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Abstract Background Latency remains a major obstacle to finding a cure for HIV despite the availability of antiretroviral therapy. Due to virus dormancy, limited biomarkers are available to identify latent HIV-infected cells. Profiling of individual HIV-infected cells is needed to explore potential latency biomarkers and to study the mechanisms of persistence that maintain the HIV reservoir. Methods Single cell spatial transcriptomic characterization using the CosMx Spatial Molecular Imager platform was conducted to analyze HIV-infected cells in formalin-fixed paraffin-embedded sections of splenic tissue surgically obtained from an HIV-infected humanized mouse model. Regulation of over a thousand human genes was quantified in both viremic and aviremic specimens. In addition, in situ hybridization and immunohistochemistry were performed in parallel to identify HIV viral RNA- and p24-containing cells, respectively. Finally, initial findings from CosMx gene profiling were confirmed by isolating RNA from CD4 + T cells obtained from a person living with HIV on antiretroviral therapy following either PMA/Ionomycin or DMSO treatment. RNA was quantified using qPCR for a panel of targeted human host genes. Results Supervised cell typing revealed that most of the HIV-infected cells in the mouse spleen sections were differentiated CD4 + T cells. A significantly higher number of infected cells, 2781 (1.61%) in comparison to 112 (0.06%), and total HIV transcripts per infected cell were observed in viremic samples compared to aviremic samples, respectively, which was consistent with the data obtained from ISH and IHC. Notably, the expression of 55 genes was different in infected cells within tissue from aviremic animals compared to viremic. In particular, both spleen tyrosine kinase (SYK) and CXCL17, were expressed approximately 100-fold higher. This data was further evaluated against bulk RNA isolated from HIV-infected human primary CD4 + T cells. A nearly 6-fold higher expression of SYK mRNA was observed in DMSO-treated CD4 + T cells compared to those stimulated with PMA/Ionomycin. Conclusion This study found that the CosMx SMI platform is valuable for assessing HIV infection and providing insights into host biomarkers associated with HIV reservoirs. Higher relative expression of the SYK gene in aviremic-infected cells from the humanized mouse HIV model was consistent with levels found in CD4 + T cells of aviremic donors.

BackgroundBiofeedback-based virtual reality (VR-BF) is a novel, nonpharmacologic method for teaching patients how to control their breathing, which in turn increases heart rate variability (HRV) and may reduce pain. Unlike traditional forms of biofeedback, VR-BF is delivered through a gamified virtual reality environment, increasing the accessibility of biofeedback. This is the first study to systematically integrate VR-BF use in the pediatric perioperative setting, with the ultimate goal of evaluating the efficacy of VR-BF to reduce pain, anxiety, and opioid consumption once feasibility and acceptability have been established. ObjectivesThe primary objective was to develop a clinical trial protocol for VR-BF use in the pediatric perioperative setting, including preoperative education and training, and postoperative application of VR-BF in children undergoing surgery. A secondary objective was to evaluate the patient and parent experience with VR-BF. MethodsA total of 23 patients (12-18 years of age) scheduled for surgery at Nationwide Children’s Hospital were recruited using purposive sampling. Following training, participants independently completed a daily, 10-minute VR-BF session for 7 days before surgery and during their inpatient stay. Participants could use VR-BF up to 2 weeks after hospital discharge. Patient- and session-level data of VR-BF usage and achievement of target HRV parameters were measured to identify the optimal frequency and duration of sessions before and after surgery for this population. Standardized questionnaires and semistructured interviews were conducted to obtain qualitative information about patients’ experiences with VR-BF. ResultsPatient-level data indicated that the highest odds of achieving 1 session under target HRV parameters was after 4 sessions (odds ratio [OR] 5.1 for 4 vs 3 sessions, 95% CI 1.3-20.6; OR 16.6 for 3 vs 2 sessions, 95% CI 1.2-217.0). Session-level data showed that a session duration of 9 to 10 minutes provided the greatest odds of achieving 1 session under target HRV parameters (OR 1.3 for 9 vs 8 min, 95% CI 1.1-1.7; OR 1.4 for 8 vs 7 min, 95% CI 1.1-1.8; OR 1 for 10 vs 9 min, 95% CI 0.9-1.2). Qualitative data revealed patient satisfaction with the VR-BF technology, particularly in managing perioperative stress (17/20, 85%). Few patients reported VR-BF as beneficial for pain (8/20, 40%). ConclusionsChildren and adolescents undergoing surgery successfully learned behavioral strategies with VR-BF with 10-minute sessions once daily for 5 days. To integrate VR-BF as a therapeutic intervention in a subsequent clinical trial, patients will be instructed to complete three 10-minute sessions a day for 7 days after surgery. Trial RegistrationClinicalTrials NCT04943874; https://clinicaltrials.gov/ct2/show/NCT04943874

