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BackgroundNeurological surgeons oftentimes educate patients and their families on complex medical conditions and treatment options. Time constraints and varied linguistic and cultural backgrounds limit the amount of information that can be disbursed. In this study, we assessed the linguistic validity of interactive educational interventions in non-English-speaking patients with traumatic brain injury (TBI) and concussion and their families.MethodsA total of 273 English-, Spanish-, Korean-, and Vietnamese-speaking neurotrauma patients (n =124) and family members (n =149) completed a presurvey to evaluate their incipient understanding, interacted with an iPad-based iBook (Apple) on concussion or TBI in their native language, completed a postsurvey to gauge changes in understanding, and then consulted with their neurosurgeon.ResultsAll participants (124 patients and 149 family members) had significantly increased (95% confidence interval [CI], P < 0.01) postsurvey scores (average pre-iBook score, 2.810; average post-iBook score, 4.109), regardless of native language or cultural background. Caucasian participants scored significantly higher than the combination of all ethnicities on both the baseline survey (95% CI, P < 0.01) and the post-iBook survey (95% CI, P

BackgroundIn order to study the genetics of diseases more accurately and effectively, one often collects large families. Different members of a large family may provide differing information about the structure and make-up of their pedigree. Thus, software is needed to facilitate reconciliation of pedigrees collected independently from multiple informants from a single large family to create a unified pedigree that is based on the best composite information available.FindingsOur implementation demonstrates that Genetic ME performs merging in terms of adding, replacing and combining information from two pedigrees. Through a tracking process, all of the changes made to the data set for the individuals can be traced back to their original source material. A new pedigree structure can be easily visualized while reconciling disparate information from multiple pedigrees.MethodsWe developed the Genetic Merging & Editing (Genetic ME) program, an open source Java application built on top of CraneFoot and Ghostscript, to support comparing, editing and merging of pedigrees collected from multiple sources in a visually-oriented manner.ConclusionsGenetic ME constitutes an ideal addition to software packages for reconciling pedigree information from multiple sources. Genetic ME provides a friendly graphical user interface, traces the changes made by users, and produces viewable merged pedigree structures able to be further used by other popular analysis programs.

Background and importance: Chronic subdural hematoma (cSDH) is a common neurosurgical pathology with a projected increase in prevalence as the elderly population grows. We describe a novel implementation of the IRRAflow irrigating drainage system after burr hole evacuation of bilateral acute-on-chronic subdural hematomas that demonstrates the benefits of this system for common post-operative challenges of these surgeries. Clinical presentation: An 83-year-old male presented to the emergency department with 3 weeks of progressive weakness, lethargy, and confusion following an unwitnessed fall. Physical examination was notable for pupillary asymmetry and altered mental status. Computed tomography (CT) of the head showed bilateral acute-on-chronic subdural hematomas. The patient underwent bilateral burr hole evacuation and placement of IRRAflow irrigating drains. Postoperative head CT demonstrated poor brain re-expansion with significant bilateral pneumocephalus. Marked improvement in brain re-expansion occurred with continuous irrigation and drainage, and the patient was discharged on postoperative day (POD) 3 with return to neurological baseline. He followed up on POD13 in stable condition. Conclusion: This report described a patient with bilateral subdural hematomas who underwent evacuation and bilateral IRRAflow dual-lumen catheter placement. Significant postoperative pneumocephalus improved rapidly with continuous irrigation and drainage, leading to improved brain re-expansion, hematoma resolution, and excellent neurological recovery.

