Low-density granulocytes display immature cells with enhanced NET formation in people living with HIV.

While the protective role of neutrophil extracellular traps (NETs) in limiting human immunodeficiency virus (HIV) spread to susceptible cells has been documented, there is comparatively little insight into whether NET formation is harmful in people living with HIV (PLWH). To gain insight into neutrophil dysregulation and the pathological role of NETs in HIV, we examined expressions of NET-associated markers [cell-free DNA (cfDNA) and citrullinated histone H3 (CitH3)] in the plasmas from a cohort of the Hawaii Aging with HIV-cardiovascular and HIV-seronegative (HIV-) individuals. In a subset of participants, circulating low-density granulocyte (LDG) levels and their maturation and activation status were analyzed via flow cytometry. We demonstrated higher plasma levels of CitH3 in PLWH compared to HIV- individuals. LDGs from PLWH had heightened CD66b, but reduced CD16 expression. The percentages and counts of CD10+ LDGs were significantly decreased in PLWH. In addition, the CD16Lo LDG subsets were enriched in PLWH, compared to HIV- group, indicating that immature LDGs are increased in PLWH. Moreover, LDGs from PLWH exhibited significantly higher NET forming capacity. In summary, our study presents evidence that LDGs from PLWH on ART display an immature and altered phenotype with increased NET formation. Among PLWH, plasma NET levels as well as LDG parameters correlated with blood markers for inflammation and coagulation, suggesting that neutrophil activation and NETs may exert proinflammatory and coagulation effects. Our data provide insights into the pathologic role of LDGs at least in part mediated through NET formation in PLWH.

Impact of antiretroviral therapy intensification with C-C motif chemokine receptor 5 antagonist maraviroc on HIV-associated neurocognitive impairment.

OBJECTIVES: Chemokine receptor CCR5 is the principal co-receptor for entry of M-tropic HIV virus into immune cells. It is expressed in the central nervous system and may contribute to neuro-inflammation. The CCR5 antagonist maraviroc (MVC) has been suggested to improve HIV-associated neurocognitive impairment (NCI). DESIGN: A double-blind, placebo-controlled, 48-week, randomized study of MVC vs. placebo in people with HIV (PWH) on stable antiretroviral therapy (ART) for more than one year in Hawaii and Puerto Rico with plasma HIV RNA less than 50 copies/ml and at least mild NCI defined as an overall or domain-specific neuropsychological z (NPZ) score less than -0.5. METHODS: Study participants were randomized 2 : 1 to intensification of ART with MVC vs. placebo. The primary endpoint was change in global and domain-specific NPZ modeled from study entry to week 48. Covariate adjusted treatment comparisons of average changes in cognitive outcome were performed using winsorized NPZ data. Monocyte subset frequencies and chemokine expression as well as plasma biomarker levels were assessed. RESULTS: Forty-nine participants were enrolled with 32 individuals randomized to MVC intensification and 17 to placebo. At baseline, worse NPZ scores were seen in the MVC arm. Comparison of 48-week NPZ change by arm revealed no differences except for a modest improvement in the Learning and Memory domain in the MVC arm, which did not survive multiplicity correction. No significant changes between arms were seen in immunologic parameters. CONCLUSION: This randomized controlled study found no definitive evidence in favor of MVC intensification among PWH with mild cognitive difficulties.

Super-multiplex imaging of cellular dynamics and heterogeneity by integrated stimulated Raman and fluorescence microscopy.

Observing multiple molecular species simultaneously with high spatiotemporal resolution is crucial for comprehensive understanding of complex, dynamic, and heterogeneous biological systems. The recently reported super-multiplex optical imaging breaks the "color barrier" of fluorescence to achieve multiplexing number over six in living systems, while its temporal resolution is limited to several minutes mainly by slow color tuning. Herein, we report integrated stimulated Raman and fluorescence microscopy with simultaneous multimodal color tunability at high speed, enabling super-multiplex imaging covering diverse molecular contrasts with temporal resolution of seconds. We highlight this technique by demonstrating super-multiplex time-lapse imaging and image-based cytometry of live cells to investigate the dynamics and cellular heterogeneity of eight intracellular components simultaneously. Our technique provides a powerful tool to elucidate spatiotemporal organization and interactions in biological systems.

IDeA States Pediatric Clinical Trials Network for Underserved and Rural Communities.