Abstract Background Joint hypermobility is a common clinical finding amongst hereditary connective tissue disorders that is observed in pediatric rheumatological settings, and often associated with chronic pain. Joint hypermobility may also contribute to deficits in physical functioning and physical activity, but previous findings have been inconsistent. It is possible that physical activity impairment in joint hypermobility may be due to chronic aberrant movement patterns subsequent to increased joint laxity. Method As part of a larger randomized pilot trial of juvenile onset fibromyalgia (JFM), a secondary analysis was conducted to explore whether adolescents with JFM and joint hypermobility differed from non-joint hypermobility peers in terms of pain, daily functioning, and biomechanics (i.e., kinetics and kinematics) during a moderately vigorous functional task. Results From the larger sample of adolescents with JFM (N = 36), 13 adolescents (36.1%) met criteria for joint hypermobility and 23 did not have joint hypermobility. Those with joint hypermobility exhibited poorer overall functioning (Md = 20, Q 1 ,Q 3 [5.8, 7.6] vs. Md = 29, Q 1 ,Q 3 [5.1, 7.6]) but there were no differences in pain (Md = 6.9, Q 1 ,Q 3 [22, 33] , vs. Md = 6.45, Q 1 ,Q 3 [15, 29.5]). Inspection of time-series plots suggests those with joint hypermobility exhibited decreased hip flexion and frontal plane hip moment (e.g., resistance to dynamic valgus) during the landing phase (early stance) and greater hip and knee transverse plane moments during the propulsion phase (late stance) of the drop vertical jump task (DVJ). No other differences in lower extremity biomechanics were observed between study groups. Conclusions In this exploratory study, there were small but notable differences in biomechanics between patients with JFM who also had joint hypermobility versus those without joint hypermobility during a landing and jumping task (e.g., DVJ). These differences may indicate decreased joint stiffness during landing, associated with increased joint laxity and decreased joint stability, which may put them at greater risk for injury. Further study with a larger sample size is warranted to examine whether these biomechanical differences in patients with JFM and joint hypermobility affect their response to typical physical therapy or exercise recommendations.

Abstract Background Hypermobile Ehlers-Danlos syndrome (hEDS) is characterized by joint and skin laxity, and often accompanied by chronic pain, dysautonomia, increased distress and, functional limitations. The journey to accurate diagnosis is often prolonged due to unclear etiology of symptoms. This manuscript is a narrative review of the literature on illness uncertainty (IU) in hEDS, highlighting the unique facets of IU in this population, as compared to the broader chronic pain population (given symptom overlap between these two disease groups), that warrant additional investigation. Additionally, we considered the unique challenges associated with IU in the context of the developmental nuances of pediatric populations. Specifically, we aimed to (1) map the extant literature of the IU experience in chronic pain conditions broadly including the pediatric and adult research to identify key concepts related to IU and incorporate potential developmental considerations in IU; (2) delineate and describe the IU experience specifically in patients with hEDS, with the goal of identifying gaps in the literature based on aspects of presentation in hEDS that do and do not differ from the broader chronic pain population; and (3) elucidate the potential areas of adverse impact of IU in both general chronic pain populations, and those with hEDS specifically, to provide actionable areas for future research and clinical care of individuals with hEDS. Results of this review indicate that IU has been well-studied in chronic pain generally, but inadequately evaluated in hEDS specifically. Specific features of hEDS (complexity of the disorder, involvement of multiple bodily systems, contribution of organic pathology) may uniquely contribute to IU in this population. This review suggests that ambiguities surrounding the diagnosis of hEDS, symptom course, and treatment recommendations, along with misdiagnosis, perceived dismissal of symptoms, or attribution of symptoms to mental health concerns might increase risk for IU and related distress in patients. Conclusion Findings from the present review suggest that distinct features of hEDS yield a set of driving factors for IU that may be somewhat different than those faced by patients with chronic pain or other medical conditions. The development of a validated measure of IU to appropriately assess this construct in patients with hEDS is a research priority. In the clinical setting, providers should be attentive to the potentially aversive diagnostic and treatment experiences reported by patients and attempt to provide clear explanations based on the extant knowledge of hEDS, and implement best-practice recommendations for multidisciplinary treatment.