Background: Chronic subdural hematomas (cSDH) are increasingly prevalent worldwide with the increased aging population and anticoagulant use. Different surgical, medical, and endovascular treatments have had varying success rates. Primary neurosurgical interventions include burr hole drainage of the cSDH and mini-craniotomies/craniotomies with or without fenestration of the inner membrane. A key assessment of the success or failure of cSDH treatments has been symptomatic recurrence rates which have historically ranged from 5 to 30%. Pre-operative prediction of the inner subdural membrane by CT scan was used to guide our decision to perform mini-craniotomies. Release of the inner membrane facilitates the expansion of the brain and likely improves glymphatic flow.Methods: Consecutive mini-craniotomies (N = 34) for cSDH evacuation performed by a single neurosurgeon at a quaternary academic medical center/Level I trauma center from July 2018-September 2020 were retrospectively reviewed. Patient characteristics [age, gender, presenting GCS, GOS, initial CTs noting the inner subdural membrane, midline shift (MLS), cSDH width, inner membrane fenestration, cSDH recurrence, post-operative seizures, infections, length of stay] were extracted from the EMR.Results: Twenty nine patients had mini-craniotomies as primary treatment of the cSDH. Mean age = 68.9 ± 19.7 years (range 22–102), mean pre-operative GCS = 14.5 ± 1.1, mean MLS = 6.75 ± 4.2 mm, and mean maximum thickness of cSDH = 17.7 ± 6.0 mm. Twenty four were unilateral, five bilateral, 34 total craniotomies were performed. Thirty three had inner membrane signs on pre-operative head CTs and an inner subdural membrane was fenestrated in all cases except for the one craniotomy that didn't show these characteristic CT findings. Mean operating time = 79.5 ± 26.0 min. Radiographic and clinical improvement occurred in all patients. Mean improvement in MLS = 3.85 ± 2.69. There were no symptomatic recurrences, re-operations, surgical site infections, or deaths during the 6 months of follow-up. One patient was treated for post-operative seizures with AEDs for 6 months.Conclusion: Pre-operative CT scans demonstrating inner subdural membranes may guide one to target the treatment to allow release of this tension band. Mini-craniotomy with careful fenestration of the inner membrane is very effective for this. Brain re-expansion and re-establishment of normal brain interstitial flow may be important in long term outcomes with cSDH and may be related to the recent interests in brain glymphatics and dural lymphatics.

Objective: Patients with medically refractory focal epilepsy can be difficult to treat surgically, especially if invasive monitoring reveals multiple ictal onset zones. Possible therapeutic options may include resection, neurostimulation, laser ablation, or a combination of these surgical modalities. To date, no study has examined outcomes associated with resection plus responsive neurostimulation (RNS, Neuropace, Inc., Mountain View, CA) implantation and we describe our initial experience in patients with multifocal epilepsy undergoing this combination therapy.Methods: A total of 43 responsive neurostimulation (RNS) devices were implanted at UCI from 2015 to 2019. We retrospectively reviewed charts of patients from the same time period who underwent both resection and RNS implantation. Patients were required to have independent or multifocal onset, undergo resection and RNS implantation, and have a minimum of six-months for follow-up to be included in the study. Demographics, location of ictal onset, location of surgery, complications, and seizure outcome were collected.Results: Ten patients met inclusion criteria for the study, and seven underwent both procedures in the same setting. The average age was 36. All patients had multifocal ictal onset on video electroencephalogram or invasive EEG with four patients undergoing subdural grid placement and four patients undergoing bilateral sEEG prior to the definitive surgery. Five patients underwent resection plus ipsilateral RNS placement and the remainder underwent resection with contralateral RNS placement. Two minor complications were encountered in this group. At six months follow up, there was an average of 81% ± 9 reduction in seizures, while four patients experienced complete seizure freedom at 1 year.Conclusion: Patients with multifocal epilepsy can be treated with partial resection plus RNS. The complication rates are low with potential for worthwhile seizure reduction.

The skin at the site of HSV-2 reactivation is enriched for HSV-2-specific T cells. To evaluate whether an immunotherapeutic vaccine could elicit skin-based memory T cells, we studied skin biopsies and HSV-2-reactive CD4+ T cells from PBMCs by T cell receptor (TCR) β chain (TRB) sequencing before and after vaccination with a replication-incompetent whole-virus HSV-2 vaccine candidate (HSV529). The representation of HSV-2-reactive CD4+ TRB sequences from PBMCs in the skin TRB repertoire increased after the first vaccine dose. We found sustained expansion after vaccination of unique, skin-based T cell clonotypes that were not detected in HSV-2-reactive CD4+ T cells isolated from PBMCs. In one participant, a switch in immunodominance occurred with the emergence of a TCR αβ pair after vaccination that was not detected in blood. This TCRαβ was shown to be HSV-2 reactive by expression of a synthetic TCR in a Jurkat-based NR4A1 reporter system. The skin in areas of HSV-2 reactivation possessed an oligoclonal TRB repertoire that was distinct from the circulation. Defining the influence of therapeutic vaccination on the HSV-2-specific TRB repertoire requires tissue-based evaluation.