The National Institutes of Health's Environmental Influences on Child Health Outcomes (ECHO) program aims to study high-priority and high-impact pediatric conditions. This broad-based health initiative is unique in the National Institutes of Health research portfolio and involves 2 research components: (1) a large group of established centers with pediatric cohorts combining data to support longitudinal studies (ECHO cohorts) and (2) pediatric trials program for institutions within Institutional Development Awards states, known as the ECHO Institutional Development Awards States Pediatric Clinical Trials Network (ISPCTN). In the current presentation, we provide a broad overview of the ISPCTN and, particularly, its importance in enhancing clinical trials capabilities of pediatrician scientists through the support of research infrastructure, while at the same time implementing clinical trials that inform future health care for children. The ISPCTN research mission is aligned with the health priority conditions emphasized in the ECHO program, with a commitment to bringing state-of-the-science trials to children residing in underserved and rural communities. ISPCTN site infrastructure is critical to successful trial implementation and includes research training for pediatric faculty and coordinators. Network sites exist in settings that have historically had limited National Institutes of Health funding success and lacked pediatric research infrastructure, with the initial funding directed to considerable efforts in professional development, implementation of regulatory procedures, and engagement of communities and families. The Network has made considerable headway with these objectives, opening two large research studies during its initial 18 months as well as producing findings that serve as markers of success that will optimize sustainability.

Medical School Location and Sex Affect the In-State Retention of Pediatric Residency Program Graduates in Hawai'i.

The objective of this study was to assess the impact of medical school, sex, career choice, and location of practice of one pediatric residency program on physician workforce. This is a retrospective study of all categorical pediatric graduates of a residency program located in Honolulu, Hawai'i from 1968 to 2015. Information on medical school training, sex, career choice (general pediatrics or specialty), and location of practice were studied by examining data into five 10-year graduation periods. The program graduated 319 residents over nearly a 50-year timespan. Of these, 181 (56.7%) residents remained in Hawai'i to practice (adjusted odds ratio [OR] = 7.46, 95% confidence interval [CI]: 3.61-15.43). There were 125 (39.1%) graduates who relocated to the continental US with the majority moving to the West (55.2%), while other graduates moved to the South, Midwest, and Northeast (25.6%, 13.6%, and 5.6%, respectively). The remaining 13 (4.1%) graduates moved internationally. Female residents steadily increased over time (P < .001), with females significantly choosing general pediatrics (OR = 3.05, 95% CI: 1.91-4.89). In the time periods with the highest percentage of University of Hawai'i medical school graduates, there was an increased percentage of graduates staying in Hawai'i. This study examined the regional and national impact of a small residency program. The results indicated that trends in gender and the impact of medical school location were important in establishing a pediatrician workforce for local communities. Support of both medical school and residency education should be considered when assessing future workforce needs.

Hawai'i IDeA Center for Pediatric and Adolescent Clinical Trials.

As one of 17 clinical sites of the Environmental influences on Child Health Outcomes (ECHO) IDeA States Pediatric Clinical Trials Network (ISPCTN), the Hawai'i IDeA Center for Pediatric and Adolescent Clinical Trials (HIPACT) was established in 2016 to participate in community-valued and scientifically-valid multi-center pediatric clinical trials to improve health and well-being of diverse multi-ethnic populations of Hawai'i. Hawai'i is home to large populations of diverse rural and underserved populations, including indigenous Hawaiian communities and immigrant populations of Pacific Islanders and Asians. Many of these communities experience significant health disparities, made worse by their geographic isolation and many socio-economic factors. In addition to providing opportunities for children and their families to participate in clinical trials, HIPACT's goal is to provide opportunities for junior faculty of the John A. Burns School of Medicine (JABSOM), University of Hawai'i at Manoa, to acquire knowledge about and to develop skills in clinical trials. HIPACT's partners include the Hawai'i Pacific Health with Kapi'olani Medical Center for Women and Children, and Waianae Coast Comprehensive Health Center. HIPACT builds on the experiences gained through partnerships with the Mountain West IDeA Clinical and Translational Research-Infrastructure Network, and Research Centers in Minority Institutions Translational Research Network. Apart from participating in ECHO ISPCTN-sponsored studies, HIPACT junior faculty serve as committee members, Working Group leaders, Protocol Study Principal Investigators (PI) and site study PIs with ECHO ISPCTN. Through participation in ECHO ISPCTN, HIPACT has successfully increased the number of pediatric and maternal-fetal medicine faculty involved in the conduct of clinical trials.

Raman-Enhanced Spectroscopy (RESpect) Probe for Childhood Non-Hodgkin Lymphoma.

