime seizure (n = 12). HHV-6 DNA was found in 40% of cases vs. 37% fever controls and 35% healthy controls, with no statistically significant differences. EBV DNA was also detected with no differences in 17% cases, 16% fever controls, and 28% healthy controls. IL-8 and IL-1β were increased in saliva of 32 random samples from cases compared with 30 fever controls: IL-8 cases mean (SD): 1158.07 pg/mL (1427.41); controls 604.92 (754.04); p = 0.02. IL-1β 185.76 (230.57); controls 86.99 (187.39); p = 0.0002. IL-1β level correlated with HHV6 viral load (p = 0.007).Conclusion: Increase in inflammatory cytokines may play a role in the onset of acute seizures and saliva could represent an inexpensive and non-invasive method for detection of viral DNA and cytokines

Human herpesvirus multiplex ddPCR detection in brain tissue from low- and high-grade astrocytoma cases and controls

BACKGROUND: Glioblastoma (GBM) is a fatal CNS malignancy, representing 50 % of all gliomas with approximately 12–18 months survival time after initial diagnosis. Recently, the human herpesvirus cytomegalovirus (CMV) has been suggested to have an oncogenic role, yet this association remains controversial. In addition, human herpesvirus 6 (HHV-6) and Epstein-Barr virus (EBV) have also been associated with low-grade gliomas, but few studies have examined HHV-6 and EBV in glioblastomas. Droplet digital PCR (ddPCR) is a highly precise diagnostic tool that enables the absolute quantification of target DNA. This study examines the association between multiple human herpesviruses and astrocytomas. METHODS: This study analyzed 112 brain tissue specimens, including 45 glioblastoma, 12 astrocytoma grade III, 2 astrocytoma grade II, 4 astrocytoma grade I, and 49 controls. All brain tissue samples were de-identified and pathologically confirmed. Each tissue block was sectioned for DNA extraction and CMV, EBV, HHV-6A and HHV-6B, and a cellular housekeeping gene were amplified by ddPCR. RESULTS: Neither CMV nor HHV-6A were detected in any of the astrocytoma samples. However, HHV-6B (p = 0.147) and EBV (p = 0.049) had a higher positivity frequency in the GBM compared to the controls. CONCLUSION: The undetectable CMV DNA in the astrocytoma cohort does not support the observation of an increased prevalence of CMV DNA in GBM, as reported in other studies. EBV has a significantly higher positivity in the GBM cohort compared to the controls, while HHV-6B has a higher but not statistically significant positivity in the case cohort. Whether these viruses play an oncogenic role in GBM remains to be further investigated

Human Herpesvirus 6 as a Viral Trigger in Mesial Temporal Lobe Epilepsy.

Although the cause(s) of epilepsy remain largely unknown, a number of neurotrop-ic viruses known to cause central nervous system (CNS) inflammation have been implicated in the development of seizures [1]. Herpesviruses are among theseagents, and seizures are thought to result from either primary infection or reactivation of latent virus [1]. More recently, human herpesvirus 6 (HHV-6), particularly the HHV-6B species known to be acquired during early childhood, continues to be associated with epilepsy, in addition to several other CNS diseases. Importantly, this specific association between epileps

Human Herpesviruses HHV-6A and 6B Accelerate Clinical and Radiological Disease in an Nonhuman Primate Model of Multiple Sclerosis

Viruses, particularly human herpesviruses, have long been suggested to be an environmental risk factor for the pathogenesis of Multiple Sclerosis (MS). Human herpesviruses HHV-6A and HHV-6B are associated with MS, in addition to several other inflammatory disorders of the central nervous system (CNS). To investigate this viral trigger hypothesis in a nonhuman primate, we asked whether marmosets previously inoculated with HHV6 exhibit an altered disease course of experimental autoimmune encephalomyelitis (EAE) compared to naïve animals. EAE is an well-accepted model of CNS inflammatory demyelination and reflects clinical and radiologic aspects of MS when induced in marmosets. To mimic a physiologically relevant route of exposure, marmosets were inoculated intranasally with HHV-6A (n=6), HHV-6B (n=4) or uninfected control material (n=6) monthly for four months. Six months after the last viral inoculation, all animals were immunized with white matter homogenate to induce EAE. All animals underwent neurologic exams, in vivo brain MRIs, and peripheral blood (PB) and saliva collection bi-monthly until predetermined clinical endpoints. Upon necropsy, the CNS and other tissues were collected for viral distribution and immunohistochemistry studies. A subset of marmosets inoculated with HHV6A or HHV6B (HHV6+EAE) mounted antiviral antibody responses and had detectable viral DNA in saliva and PB. Following EAE induction, HHV6+EAE marmosets exhibited accelerated and more aggressive clinical disease compared to controls, with significantly shorter survival times (p=0.01). HHV6+EAE marmosets also had an earlier onset of brain lesions (p=0.04) and mounted earlier and more robust anti-myelin antibody responses. Following HHV-6 inoculations, there was no detectable increase in serum anti-myelin antibodies, and no evidence of increased T cell responsiveness to myelin antigens, suggesting that a direct mechanism such as molecular mimicry was not underlying the observation of accelerated disease in the virus inoculated marmosets. However, we observed increased cellular immune responses in HHV-6-inoculated marmosets following the viral inoculations. These data suggest that EAE acceleration may have resulted from a more indirect mechanism of inflammatory-mediated blood brain barrier breakdown, possibly due to a viral ‘priming’ of peripheral immune cells. This study provides an experimental counterpart to the fertile field hypothesis, which puts forth that autoimmune diseases may be induced and/or exacerbated by microbial infections, and provides mechanistic insights into the interplay of viral and autoimmune components, which are believed to be involved in the complex pathophysiology of MS