Background: Coronavirus disease 2019 (COVID-19) patients with sleep disorders may be at greater risk for respiratory exacerbation or death compared to those without. After being infected with COVID-19, patients have many symptoms related to sleep disorders, especially those with severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) infection. This study aimed to evaluate sleep disturbances in patients with severe SARS-CoV-2 infection who were treated in the Intensive Care Unit (ICU)., Methods: This cross-sectional study used the questionnaire provided by the Vietnam Sleep Disorder Study (ViSDiS) research, elaborated by the Vietnam Society of Sleep Medicine (VSSM). Seventy-seven COVID-19 patients were included., Results: There was a significant difference in sleep status before and after SARS-CoV-2 infection among participants. Up to 83% of them reported experiencing insomnia after illness, 60% reported having frequent nightmares, and more than half of participants reported nocturia ( p < 0.0001). More than 81.8% of patients with severe SARS-CoV-2 infection were unsatisfied with their sleep quality during hospitalization After SARS-CoV-2 infection, only 2.6% of participants felt they had good quality sleep ( p < 0.0001). The majority of patients suffered from fatigue after SARS-CoV-2 infection, including a lack of energy, feeling heaviness in their limbs, aggravation of pre-existing sleep disorders, idleness, constant fatigue throughout the day, and difficulty concentrating., Conclusion: Sleep problems are highly prevalence among hospitalized patients with severe COVID-19 in the ICU. Healthcare providers should pay attention to sleep problems and their associated symptoms to initiate appropriate treatment to improve severe COVID-19 patients' health status and minimize the risk of death.

Difficulty falling asleep is one of the typical insomnia symptoms. However, intervention therapies available nowadays, ranging from pharmaceutical to hi-tech tailored solutions, remain ineffective due to their lack of precise real-time sleep tracking, in-time feedback on the therapies, and an ability to keep people asleep during the night. This paper aims to enhance the efficacy of such an intervention by proposing a novel sleep aid system that can sense multiple physiological signals continuously and simultaneously control auditory stimulation to evoke appropriate brain responses for fast sleep promotion. The system, a lightweight, comfortable, and user-friendly headband, employs a comprehensive set of algorithms and dedicated own-designed audio stimuli. Compared to the gold-standard device in 883 sleep studies on 377 subjects, the proposed system achieves (1) a strong correlation (0.89 ± 0.03) between the physiological signals acquired by ours and those from the gold-standard PSG, (2) an 87.8% agreement on automatic sleep scoring with the consensus scored by sleep technicians, and (3) a successful non-pharmacological real-time stimulation to shorten the duration of sleep falling by 24.1 min. Conclusively, our solution exceeds existing ones in promoting fast falling asleep, tracking sleep state accurately, and achieving high social acceptance through a reliable large-scale evaluation., (© 2023. Springer Nature Limited.)

Purpose: In this study, we described "PT for Sleep Apnea", a smartphone application for home-based physical therapy of patients with Obstructive Sleep Apnea (OSA)., Methods: The application was created in a joint program between the University of Medicine and Pharmacy at Ho Chi Minh City (UMP), Vietnam, and National Cheng Kung University (NCKU), Taiwan. Exercises maneuvers were derived from the exercise program previously published by the partner group at National Cheng Kung University. They included exercises for upper airway and respiratory muscle training and general endurance training., Results: The application provides video and in-text tutorials for users to follow at home and a schedule function to assist the user in organizing the training program, which may improve the efficacy of home-based physical therapy in patients with Obstructive Sleep Apnea., Conclusion: In the future, our group plans to conduct a user study and randomized-controlled trials to investigate whether our application can benefit patients with OSA., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Bui-Diem, Hung, Zhu, Tho, Nguyen-Binh, Vu-Tran-Thien, To-Truong, Ngo-Thanh and Duong-Quy.)

