James G. Taylor

RNA sequencing (RNA-Seq) and analysis methods like gene set enrichment analysis (GSEA) stand at the forefront of modern molecular biology, offering unparalleled insights into gene expression dynamics and transcriptomic landscapes. In this chapter, we focus on the application of RNA-Seq technology and the GSEA method. First, we provide a real-world workflow for RNA-Seq to unravel the intricate interplay between host factors and HIV-1 infection within peripheral blood mononuclear cells (PBMCs). We elucidate the core principles of RNA-Seq and its pivotal role in identifying HIV-1 restriction factors and understanding their regulatory mechanisms in PBMCs. We provide the essential steps of the RNA-Seq workflow tailored to PBMCs, encompassing sample preparation, library construction, sequencing, and advanced bioinformatics. We underscore how RNA-Seq enables precise quantification of gene expression levels, detection of alternative splicing, and exploration of novel transcripts pertinent to HIV-1 restriction. Moreover, we delve into the intricacies of HIV-1 restriction factor discovery using RNA-Seq data, shedding light on the diverse repertoire of PBMC host defense mechanisms. We discuss the challenges of RNA-Seq data analysis, including normalization, differential expression analysis, and the integration of multi-omics datasets, and propose strategies to mitigate these barriers. Next, we provide an overview of GSEA, a powerful computational method that extends beyond traditional differential gene expression analysis. This tool can provide deeper insights into the functional significance of gene expression changes. By leveraging curated gene sets representing biological pathways, molecular functions, or cellular processes, GSEA enables the identification of coordinated changes in gene expression within predefined gene sets. In our example, we applied GSEA in transcriptomic studies, highlighting its ability to uncover subtle yet coordinated changes in gene expression that may not be evident by individual gene analysis. We demonstrate how GSEA enhances our understanding of complex biological phenomena, informs hypothesis generation, and identifies potential therapeutic targets. Overall, RNA-Seq and GSEA are indispensable tools for elucidating the complex interactions in host cells altered by physiologic processes, disease states or infectious agents like HIV-1, thereby advancing our ability to identify potential targets for therapeutic interventions.

The efficacy and safety of rivipansel, a predominantly E-selectin antagonist, were studied in a phase 3, randomized, controlled trial for vaso-occlusive crisis (VOC) requiring hospitalization (RESET). A total of 345 subjects (204 adults and 141 children) were randomized and 320 were treated (162 with rivipansel, 158 with placebo) with an IV loading dose, followed by up to 14 additional 12-hourly maintenance doses of rivipansel or placebo, in addition to standard care. Rivipansel was similarly administered during subsequent VOCs in the Open-label Extension (OLE) study. In the full analysis population, the median time to readiness for discharge (TTRFD), the primary end point, was not different between rivipansel and placebo (−5.7 hours, P = .79; hazard ratio, 0.97), nor were differences seen in secondary end points of time to discharge (TTD), time to discontinuation of IV opioids (TTDIVO), and cumulative IV opioid use. Mean soluble E-selectin decreased 61% from baseline after the loading dose in the rivipansel group, while remaining unchanged in the placebo group. In a post hoc analysis, early rivipansel treatment within 26.4 hours of VOC pain onset (earliest quartile of time from VOC onset to treatment) reduced median TTRFD by 56.3 hours, reduced median TTD by 41.5 hours, and reduced median TTDIVO by 50.5 hours, compared with placebo (all P < .05). A similar subgroup analysis comparing OLE early-treatment with early-treatment RESET placebo showed a reduction in TTD of 23.1 hours (P = .062) and in TTDIVO of 30.1 hours (P = .087). Timing of rivipansel administration after pain onset may be critical to achieving accelerated resolution of acute VOC. Trial Registration: Clinicaltrials.gov, NCT02187003 (RESET), NCT02433158 (OLE).

Patients with sickle cell disease (SCD) have a lower risk for HIV-1 infection. We reported restriction of ex vivo HIV-1 infection in SCD peripheral blood mononuclear cells (PBMCs) that was due, in part, to the upregulation of antiviral, inflammatory, and hemolytic factors, including heme oxygenase-1 (HO-1). Here, we investigated whether individuals with sickle cell trait (SCT), who develop mild hemolysis, also restrict HIV-1 infection. Ex vivo infection of SCT PBMCs exhibited an approximately twofold reduction of HIV-1 replication and lower levels of HIV-1 reverse transcription products, 2-long terminal repeat circle, HIV-1 integration, and gag RNA expression. SCT PBMCs had higher HO-1 messenger RNA (mRNA) and protein levels and reduced ribonucleotide reductase 2 (RNR2) protein levels. HO-1 inhibition by tin porphyrin eliminated ex vivo HIV-1 restriction. Among Howard University clinic recruits, higher levels of HO-1 and RNR2 mRNA and lower HIV-1 env mRNA levels were found in SCT individuals living with HIV-1. To determine the population-level effect of SCT on HIV-1 prevalence, we assessed SCT among women living with HIV (WLH) in the WIHS (Women Interagency HIV-1 Study). Among WIHS African-American participants, the prevalence of SCT was lower among women with HIV compared with uninfected women (8.7% vs 14.2%; odds ratio, 0.57; 95% confidence interval, 0.36-0.92; P = .020). WIHS WLH with SCT had higher levels of CD4+/CD8+ ratios over 20 years of follow-up (P = .003) than matched WLH without SCT. Together, our findings suggest that HIV-1 restriction factors, including HO-1 and RNR2, might restrict HIV-1 infection among individuals with SCT and limit the pathogenicity of HIV.