We demonstrate that human immunodeficiency virus (HIV) gag p24 protein is more readily detected in gut and lymph node tissues than in blood CD4+ T cells and correlates better with CD4 count during antiretroviral therapy (ART). Gut p24 levels also measurably decline with ART in natural controllers. During ART, gut p24 expression is more strongly associated both with HIV-specific CD8+ T-cell frequency and plasma soluble CD14 levels than gut HIV RNA expression. This study supports using gag p24 as a marker of HIV expression in HIV+ tissues to study effects of viral persistence and to monitor efficacy of treatment in HIV-based clearance studies.

BackgroundFunctional abdominal pain disorders (FAPD) are the most common chronic pain conditions of childhood and are made worse by co-occurring anxiety. Our research team found that the Aim to Decrease Pain and Anxiety Treatment (ADAPT), a six-session coping skills program using cognitive behavioral therapy strategies, was effective in improving pain-related symptoms and anxiety symptoms compared to standard care. In follow-up, this current randomized clinical trial (RCT) aims to test potential neural mechanisms underlying the effect of ADAPT. Specifically, this two-arm RCT will explore changes in amygdalar functional connectivity (primary outcome) following the ADAPT protocol during the water loading symptom provocation task (WL-SPT). Secondary (e.g., changes in regional cerebral blood flow via pulsed arterial spin labeling MRI) and exploratory (e.g., the association between the changes in functional connectivity and clinical symptoms) outcomes will also be investigated.MethodsWe will include patients ages 11 to 16 years presenting to outpatient pediatric gastroenterology care at a midwestern children's hospital with a diagnosis of FAPD plus evidence of clinical anxiety based on a validated screening tool (the Generalized Anxiety Disorder-7 [GAD-7] measure). Eligible participants will undergo baseline neuroimaging involving the WL-SPT, and assessment of self-reported pain, anxiety, and additional symptoms, prior to being randomized to a six-week remotely delivered ADAPT program plus standard medical care or standard medical care alone (waitlist). Thereafter, subjects will complete a post assessment neuroimaging visit similar in nature to their first visit.ConclusionsThis small scale RCT aims to increase understanding of potential neural mechanisms of response to ADAPT.Trial registrationClinicalTrials.gov registration: NCT03518216.

Nearshore sandbars are common features along sandy coasts. However, identifying sandbars within a beach profile traditionally requires a large historical dataset or subjective input from an observer. Several existing methodologies rely on reference profiles, which is problematic for new study sites with limited data sets and for nourished beaches that have drastic fluctuations in the cross-shore. This novel technique is suitable for beaches where a reference profile does not exist, as it identifies morphological sandbar features by a quantitative automated process. The technique identifies sandbars with a minimum steepness of 2% grade and a minimum height of 0.2 m. The morphological boundaries of sandbars were previously not well-defined, especially the seaward limit of the sandbar, contributing to difficulty in comparing surveys and sandbar morphologies. This technique standardizes the definitions of the bar limits mathematically via standard MATLAB functions, thus removing subjectivity and allowing results to be replicated. Bar identification is focused on the beach profile below the mean high water line, not cross on-shore positions, making the technique appropriate for nourished shorelines as well as those with large seasonal fluctuations. The automated technique was tested on 840 profiles collected near a recently completed beach nourishment project in Long Branch, NJ, USA. Results indicate success in identifying prominent sandbars within the test data set.

Accurate characterization of the HIV reservoir is imperative to develop an effective cure. HIV was measured in antiretroviral therapy-suppressed individuals using the intact proviral DNA assay (IPDA), along with assays for total or integrated HIV DNA, and inducible HIV RNA or p24. Intact provirus correlated with total and integrated HIV.

Productively infected cells are generally thought to arise from HIV infection of activated CD4+ T cells, and these infected activated cells are thought to be a recurring source of latently infected cells when a portion of the population transitions to a resting state. We discovered and report here that productively and latently infected cells can instead originate from direct infection of resting CD4+ T cell populations in lymphoid tissues in Fiebig I, the earliest stage of detectable HIV infection. We found that direct infection of resting CD4+ T cells was correlated with the availability of susceptible target cells in lymphoid tissues largely restricted to resting CD4+ T cells in which expression of pTEFb enabled productive infection, and we documented persistence of HIV-producing resting T cells during antiretroviral therapy (ART). Thus, we provide evidence of a mechanism by which direct infection of resting T cells in lymphoid tissues to generate productively and latently infected cells creates a mechanism by which the productively infected cells can replenish both populations and maintain two sources of virus from which HIV infection can rebound, even if ART is instituted at the earliest stage of detectable infection.