Obstructive sleep apnea (OSA) is a common disease that is often under-diagnosed and under-treated in all ages. This is due to differences in morphology, diversity in clinical phenotypes, and differences in diagnosis and treatment of OSA in children and adults, even among individuals of the same age. Therefore, a personalized medicine approach to diagnosis and treatment of OSA is necessary for physicians in clinical practice. In children and adults without serious underlying medical conditions, polysomnography at sleep labs may be an inappropriate and inconvenient testing modality compared to home sleep apnea testing. In addition, the apnea-hypopnea index should not be considered as a single parameter for making treatment decisions. Thus, the treatment of OSA should be personalized and based on individual tolerance to sleep-quality-related parameters measured by the microarousal index, harmful effects of OSA on the cardiovascular system related to severe hypoxia, and patients' comorbidities. The current treatment options for OSA include lifestyle modification, continuous positive airway pressure (CPAP) therapy, oral appliance, surgery, and other alternative treatments. CPAP therapy has been recommended as a cornerstone treatment for moderate-to-severe OSA in adults. However, not all patients can afford or tolerate CPAP therapy. This narrative review seeks to describe the current concepts and relevant approaches towards personalized management of patients with OSA, according to pathophysiology, cluster analysis of clinical characteristics, adequate combined therapy, and the consideration of patients' expectations.

Background: The COVID-19 outbreak witnessed in the autumn of 2021 led to unprecedented changes in healthcare systems in some emerging countries. Many field-hospitals, temporary sites of care for COVID-19 patients, were built around the country and followed by the healthcare workers who were mobilized. This study aimed to measure sleep disorders, depression, and fatigue in volunteers working at field hospitals during the COVID-19 outbreak., Methods: This was a cross-sectional study. The self-report questionnaire was used for each study subject. Sleep characters, including STOP's elements were questioned. Healthcare workers' burnout was detected by using Pichot's questionnaire., Results: One hundred front-line healthcare workers (FHWs), predominantly last year and graduated medical students, were included in the study (86% female subjects). The mean sleep-time of FHWs before, while working, and during the isolation period after working at COVID-19 field hospitals were: 7.78 ± 1.48, 5.71 ± 1.40, and 8.78 ± 2.31 h per day, respectively. Burnout was not a crucial issue for these volunteer subjects. The mean scores of Pichot's Fatigue Scale and Pichot's Depression Scale, measured after 4 weeks working at field hospitals, were 4.18 ± 5.42 and 2.54 ± 3.36, respectively. Thirteen participants were suspected of depression. The fatigue scores decreased significantly in the group who claimed short sleep latency. The factor that increased the depression score was "anxious feeling" ( p = 0.001). Other significant factors were "short sleep latency," "observed sleep apnea," "tiredness, daily sleepiness" and "snoring.", Conclusion: Appropriate work schedule, better sleep conditions, and mental health support could be helpful for FHWs. The mandatory 2 weeks of isolation after working in field hospitals provided opportunity for FHWs' recovery., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Duong-Quy, Tran-Duc, Hoang-Chau-Bao, Bui-Diem, Vu-Tran-Thien and Nguyen-Nhu.)