Psychosocial stress is increasingly acknowledged for families having children with chronic illness. 1 Pediatric chronic illness impacts family structure, parental functioning, and health outcomes of the child. 2 Sickle cell disease (SCD) is a chronic disease that can progress to a spectrum of complications, including vaso-occlusive pain crisis and cardiopulmonary dysfunction. 3 The disease begins to manifest in early childhood, during which life-threatening conditions can develop rapidly. Chronic disease management, frequent infections, and unpredictable pain episodes cause anxiety and depression in children with SCD. 4,5 Families, especially mothers, are a primary resource for stress management and mental health needs. 6 The demands of caring for a chronically ill child and the feeling of powerlessness over the child's illness often associate with emotional stress in parents and impact family conflicts. 7 Single parenthood, often single motherhood, is more common among families having children with SCD (60%) than the general African-American population (46%). 8 Parental distress related to SCD is considerably more difficult to manage for single parents. 7,9 The parent-child relationship, in turn, significantly affects a child's perception of stress in life. 5,10 Recent studies have begun to investigate the impact of single parenthood on SCD outcomes. 11

In the US, mortality in sickle cell disease (SCD) increases after age 18‐20 years. Biomarkers of mortality risk can identify patients who need intensive follow‐up and early or novel interventions. We prospectively enrolled 510 SCD patients aged 3‐20 years into an observational study in 2006‐2010 and followed 497 patients for a median of 88 months (range 1‐105). We hypothesized that elevated pulmonary artery systolic pressure as reflected in tricuspid regurgitation velocity (TRV) would be associated with mortality. Estimated survival to 18 years was 99% and to 25 years, 94%. Causes of death were known in seven of 10 patients: stroke in four (hemorrhagic two, infarctive one, unspecified one), multiorgan failure one, parvovirus B19 infection one, sudden death one. Baseline TRV ≥2.7 m/second (>2 SD above the mean in age‐matched and gender‐matched non‐SCD controls) was observed in 20.0% of patients who died vs 4.6% of those who survived (P = .012 by the log rank test for equality of survival). The baseline variable most strongly associated with an elevated TRV was a high hemolytic rate. Additional biomarkers associated with mortality were ferritin ≥2000 μg/L (observed in 60% of patients who died vs 7.8% of survivors, P < .001), forced expiratory volume in 1 minute to forced vital capacity ratio (FEV1/FVC) P < .001), and neutrophil count ≥10x10⁹/L (30.0% of patients who died vs 7.9% of survivors, P = .018). In SCD children, adolescents and young adults, steady‐state elevations of TRV, ferritin and neutrophils and a low FEV1/FVC ratio may be biomarkers associated with increased risk of death.

Pain is the hallmark of sickle cell anemia (SCA), presenting as recurrent acute events or chronic pain. Central sensitization, or enhanced excitability of the central nervous system, alters pain processing and contributes to the maintenance of chronic pain. Individuals with SCA demonstrate enhanced sensitivity to painful stimuli however central mechanisms of pain have not been fully explored. We hypothesized that adults with SCA would show evidence of central sensitization as observed in other diseases of chronic pain. Methods: We conducted a prospective study of static and dynamic quantitative sensory tests in 30 adults with SCA and 30 matched controls. Results: Static thermal testing using cold stimuli showed lower pain thresholds ( p = 0.04) and tolerance ( p = 0.04) in sickle cell subjects, but not for heat. However, SCA subjects reported higher pain ratings with random heat pulses ( p < 0.0001) and change in scores with temporal summation at the heat pain threshold ( p = 0.002). Similarly, with the use of pressure pain stimuli, sickle cell subjects reported higher pain ratings ( p = 0.04), but not higher pressure pain tolerance/thresholds or allodynia to light tactile stimuli. Temporal summation pain score changes using 2 pinprick probes (256 and 512 mN) were significantly greater ( p = 0.004 and p = 0.008) with sickle cell, and delayed recovery was associated with lower fetal hemoglobin ( p = 0.002 and 0.003). Conclusions: Exaggerated temporal summation responses provide evidence of central sensitization in SCA. Implications: The association with fetal hemoglobin suggests this known SCA modifier may have a therapeutic role in modulating central sensitization.