Giovanni Schifitto

Background: People with HIV (PWH) experience excess cardiovascular disease risk with persistent inflammation considered a key driver, despite antiretroviral therapy. Low-level viremia (LLV) may contribute to inflammation and vascular injury, but data in sub-Saharan Africa are limited. We assessed correlates of endothelial function among Black African PWH with a history of viremia despite antiretroviral therapy use. Methods: We conducted a cross-sectional substudy within the South African UTRA trial (NCT05333679). Endothelial function was measured with EndoPAT using the natural log-transformed Reactive Hyperemia Index (LnRHI); endothelial dysfunction was defined as LnRHI ≤ 0.51. The primary exposure was 12-month cumulative HIV viral load (VL) area under the curve. Body mass index, waist-to-hip ratio, and Body Roundness Index were modeled using adjusted natural cubic splines with robust standard errors. We evaluated associations of various covariates, including these body composition metrics, with LnRHI via multivariable regression. Results: Of 120 participants (median age 46 years, 69% women), 26% met criteria for endothelial dysfunction despite low 10-year cardiovascular disease risk. Median LnRHI was 0.69 (interquartile range 0.50–0.94). Mean cumulative VL area under the curve was 1.55 log ₁₀ copy-yr/mL (interquartile range 1.30–2.20), consistent with LLV. Cumulative VL was not associated with endothelial function after adjustment. By contrast, LnRHI varied significantly and nonlinearly across body mass index, waist-to-hip ratio, and Body Roundness Index, with an overall downward trend with increasing adiposity. Conclusions: In this cross-sectional analysis of Black African PWH with LLV, no association between cumulative VL and endothelial dysfunction was observed. However, higher adiposity was consistently associated with declining endothelial function. Our data add to the growing evidence on the importance of addressing body composition as a multifactorial risk determinant and important correlate of early vascular risk in PWH.

High prevalence of HIV-associated neurocognitive impairment persists despite effective antiretroviral therapy. While conventional neuroimaging has been previously used to explain cognitive deficits in virally suppressed people with HIV (PWH), magnetic resonance elastography (MRE) offers a novel approach to quantify microstructural tissue coherence through brain biomechanics. This study investigated whether MRE-derived biomechanical metrics exhibit a pathological, group-specific relationship with cognitive performance in PWH, and compared their sensitivity against advanced diffusion MRI and peripheral biomarkers of neuronal injury and inflammation. In a cross-sectional cohort of 27 virally suppressed PWH and 30 matched healthy controls, participants underwent comprehensive cognitive testing, multi-frequency MRE (measuring stiffness, viscosity, and strain), multi-shell diffusion MRI (including NODDI metrics), and blood sampling for plasma neurofilament light chain and glial fibrillary acidic protein. PWH exhibited significantly lower cognitive scores than controls, particularly in learning and memory. Although baseline mean values across imaging and peripheral biomarkers did not notably differ between the groups, a significant group-by-cognition interaction emerged exclusively for MRE parameters. Specifically, in PWH, elevated viscosity and reduced strain correlated with better attention, whereas lower viscosity and strain correlated with poorer memory. These biomechanical-behavioral relationships were absent in healthy controls, and no notable interactions were observed for advanced diffusion metrics or blood biomarkers. Ultimately, brain biomechanics measured by MRE demonstrate greater sensitivity to altered cognitive function in PWH than advanced diffusion MRI and peripheral biomarkers, highlighting their potential as functional correlates of neurocognitive impairment that warrant further longitudinal validation.