Determinants of protective immunity against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection require the development of well-standardized, reproducible antibody assays. This need has led to the emergence of a variety of neutralization assays. Head-to-head evaluation of different SARS-CoV-2 neutralization platforms could facilitate comparisons across studies and laboratories. Five neutralization assays were compared using 40 plasma samples from convalescent individuals with mild to moderate coronavirus disease 2019 (COVID-19): four cell-based systems using either live recombinant SARS-CoV-2 or pseudotyped viral particles created with lentivirus (LV) or vesicular stomatitis virus (VSV) packaging and one surrogate enzyme-linked immunosorbent assay (ELISA)-based test that measures inhibition of the spike protein receptor binding domain (RBD) binding its receptor human angiotensin converting enzyme 2 (hACE2). Vero cells, Vero E6 cells, HEK293T cells expressing hACE2, and TZM-bl cells expressing hACE2 and transmembrane serine protease 2 were tested. All cell-based assays showed 50% neutralizing dilution (ND 50 ) geometric mean titers (GMTs) that were highly correlated (Pearson r  = 0.81 to 0.89) and ranged within 3.4-fold. The live virus assay and LV pseudovirus assays with HEK293T/hACE2 cells showed very similar mean titers, 141 and 178, respectively. ND 50 titers positively correlated with plasma IgG targeting SARS-CoV-2 spike protein and RBD ( r  = 0.63 to 0.89), but moderately correlated with nucleoprotein IgG ( r  = 0.46 to 0.73). ND 80 GMTs mirrored ND 50 data and showed similar correlation between assays and with IgG concentrations. The VSV pseudovirus assay and LV pseudovirus assay with HEK293T/hACE2 cells in low- and high-throughput versions were calibrated against the WHO SARS-CoV-2 IgG standard. High concordance between the outcomes of cell-based assays with live and pseudotyped virions enables valid cross-study comparison using these platforms.

Tissue-resident-memory T cells (TRM) populate the body's barrier surfaces, functioning as frontline responders against reencountered pathogens. Understanding of the mechanisms by which CD8TRM achieve effective immune protection remains incomplete in a naturally recurring human disease. Using laser capture microdissection and transcriptional profiling, we investigate the impact of CD8TRM on the tissue microenvironment in skin biopsies sequentially obtained from a clinical cohort of diverse disease expression during herpes simplex virus 2 (HSV-2) reactivation. Epithelial cells neighboring CD8TRM display elevated and widespread innate and cell-intrinsic antiviral signature expression, largely related to IFNG expression. Detailed evaluation via T-cell receptor reconstruction confirms that CD8TRM recognize viral-infected cells at the specific HSV-2 peptide/HLA level. The hierarchical pattern of core IFN- γ signature expression is well-conserved in normal human skin across various anatomic sites, while elevation of IFI16, TRIM 22, IFITM2, IFITM3, MX1, MX2, STAT1, IRF7, ISG15, IFI44, CXCL10 and CCL5 expression is associated with HSV-2-affected asymptomatic tissue. In primary human cells, IFN- γ pretreatment reduces gene transcription at the immediate-early stage of virus lifecycle, enhances IFI16 restriction of wild-type HSV-2 replication and renders favorable kinetics for host protection. Thus, the adaptive immune response through antigen-specific recognition instructs innate and cell-intrinsic antiviral machinery to control herpes reactivation, a reversal of the canonical thinking of innate activating adaptive immunity in primary infection. Communication from CD8TRM to surrounding epithelial cells to activate broad innate resistance might be critical in restraining various viral diseases., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The reviewer has declared a shared affiliation with some of the authors CS, MM, DK to the handling Editor at the time of review., (Copyright © 2021 Peng, Phasouk, Sodroski, Sun, Hwangbo, Layton, Jin, Klock, Diem, Magaret, Jing, Laing, Li, Huang, Mertens, Johnston, Jerome, Koelle, Wald, Knipe, Corey and Zhu.)

Chimeric antigen receptor (CAR) T cells are engineered cells used in cancer therapy and are studied to treat infectious diseases. Trafficking and persistence of CAR T cells is an important requirement for efficacy to target cancer. Here, we describe a CAR RNA FISH histo-cytometry platform combined with a random reaction seed image analysis algorithm to quantitate spatial distribution and in vivo functional activity of a CAR T cell population at a single cell resolution for preclinical models. In situ, CAR T cell exhibited a heterogenous effector gene expression and this was related to the distance from tumor cells, allowing a quantitative assessment of the potential in vivo effectiveness. The platform offers the potential to study immune functions of genetically engineered cells in situ with their target cells in tissues with high statistical power and thus, can serve as an important tool for preclinical assessment of CAR T cell effectiveness.