Accurate characterization of the HIV reservoir is imperative to develop an effective cure. HIV was measured in antiretroviral therapy-suppressed individuals using the intact proviral DNA assay (IPDA), along with assays for total or integrated HIV DNA, and inducible HIV RNA or p24. Intact provirus correlated with total and integrated HIV.

Productively infected cells are generally thought to arise from HIV infection of activated [CD4.sup.+] T cells, and these infected activated cells are thought to be a recurring source of latently infected cells when a portion of the population transitions to a resting state. We discovered and report here that productively and latently infected cells can instead originate from direct infection of resting [CD4.sup.+] T cell populations in lymphoid tissues in Fiebig I, the earliest stage of detectable HIV infection. We found that direct infection of resting [CD4.sup.+] T cells was correlated with the availability of susceptible target cells in lymphoid tissues largely restricted to resting [CD4.sup.+] T cells in which expression of pTEFb enabled productive infection, and we documented persistence of HIV-producing resting T cells during antiretroviral therapy (ART). Thus, we provide evidence of a mechanism by which direct infection of resting T cells in lymphoid tissues to generate productively and latently infected cells creates a mechanism by which the productively infected cells can replenish both populations and maintain two sources of virus from which HIV infection can rebound, even if ART is instituted at the earliest stage of detectable infection., Introduction From the beginning of HIV research, HIV has been mainly propagated in vitro in tissue cultures of activated [CD4.sup.+] T cells (1), leading to the prevailing view that activated [...]

There is a great need for nonpharmacologic pain management strategies, given the catastrophic effects of the opioid epidemic and the role of opioid prescription in precipitating addiction [1], particularly in children and adolescents at risk of chronic pain and opioid use after surgery [2–4]. Biofeedback-based virtual reality (VR-BF) is an innovative approach to managing pain that compliments and may even increase accessibility [5] and acceptability [6] of existing mind-body therapies for pain management, like biofeedback (BF). BF teaches patients behavioral modification techniques that impact involuntary processes [7,8]. For example, slow breathing increases heart rate variability (HRV) [9] to reduce pain through the downregulation of the sympathetic nervous system [10,11]. However, barriers to widespread use, such as the need for trained personnel and high costs of direct intervention, have hindered its widespread clinical use and access to this therapy [5,12]. VR-BF has not yet been integrated into perioperative care, and as such, no defined treatment protocols for preoperative training and postoperative application of VR-BF exist, particularly in children. The dataset presented in this article may help fill the unmet, critical need for accessible, effective, alternative therapeutic options for reducing postoperative pain and opioid exposure in children.This investigation aimed to establish measurable outcomes impacting a perioperative treatment protocol of VR-BF, a novel VR-based therapy that teaches patients relaxation techniques and monitors the sensitivity of heart rate variability (HRV) to different frequencies and durations of VR-BF sessions. Achievement of target physiological parameters, including HRV, was measured in children and adolescents undergoing surgery anticipated to cause moderate to severe pain (e.g., orthopedic, chest) requiring postoperative pain management by the Acute Pain Services at Nationwide Children's Hospital (NCH).This dataset included 23 surgical patients evaluated quantitatively and qualitatively to refine a treatment protocol for the feasibility and acceptability of (a) preoperative education and training in relaxation, and (b) postoperative application of a VR-BF intervention for pain management [13]. Qualitative data was collected using an investigator-derived questionnaire to obtain feedback and understand the patient and family experience using VR-BF. Descriptive statistics (mean±SD or median with interquartile range [IQR] for continuous variables; frequencies and percentages for categorical variables) and exploratory spline regression analyses were generated to define measurable outcomes for a future pilot, randomized clinical trial protocol.