Time-of-flight magnetic resonance angiography (TOF-MRA) is widely used for noninvasive visualization of arterial anatomy, but extracting hemodynamics like blood velocity typically requires supplementary phase-contrast scans, tagging or multi-TE images. This study proposes a novel, physics-informed computational framework to extract variable fluid velocity directly from standard TOF-MRA signal profiles. We analytically expand the approach-to-steady-state Bloch equations to include convective flow, establishing a mathematical relationship between the spatial decay of longitudinal magnetization and fluid velocity. The velocity derivation was further extended to pointwise estimation over a 1-D centerline, overcoming the limitations of constant-velocity assumptions. To validate and solve this problem, a MATLAB (R2025b) simulation framework was developed to model fluid flow in two variable-geometry flowing tube cases, i.e., continuous narrowing and focal stenosis, under synthetic scanner noise. A global inverse optimization approach utilizing Dual-Tikhonov regularization was applied to stably invert the ill-posed transit time integral, actively penalizing high-frequency numerical ringing while preserving structural curves. The computational simulations successfully recovered ground-truth point-wise velocities, tracking gradual hemodynamic accelerations and sharp stenotic jets. This theoretical framework and the example centerline TOF-MRA signal intensity provide a robust mathematical proof-of-concept that quantitative, localized functional hemodynamic metrics can be extracted from standard structural MRA imaging, establishing a foundation for advanced flow quantification without requiring additional scan time.

BACKGROUND: In people living with HIV (PLWH), persistent systemic inflammation drives comorbidities, including cardiovascular and cerebrovascular diseases. Monocyte-mediated inflammation, rather than T-cell activation, is a major contributor to chronic inflammation. However, the underlying mechanisms remain elusive. Here, we investigated the role of Dll4 (delta-like 4)-Notch signaling in monocyte-mediated inflammation. METHODS: In clinical specimens, we quantified monocyte mDll4 (membrane-bound Dll4) expression and plasma levels of exDll4 (extracellular Dll4). We also analyzed the association between circulating exDll4 and monocyte subsets in PLWH. In vitro, human primary monocytes, endothelial cells, and T cells were stimulated with lipopolysaccharide or TNF-α (tumor necrosis factor-α) to assess Dll4 mRNA and protein expression and exDll4 secretion. RNA sequencing was performed after Dll4 silencing to determine downstream signaling pathways. In addition, molecular and functional studies examined the effects of Dll4-Notch1 signaling on interferon-related gene expression, monocyte adhesion, and transmigration. RESULTS: Both male and female PLWH exhibited significantly increased mDll4 expression, activation of Dll4-Notch1 signaling, and increased Notch-targeted inflammatory factors. Although monocyte mDll4 did not differ by sex in PLWH, plasma exDll4 was significantly elevated in males, but not females, compared with HIV-uninfected individuals. In male PLWH, plasma exDll4 levels paralleled monocyte mDll4 expression. Circulating exDll4 was positively associated with proinflammatory monocytes and negatively associated with classical monocytes in male PLWH. Mechanistically, lipopolysaccharide or TNF-α robustly increased Dll4 mRNA and protein expression in human primary monocytes, along with exDll4 secretion from monocytes, but not from endothelial cells or T cells. RNA sequencing after Dll4 silencing showed that Dll4-Notch signaling suppresses a broad anti-pathogen program regulated by interferon and NF-κB, as well as cell-mobility-related genes. At the molecular level, Dll4 activated Notch1, which upregulated Hes1, forming a heterotrimer with STATs (signal transducers and activators of transcription), and repressed interferon pathway-related gene expression. Functionally, loss of Dll4 significantly reduced monocyte adhesion and transmigration. CONCLUSIONS: Proinflammatory cues upregulate Dll4 in monocytes and activate Dll4-Notch signaling, thereby suppressing antiviral responses and enhancing monocyte transmigration. Together, these effects sustain systemic inflammation in PLWH of both sexes. Our data identify monocyte-derived Dll4 as a promising biomarker and therapeutic target for systemic inflammation.