The efficacy of short-term treatment with mifepristone (MIFE), a high-affinity, nonselective glucocorticoid receptor antagonist, to reduce ethanol drinking was tested in a rhesus macaque model. Stable individual daily ethanol intakes were established, ranging from 1.6 to 4.0 g/kg per day ( n = 9 monkeys). After establishment of chronic ethanol intake, a MIFE dosing regimen that modeled a study of rodent drinking and human alcohol craving was evaluated. Three doses of MIFE (17, 30, and 56 mg/kg per day) were each administered for four consecutive days. Both 30 and 56 mg/kg decreased ethanol intake compared with baseline drinking levels without a change in water intake. The dose of 56 mg/kg per day of MIFE produced the largest reduction in ethanol self-administration, with the average intake at 57% of baseline intakes. Cortisol was elevated during MIFE dosing, and a mediation analysis revealed that the effect on ethanol drinking was fully mediated through cortisol. During a forced abstinence phase, access to 1.5 g/kg ethanol resulted in relapse in all drinkers and was not altered by treatment with 56 mg/kg MIFE. Overall, these results show that during active drinking MIFE is efficacious in reducing heavy alcohol intake in a monkey model, an effect that was related to MIFE-induced increase in cortisol. However, MIFE treatment did not eliminate ethanol drinking. Further, cessation of MIFE treatment resulted in a rapid return to baseline intakes, and MIFE was not effective in preventing a relapse during early abstinence. SIGNIFICANCE STATEMENT: Mifepristone reliably decreases average daily ethanol self-administration in a nonhuman primate model. This effect was mediated by cortisol, was most effective during open-access conditions, and did not prevent or reduce relapse drinking., Competing Interests: H.H. is an employee at Corcept Therapeutics., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Secretion of pulmonary surfactant in the alveoli of the lungs is essential to maintain lung function. Stretching of alveoli during lung inflation is the main trigger for surfactant secretion. Yet, the molecular mechanisms how mechanical distension of alveoli results in surfactant secretion are still elusive. The alveolar epithelium consists of alveolar epithelial type I (ATI) and surfactant secreting type II (ATII) cells. ATI, but not ATII cells, express caveolae, small plasma membrane invaginations that can respond to plasma membrane stresses and serve mechanotransductive roles. Within this study, we investigated the role of caveolae as mechanosensors in the alveolus. We generated a human caveolin-1 knockout ATI cell (hAELVi cav-/- ) using CRISPR/Cas9. Wildtype (hAELVi wt ) and hAELVi cav-/- cells grown on flexible membranes responded to increasing stretch amplitudes with rises in intracellular Ca 2+ . The response was less frequent and started at higher stretch amplitudes in hAELVi cav-/- cells. Stretch-induced Ca 2+ -signals depended on Ca 2+ -entry via piezo1 channels, localized within caveolae in hAELVi wt and primary ATI cells. Ca 2+ -entry via piezo1 activated pannexin-1 hemichannels resulting in ATP release from ATI cells. ATP release was reduced in hAELVi cav-/- cells. In co-cultures resembling the alveolar epithelium, released ATP stimulated Ca 2+ signals and surfactant secretion from neighboring ATII cells when co-cultured with hAELVi wt but not hAELVi cav-/- cells. In summary, we propose that caveolae in ATI cells are mechanosensors within alveoli regulating stretch-induced surfactant secretion from ATII cells., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Keratin filaments (KFs) comprise the intermediate filaments of epithelial cells and are well known for their cytoprotective properties and their mechanical resilience. Although, several studies have demonstrated KFs' remarkable tensile properties relatively little is known about acute implications of mechanical stretch on KFs in living cells. This includes structural effects on the KFs and their higher level assembly structures as well as posttranslational response mechanisms to possibly modify KF's properties. We subjected simple epithelial A549 lung cells to 30% unidirectional stretch and already after 10 seconds we observed morphological changes of the KF-network as well as structural effects on their desmosomal anchor sites-both apparently caused by the tensile strain. Interestingly, the effect on the desmosomes was attenuated after 30 seconds of cell stretch with a concomitant increase in phosphorylation of keratin8-S432, keratin18-S53, and keratin18-S34 without an apparent increase in keratin solubility. When mimicking the phosphorylation of keratin18-S34 the stretch-induced effect on the desmosomes could be diminished and probing the cell surface with atomic force microscopy showed a lowered elastic modulus. We conclude that the stretch-induced KF phosphorylation affects KF's tensile properties, probably to lower the mechanical load on strained desmosomal cell-cell contacts, and hence, preserve epithelial integrity., (© 2020 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Background: Quantitative estimation of the extent to which the immune system's protective effect against one herpes simplex virus type 2 (HSV-2) infection protects against infection with additional HSV-2 strains is important for understanding the potential for HSV-2 vaccine development. Using viral genotyping, we estimated the prevalence of HSV-2 dual-strain infection and identified risk factors., Methods and Findings: People with and without HIV infection participating in HSV-2 natural history studies (University of Washington Virology Research Clinic) and HIV prevention trials (HIV Prevention Trials Network 039 and Partners in Prevention HSV/HIV Transmission Study) in the US, Africa, and Peru with 2 genital specimens each containing ≥105 copies herpes simplex virus DNA/ml collected a median of 5 months apart (IQR: 2-11 months) were included. It is unlikely that 2 strains would be detected in the same sample simultaneously; therefore, 2 samples were required to detect dual-strain infection. We identified 85 HSV-2 SNPs that, in aggregate, could determine whether paired HSV-2 strains were the same or different with >90% probability. These SNPs were then used to create a customized high-throughput array-based genotyping assay. Participants were considered to be infected with more than 1 strain of HSV-2 if their samples differed by ≥5 SNPs between the paired samples, and dual-strain infection was confirmed using high-throughput sequencing (HTS). We genotyped pairs of genital specimens from 459 people; 213 (46%) were men, the median age was 34 years (IQR: 27-44), and 130 (28%) were HIV seropositive. Overall, 272 (59%) people were from the US, 59 (13%) were from Peru, and 128 (28%) were from 8 countries in Africa. Of the 459 people, 18 (3.9%) met the criteria for dual-strain infection. HTS and phylogenetic analysis of paired specimens confirmed shedding of 2 distinct HSV-2 strains collected at different times in 17 pairs, giving an estimated dual-strain infection prevalence of 3.7% (95% CI = 2.0%-5.4%). Paired samples with dual-strain infection differed by a median of 274 SNPs in the UL&#95;US region (range 129-413). Matching our observed dual-strain infection frequency to simulated data of varying prevalences and allowing only 2 samples per person, we inferred the true prevalence of dual-strain infection to be 7%. In multivariable analysis, controlling for HIV status and continent of origin, people from Africa had a higher risk for dual-strain infection (risk ratio [RR] = 9.20, 95% CI = 2.05-41.32), as did people who were HIV seropositive (RR = 4.06, 95% CI = 1.42-11.56)., Conclusions: HSV-2 dual-strain infection was detected in 3.7% of paired samples from individual participants, and was more frequent among people with HIV infection. Simulations suggest that the true prevalence of dual-strain infection is 7%. Our data indicate that naturally occurring immunity to HSV-2 may be protective against infection with a second strain. This study is limited by the inability to determine the timing of acquisition of the second strain.