Raman-enhanced spectroscopy (RESpect) probe, which enhances Raman spectroscopy technology through a portable fiber-optic device, characterizes tissues and cells by identifying molecular chemical composition showing distinct differences/similarities for potential tumor markers or diagnosis. In a feasibility study with the ultimate objective to translate the technology to the clinic, a panel of pediatric non-Hodgkin lymphoma tissues and non-malignant specimens had RS analyses compared between standard Raman spectroscopy microscope instrument and RESpect probe. Cryopreserved tissues were mounted on front-coated aluminum mirror slides and analyzed by standard Raman spectroscopy and RESpect probe. Principal Component Analysis revealed similarities between non-Hodgkin lymphoma subtypes but not follicular hyperplasia. Standard Raman spectroscopy and RESpect probe fingerprint comparisons demonstrated comparable primary peaks. Raman spectroscopic fingerprints and peaks of pediatric non-Hodgkin lymphoma subtypes and follicular hyperplasia provided novel avenues to pursue diagnostic approaches and identify potential new therapeutic targets. The information could inform new insights into molecular cellular pathogenesis. Translating Raman spectroscopy technology by using the RESpect probe as a potential point-of-care screening instrument has the potential to change the paradigm of screening for cancer as an initial step to determine when a definitive tissue biopsy would be necessary.

Relationship between Circulating Inflammatory Monocytes and Cardiovascular Disease Measures of Carotid Intimal Thickness.

AIMS: Cardiovascular disease (CVD) remains the leading cause of death worldwide despite improvements in the treatment of atherosclerosis, an inflammatory disease and major underlying cause of CVD. Monocytes, an innate immune cell type, are linked to CVD progression; however, given their heterogeneity, the association between distinct monocyte subsets and increased risk of CVD remains unclear. This study investigated the association between peripheral monocyte subpopulation numbers and carotid intima-media thickness (cIMT), a sensitive measure of CVD risk, in a cohort of adults recruited from the general population. METHODS: We used clinical data and peripheral blood mononuclear cell (PBMC) specimens from 67 individuals. cIMT was measured by high-resolution, B-mode, ultrasound images of the right carotid artery. PBMCs were stained with conjugated monoclonal antibodies to define monocyte subpopulations based on CD14 and CD16 co-expressions into classical (CD14＋＋CD16－), intermediate/inflammatory (CD14＋＋CD16＋), and non-classical/patrolling (CD14low/＋CD16＋＋) monocytes. RESULTS: We found a higher intermediate monocyte count was significantly correlated with increased right common carotid artery (RCCA) and right carotid bifurcation (RBIF) intima-media thickness (IMT) (p=0.004 and 0.006,respectively), even after adjusting for CVD-associated clinical data (p=0.006 and 0.004, respectively). CONCLUSION: Our study demonstrated a strong correlation between inflammatory monocyte counts and cIMT. These results suggest that, in the general population, there is a relationship between intermediate monocyte expansion and elevated predictors for CVD risk, and intermediate monocytes may be involved in the development of atherosclerosis and metabolic diseases. Strategies targeting inflammatory monocytes may be needed to slow CVD progression.

The Research Centers in Minority Institutions (RCMI) Translational Research Network: Building and Sustaining Capacity for Multi-Site Basic Biomedical, Clinical and Behavioral Research.

The Research Centers in Minority Institutions (RCMI) program was established by the US Congress to support the development of biomedical research infrastructure at minority-serving institutions granting doctoral degrees in the health professions or in a health-related science. RCMI institutions also conduct research on diseases that disproportionately affect racial and ethnic minorities (ie, African Americans/Blacks, American Indians and Alaska Natives, Hispanics, Native Hawaiians and Other Pacific Islanders), those of low socioeconomic status, and rural persons. Quantitative metrics, including the numbers of doctoral science degrees granted to underrepresented students, NIH peer-reviewed research funding, peer-reviewed publications, and numbers of racial and ethnic minorities participating in sponsored research, demonstrate that RCMI grantee institutions have made substantial progress toward the intent of the Congressional legislation, as well as the NIH/NIMHD-linked goals of addressing workforce diversity and health disparities. Despite this progress, nationally, many challenges remain, including persistent disparities in research and career development awards to minority investigators. The continuing underrepresentation of minority investigators in NIH-sponsored research across multiple disease areas is of concern, in the face of unrelenting national health inequities. With the collaborative network support by the RCMI Translational Research Network (RTRN), the RCMI community is uniquely positioned to address these challenges through its community engagement and strategic partnerships with non-RCMI institutions. Funding agencies can play an important role by incentivizing such collaborations, and incorporating metrics for research funding that address underrepresented populations, workforce diversity and health equity.

Raman-Enhanced Spectroscopy Distinguishes Anal Squamous Intraepithelial Lesions in Human Immunodeficiency Virus-Serodiscordant Couples.