Background: Despite the success of combination antiretroviral therapy (cART), vascular comorbidities, including cerebrovascular disease, are more prominent in people living with HIV (PLWH) compared to people without HIV (PWOH). However, quantitative assessments of cerebrovascular morphometry and their associations with cognitive outcomes in the context of HIV are still limited. In this study, we explore this missing link. Methods: Magnetic Resonance Angiography (MRA) data, blood markers, and neurocognitive assessments were collected from 73 PWOH subjects ( male : 57, female : 16; age : 53 ± 16) and 99 PLWH subjects ( male : 66, female : 30, age : 53 ± 11). Vessel morphometric features were quantified using intraCranial Artery Feature Extraction (iCafe) to investigate associations between vessel morphometry, markers of monocytes, endothelial cell activation, and cognitive performance. Results: HIV status predicted a lower total number of branches ( β = − 0.224, p = 0.001, d = − 0.517) and shorter total distal length ( β = − 0.173, p = 0.021, d = –0.370) with a moderate effect size. Total branch number was found to be negatively associated with plasma levels of monocyte markers (sCD14: r = –0.167, p = 0.033; sCD163: r = –0.157, p = 0.045) and positively correlated with white matter cerebral blood flow ( r = 0.550; p ≤ 0.05). HIV status was the strongest predictor of overall cognitive performance in ANCOVA model ( β = –0.219, p = 0.006, d = –0.453). Conclusions: Our results suggest that cognitive impairment in PLWH is associated with vessel morphology metrics. Monocyte immune activation may contribute to changes in vessel morphology.

Introduction: Combination antiretroviral therapy (cART) has been shown to reduce inflammation in persons with HIV (PWH), leading to overall improvements in cognition. However, these improvements are patient-dependent and not always observable over short treatment periods. Methods: We applied a multimodal integrative model to associate various baseline MR neuroimaging metrics with baseline neurocognitive performance and their longitudinal changes over 12 weeks of cART treatment. Features in our model included volumetric data, cerebral blood flow metrics, cerebrovascular reactivity, and diffusion MRI data from cortical, subcortical, and white matter regions of the brain. Our integrative model, which includes multilayered principal component analysis, penalized regression, and feature weight back-propagation, is designed for “large p, small n” data and offers better interpretability than deep-learning methods. Results: There is a modest association between imaging metrics and baseline neurocognitive scores for both PWH and age-matched healthy controls, driven primarily by subcortical regions. In contrast, baseline imaging features exhibited stronger associations with longitudinal changes in cognitive performance over 12 weeks of cART in PWH than with baseline cognitive scores. The multimodal integrative model outperformed all comparable unimodal models in explaining longitudinal cognitive change. Among unimodal analyses, models based on cerebral blood flow and free-water-corrected fractional anisotropy demonstrated the strongest associations. Frequently selected predictors included the frontal pole (cortical gray matter); the amygdala, putamen, and hippocampus (subcortical gray matter); and the posterior limb of the internal capsule (white matter). Discussion: Our approach provides an interpretable statistical framework that integrates complementary information across various imaging modalities into a robust and interpretable model for short-term cognitive trajectories in PWH undergoing cART.

Cognitive impairment persists in people with HIV (PWH) despite effective combination antiretroviral therapy, possibly as a result of persistent alterations in white matter microstructural abnormalities in the brain. Noninvasive tensor-valued diffusion MRI (dMRI) is sensitive to microstructural integrity; thus, it may contribute to the understanding of HIV-associated cognitive impairment. In this exploratory cross-sectional study, 31 healthy controls (HCs) and 24 PWH underwent 3T MRI and neurocognitive assessment. Tensor-valued dMRI metrics, including microscopic fractional anisotropy (µFA) and isotropic, anisotropic, and total mean kurtosis (MKi, MKa, MKt), and conventional DTI and DKI metrics (FA, MD, and MK) were evaluated across six functionally defined brain networks. Compared with HCs, PWH exhibited reduced FA, µFA, and MKa in the dorsal default mode and anterior salience networks, along with increased MKi in the salience network and decreased MKi in the executive control network, with moderate effect sizes. Compared with HCs, PWH performed significantly worse on measures of learning, memory, and language, but showed no differences in executive function, attention, or processing speed. Additionally, significant associations and interactions between dMRI metrics and HIV status were observed, particularly for MKi and attention, executive function, and processing speed across the default mode, salience, and executive control networks. These preliminary findings underscore tensor-valued dMRI as a sensitive biomarker of network-specific neurocognitive vulnerability in HIV.