Introduction: Understanding the variability in circulating herpes simplex virus type 2 (HSV-2) genomic sequences is critical to the development of HSV-2 vaccines., Methods: Genital lesion swabs containing ≥ 10 7 log 10 copies HSV DNA collected from Africa, the USA, and South America underwent next-generation sequencing, followed by K-mer based filtering and de novo genomic assembly. Sites of heterogeneity within coding regions in unique long and unique short (U L &#95;U S ) regions were identified. Phylogenetic trees were created using maximum likelihood reconstruction., Results: Among 46 samples from 38 persons, 1468 intragenic base-pair substitutions were identified. The maximum nucleotide distance between strains for concatenated U L&#95; U S segments was 0.4%. Phylogeny did not reveal geographic clustering. The most variable proteins had non-synonymous mutations in < 3% of amino acids., Conclusions: Unenriched HSV-2 DNA can undergo next-generation sequencing to identify intragenic variability. The use of clinical swabs for sequencing expands the information that can be gathered directly from these specimens., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The ability to genetically manipulate trigeminal ganglion (TG) neurons would be useful in the study of the craniofacial nervous system and latent alphaherpesvirus infections. We investigated adeno-associated virus (AAV) vectors for gene delivery to the TG after intradermal whiskerpad delivery in mice. We demonstrated that AAV vectors of serotypes 1, 7, 8, and 9 trafficked from the whiskerpad into TG neurons and expressed transgenes within cell bodies and axons of sensory neurons in all three branches of the TG. Gene expression was highest with AAV1, and steadily increased over time up to day 28. Both constitutive and neuronal-specific promoters were able to drive transgene expression in TG neurons. Levels of vector genomes in the TG increased with input dose, and multiple transgenes could be co-delivered to TG neurons by separate AAV vectors. In conclusion, AAV1 vectors are suitable for gene delivery to TG sensory neurons following intradermal whiskerpad injection.