ABSTRACT Cholera has been a human scourge since the early 1800s and remains a global public health challenge, caused by the toxigenic strains of the bacterium Vibrio cholerae. In its aquatic reservoirs, V. cholerae has been shown to live in association with various arthropod hosts, including the chironomids, a diverse insect family commonly found in wet and semiwet habitats. The association between V. cholerae and chironomids may shield the bacterium from environmental stressors and amplify its dissemination. However, the interaction dynamics between V. cholerae and chironomids remain largely unknown. In this study, we developed freshwater microcosms with chironomid larvae to test the effects of cell density and strain on V. cholerae-chironomid interactions. Our results show that chironomid larvae can be exposed to V. cholerae up to a high inoculation dose (109 cells/mL) without observable detrimental effects. Meanwhile, interstrain variability in host invasion, including prevalence, bacterial load, and effects on host survival, was highly cell density-dependent. Microbiome analysis of the chironomid samples by 16S rRNA gene amplicon sequencing revealed a general effect of V. cholerae exposure on microbiome species evenness. Taken together, our results provide novel insights into V. cholerae invasion dynamics of the chironomid larvae with respect to various doses and strains. The findings suggest that aquatic cell density is a crucial driver of V. cholerae invasion success in chironomid larvae and pave the way for future work examining the effects of a broader dose range and environmental variables (e.g., temperature) on V. cholerae-chironomid interactions. IMPORTANCE Vibrio cholerae is the causative agent of cholera, a significant diarrheal disease affecting millions of people worldwide. Increasing evidence suggests that the environmental facets of the V. cholerae life cycle involve symbiotic associations with aquatic arthropods, which may facilitate its environmental persistence and dissemination. However, the dynamics of interactions between V. cholerae and aquatic arthropods remain unexplored. This study capitalized on using freshwater microcosms with chironomid larvae to investigate the effects of bacterial cell density and strain on V. cholerae-chironomid interactions. Our results suggest that aquatic cell density is the primary determinant of V. cholerae invasion success in chironomid larvae, while interstrain variability in invasion outcomes can be observed under specific cell density conditions. We also determined that V. cholerae exposure generally reduces species evenness of the chironomid-associated microbiome. Collectively, these findings provide novel insights into V. cholerae-arthropod interactions using a newly developed experimental host system.

Ian Boggero,1,2 Cecelia Valrie,3,4 Krystal Morgan,5 Nao Hagiwara,3 Susmita Kashikar-Zuck,2,6 Christopher King2,6 1Department of Oral Health Science, Division of Orofacial Pain, University of Kentucky College of Dentistry, Lexington, KY, USA; 2Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH, USA; 3Department of Psychology, Virginia Commonwealth University, Richmond, VA, USA; 4Institute for Inclusion, Inquiry, and Innovation, Virginia Commonwealth University, Richmond, VA, USA; 5Division of Transplant, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA; 6Division of Behavioral Medicine and Clinical Psychology, Cincinnati Children’s Hospital Medical Center, Cincinnati, OH, USACorrespondence: Ian Boggero, 740 S Limestone, Lexington, KY, 40536, USA, Tel +859-562-3291, Email ian.boggero@uky.eduContext: General fatigue, sleep-related fatigue, and cognitive fatigue are prevalent and disruptive in adults with chronic musculoskeletal (MSK) pain, but little is known about these fatigue subtypes in pediatric musculoskeletal pain.Objective: To compare fatigue and its subtypes between adolescents with chronic MSK pain and pain-free controls and to test if fatigue subtypes were associated with concurrent pain and its impact (pain intensity, number of pain sites, pain interference, and functional disability) or experimental pain (intensity and tolerance) in adolescents with chronic MSK pain. Finally, we sought to explore adolescents’ qualitative characterizations of their fatigue.Methods: Adolescents with chronic MSK pain (12– 17 y.o., n = 26) and pain-free controls (n = 26) completed validated self-report measures of fatigue, pain, and functional disability, underwent an experimental pain tolerance task (cold water immersion of the hand), and provided qualitative descriptions of their fatigue (pain group only).Results: Adolescents with chronic MSK pain reported significantly greater general, sleep-related, and cognitive fatigue than pain-free controls (all p’s < 0.001). In adolescents with chronic MSK pain, fatigue subtypes were associated with clinical pain and pain impact (r’s = 0.43– 0.84) but not experimental pain measures (p’s > 0.05). Adolescents with chronic MSK pain qualitatively described the negative implications of the different fatigue subtypes, particularly when perceived as long-lasting.Conclusion: This preliminary study suggests that fatigue subtypes are prevalent and impactful in pediatric patients with chronic MSK pain. When planning multi-disciplinary treatment for pediatric MSK pain, providers should recognize fatigue as another disabling symptom.Keywords: disability, fatigue, pediatric, musculoskeletal pain, widespread bodily pain