HIV-positive individuals are at increased risk for precancerous anal squamous intraepithelial lesions (SILs). Anal cytology and digital rectal examination are performed as screening tools, but extensive training and appropriate instruments are required to follow up on an abnormal anal cytology. Thus, novel approaches to SIL evaluation could improve better health care follow-up by efficient and timely diagnosis to offer treatment options. Recently, Raman-enhanced spectroscopy (RESpect) has emerged as a potential new tool for early identification of SIL. RESpect is a noninvasive, label-free, laser-based technique that identifies molecular composition of tissues and cells. HIV-serodiscordant couples had anal biopsies obtained during high-resolution anoscopy. RESpect was performed on the specimens. Principal component analysis of the data identified differences between normal and abnormal tissue as well as HIV-positive and HIV-negative individuals of each couple even with similar pathologies. RESpect has the potential to change the paradigm of anal pathology diagnosis and could provide insight into different pathways leading to SIL in HIV-serodiscordant couples.

Elevated cerebrospinal fluid Galectin-9 is associated with central nervous system immune activation and poor cognitive performance in older HIV-infected individuals.

We previously reported that galectin-9 (Gal-9), a soluble lectin with immunomodulatory properties, is elevated in plasma during HIV infection and induces HIV transcription. The link between Gal-9 and compromised neuronal function is becoming increasingly evident; however, the association with neuroHIV remains unknown. We measured Gal-9 levels by ELISA in cerebrospinal fluid (CSF) and plasma of 70 HIV-infected (HIV+) adults stratified by age (older > 40 years and younger < 40 years) either ART suppressed or with detectable CSF HIV RNA, including a subgroup with cognitive assessments, and 18 HIV uninfected (HIV-) controls. Gal-9 tissue expression was compared in necropsy brain specimens from HIV- and HIV+ donors using gene datasets and immunohistochemistry. Among older HIV+ adults, CSF Gal-9 was elevated in the ART suppressed and CSF viremic groups compared to controls, whereas in the younger group, Gal-9 levels were elevated only in the CSF viremic group (p < 0.05). CSF Gal-9 positively correlated with age in all groups (p < 0.05). CSF Gal-9 tracked with CSF HIV RNA irrespective of age (ß = 0.33; p < 0.05). Higher CSF Gal-9 in the older viremic HIV+ group correlated with worse neuropsychological test performance scores independently of age and CSF HIV RNA (p < 0.05). Furthermore, CSF Gal-9 directly correlated with myeloid activation (CSF-soluble CD163 and neopterin) in both HIV+ older groups (p < 0.05). Among HIV+ necropsy specimens, Gal-9 expression was increased in select brain regions compared to controls (p < 0.05). Gal-9 may serve as a novel neuroimmuno-modulatory protein that is involved in driving cognitive deficits in those aging with HIV and may be valuable in tracking cognitive abnormalities.

Red blood cell distribution width as an easily measurable biomarker of persistent inflammation and T cell dysregulation in antiretrovirally treated HIV-infected adults.

BACKGROUND: Chronic inflammation and immune dysfunction occur in human immunodeficiency virus (HIV)-infection despite stable antiretroviral therapy (ART). Red blood cell distribution width (RDW) has been shown to correlate with markers of inflammation in non-HIV conditions. The study objective was to determine associations between RDW with cellular markers of immune activation and immune dysfunction including soluble inflammatory mediators in ART treated HIV infection. METHODS: We performed a cross-sectional analysis of the Hawaii Aging with HIV-Cardiovascular study. RDW was defined as one standard deviation of RBC size divided by mean corpuscular volume multiplied by 100%. Correlations were analyzed between RDW, soluble inflammatory biomarkers and T cell activation (CD38 + HLA-DR+), senescence (CD28-CD57+), and immune exhaustion (PD-1, TIGIT, TIM-3 expression). RESULTS: Of 158 participants analyzed, median age was 50 years, duration of ART 12.6 years, virally suppressed 84.4%, and CD4 count 503 cells/mm3. Significant positive correlations were identified between RDW and soluble biomarkers including sICAM, IL-8, IL-6, SAA, TNF-α, sE-selection, fibrinogen, D-dimer, CRP, CD4/CD8 ratio, and frequency of multiple CD8 T-cell populations such as CD38 + HLA-DR + T-cells, single TIGIT+, and dual expressing of TIGIT + PD1+, TIGIT + TIM3+, and TIM3 + PD1+ CD8+ T-cell subsets (p < .05). Frequencies of CD38 + HLA-DR + CD8+ T-cells and TIGIT + CD8+ T-cells remained significant adjusting for baseline variables (p < .01). CONCLUSION: Our study revealed correlations between RDW with systemic inflammatory biomarkers and CD8+ T-cell populations related to immune activation and exhaustion in HIV-infected individuals on ART. Further studies are warranted to determine the utility of RDW as a marker of immune dysregulation in HIV.