Homo sapiens harbor two distinct, medically significant species of simplexviruses, herpes simplex virus (HSV)-1 and HSV-2, with estimated divergence 6-8 million years ago (MYA). Unexpectedly, we found that circulating HSV-2 strains can contain HSV-1 DNA segments in three distinct genes. Using over 150 genital swabs from North and South America and Africa, we detected recombinants worldwide. Common, widely distributed gene UL39 genotypes are parsimoniously explained by an initial >457 basepair (bp) HSV-1 × HSV-2 crossover followed by back-recombination to HSV-2. Blocks of >244 and >539 bp of HSV-1 DNA within genes UL29 and UL30, respectively, have reached near fixation, with a minority of strains retaining sequences we posit as ancestral HSV-2. Our data add to previous in vitro and animal work, implying that in vivo cellular co-infection with HSV-1 and HSV-2 yields viable interspecies recombinants in the natural human host.

Background: Pritelivir is a novel helicase-primase inhibitor in clinical development for treatment of herpes simplex virus type 2 (HSV-2) infections. In preclinical work, resistance-mediating mutations were identified in the HSV-2 genome at 3 loci in the UL5 gene and 1 locus in UL52., Methods: To evaluate whether daily pritelivir treatment results in emergence of resistance-mediating mutations, we analyzed HSV-2 strains detected in genital swab specimens from trial participants who were randomly assigned to receive different dosages of pritelivir. We sequenced resistance regions from 87 participants' samples, the UL5 gene in 73 samples from 44 participants, and the UL52 gene in 71 samples from 43 participants., Results: We found no evidence that pritelivir induced known resistance-mediating mutations or for amino acid variation at other loci. In one participant's HSV-2 isolate, we found a previously unidentified mutation close to the putative resistance-mediating region in UL5 and subsequently determined in vitro susceptibility to pritelivir. We characterized mutations from 32 cultivated HSV-2 isolates previously found to be susceptible to pritelivir in vitro and identified several novel mutations that most likely reflect preexisting variation in circulating HSV-2., Conclusions: This study demonstrates evidence of retained susceptibility of HSV-2 to pritelivir in immunocompetent persons following daily therapy for up to 28 days., (© The Author 2016. Published by Oxford University Press for the Infectious Diseases Society of America.)

In situ detection of specific cells offers a unique perspective on the spatial interactions between host immune cells and specific viral pathogens or cancers. Most immunohistochemistry techniques require manual cell counting on biopsied and fixed tissue sections. The availability of sophisticated software packages for analyzing fluorescently labeled tissue has made it possible to quickly and accurately quantitate the number of positive cells on such slides. Manual cell counting was compared to automatic cell counting using the program CellProfiler. The two techniques were used to count CD4+ and CD8+ T cells in human genital skin biopsies from herpesvirus type 2 (HSV-2) infected subjects. Manual counting and CellProfiler demonstrated high correlation both in cell counting as well as detection of immune cell "clustering" in tissue, an important visceral component of localized inflammation and characteristic of most chronic infections. Overall, CellProfiler is an effective and accurate method in addition to or replacement of manual cell counting of fluorescently labeled biopsies., (Copyright © 2015 Elsevier B.V. All rights reserved.)