In Vitro Blood-Brain Barrier Modeling adapted for Peripheral Blood Mononuclear Cell Transmigration from HIV-Positive Patients for Clinical Research on Therapeutic Drug Intervention.

OBJECTIVE: HIV-associated cognitive impairment (HACI) continues to persist for HIV-seropositive individuals who are on antiretroviral therapy (ART). HACI develops in part when HIV-infected monocytes (MOs) transmigrate through the blood-brain barrier (BBB) and secrete pro-inflammatory cytokines and chemokines, which leads to neuronal damage. In vitro BBB models are important tools that can elucidate mechanisms of MO transmigration. Previously described in vitro BBB models relied on pathology specimens, resulting in potentially variable and inconsistent results. This project reports on a reliable and consistent alternative in vitro BBB model that has the potential to be used in clinical research intervention studies analyzing the effects of ART on the BBB and on MO transmigration. METHODS: A bilayer BBB model was established with commercially available astrocytes and endothelial cells on a 3µm PET membrane insert to allow the contact of astrocytic foot processes with endothelial cells. Inserts were cultured in growth medium for 7 days before exposure to HIV- or HIV+ peripheral blood mononuclear cells (PBMCs). PBMCs were allowed to transmigrate across the BBB for 24 hours. RESULTS: Confluency and integrity measurements by trans-endothelial electrical resistance (TEER) (136.7 ± 18.3Ω/cm2) and permeability (5.64 ± 2.20%) verified the integrity of the in vitro BBB model. Transmigrated MOs and non-MOs were collected and counted (6.0x104 MOs; 1.1x105 non-MOs). Markers indicative of glial fibrillary acidic protein (GFAP), von Willebrand factor (vWF), and p-glycoprotein (Pgp) were revealed in immunofluorescence staining (IF), indicating BBB phenotype and functionality. CONCLUSION: Potential applications for this model include assessing the HIV DNA copy numbers of transmigrated cells (pre- and post-targeted ART) and understanding the role of oxidative stress related to HIV DNA and HACI.

Human Papillomavirus-16 DNA Quantitation Differentiates High-Grade Anal Neoplasia.

Background: Due to their higher rates of anal dysplasia/cancer, human immunodeficiency virus (HIV)-positive individuals are recommended to undergo anal dysplasia screening, which consists of anal cytology (AC) and high resolution anoscopy (HRA) with anal biopsy (AB) after abnormal AC result. However, AC variability limits its usefulness. Our objective was to evaluate human papillomavirus (HPV)-16 DNA quantitation as part of the screening algorithm. Methods: HPV-16 was detected in AC specimens from 75 HIV-positive participants using quantitative real-time polymerase chain reaction. AB results were available from 18/44 patients who had abnormal AC. Statistical tests included Mann-Whitney U, Kruskal-Wallis, receiver operating characteristic (ROC) analysis and Kappa coefficient tests. Results: HPV-16 copy numbers differed significantly across AC (p = 0.001) and AB grades (p = 0.009). HPV-16 ≥ 65 copies/cell predicted high-grade AB (p = 0.04). Using this cut-off in comparison to AB, it had better specificity (1.00) than AC (0.75) and specificity (0.77) than qualitative HPV-16 detection (0.38). Also, the Kappa coefficient of the cut-off (κ = 0.649) was higher than AC (κ = 0.557) and qualitative HPV-16 detection (κ = 0.258) to AB. Conclusion: Higher HPV-16 copy numbers corresponded to higher AC and AB grades, suggesting the importance of HPV burden on disease stage. Furthermore, HPV-16 ≥ 65 copies/cell distinguished high-grade disease and demonstrated better sensitivity, specificity, and agreement with AB than AC or qualitative HPV-16 detection. These results support the potential use of HPV quantitation in conjunction with AC in anal dysplasia screening.

Paroxysmal Nocturnal Dyspnea Secondary to Right Ventricular Myxoma: A Novel Presentation of an Unusual Tumor.

A 14-month-old male presented with paroxysmal nocturnal dyspnea and grade III/VI systolic ejection murmur at the upper left sternal border with an S4 gallop and was subsequently found to have a right ventricular cardiac myxoma. Prior presentations of these tumors have been with exertional syncope and murmur, asymptomatic murmur, or exertional dyspnea; the presentation of such a tumor with paroxysmal nocturnal dyspnea is novel.