Background: Monocytes contribute to atherosclerosis by migrating into inflamed endothelium and differentiating into lipid‐laden macrophages. In people living with HIV, chronic inflammation increases atherosclerosis risk, yet the role of specific monocyte subsets remains unclear. We investigated how distinct monocyte populations contribute to vascular pathology in early HIV‐associated atherosclerosis. Methods: We profiled 123 965 circulating monocytes using single‐cell RNA sequencing and integrated plasma microparticle proteomics in 32 individuals stratified by HIV and atherosclerosis status. Supervised learning identified cluster‐ and disease‐specific signatures, validated by platelet‐monocyte cocultures, reverse transcription–quantitative polymerase chain reaction, bulk RNA sequencing, flow cytometry, and ELISA. Results: Seven monocyte clusters were identified, including a subset characterized by platelet–monocyte complexes. Bulk RNA sequencing of platelet–monocyte cocultures revealed platelet‐driven upregulation of genes involved in inflammation, lipid metabolism, oxidative stress, and endothelial adhesion. Platelet–monocyte complex–derived macrophages secreted higher levels of TGF‐β (transforming growth factor‐β) and IL‐10 (interleukin‐10), displayed decreased CD14 and increased CD80/CD86 while retaining CD36, and promoted endothelial‐to‐mesenchymal transition (decreased expression of CDH5 and PECAM1 ; increased expression of S100A4 and markers of vascular inflammation ( ICAM1 ), VCAM ), and IL‐6 (interleukin‐6), and CCL2 (C‐C motif chemokine ligand 2) secretion). Additionally, CD14 ⁺ monocytes from HIV ⁺ atherosclerosis‐negative and HIV ⁺ atherosclerosis‐positive groups showed enhanced ROS‐NRF2 (reactive oxygen species–nuclear factor erythroid 2–related factor 2) pathway activities, supported by increased basal and H ₂ O ₂ ‐induced p90RSK phosphorylation, indicating oxidative stress priming. Conclusions: Two monocyte clusters contribute independently to vascular immune dysregulation in people living with HIV. Platelet–monocyte complex–derived macrophages promote endothelial dysfunction while adopting a profibrotic cytokine profile. CD14 ⁺ monocytes show heightened oxidative signaling and stress responses, consistent with vascular activation. Together, these mechanisms may accelerate atherosclerosis development in HIV, even in the absence of traditional cardiovascular risk factors.

Background: Vascular disease is a known risk factor for the development of leukoaraiosis. Assessment of cerebral blood flow (CBF) was performed at baseline and after acetazolamide (AZM) challenge to evaluate for vascular reserve and steal within the brain. Little has been reported on the physiological reserve in the non-flow-limited hemispheres. This study attempts to evaluate for vascular steal in areas commonly involved in leukoaraiosis, in the setting of pharmaceutically induced states of increased CBF. Methods: Patients who underwent AZM challenge MRI from 2014 to 2021 and a cerebral angiogram within one year were included. Patients with bilateral disease or non-diagnostic imaging artifacts were excluded. MRIs were obtained after 1 g of AZM was administered 5 and 10 min prior to acquisition. Augmentation and steal maps were generated. Regression analysis, Pearson correlation coefficient, two-sample t-test, Spearman and Mann–Whitney U analyses were utilized for statistical evaluation. Results: A total of 38 patients with unilateral cerebral vaso-occlusive disease underwent the AZM challenge. Vascular steal and T2 hyperintensities were assessed in non-flow-limited hemispheres (NFLH) and flow-limited hemispheres (FLH). A moderate correlation was demonstrated between NFLH steal and NFLH T2 hyperintensities (rs = 0.48, p = 0.0020). A weak correlation without statistical significance was demonstrated between ipsilateral T2 and contralateral T2 hyperintensities (rs = 0.27, p = 0.10). Conclusions: The vascular steal phenomenon was demonstrated in the distal cerebral vasculature of cerebral white matter even in the absence of upstream flow-limiting stenosis, suggesting an inherent vulnerability of these structures to hemodynamic fluctuations and possiblly contributing etiology to leukoaraiosis.

Chemical Exchange Saturation Transfer (CEST) imaging has emerged as a promising non-invasive molecular MRI technique for investigating neuroinflammation. It offers unique insights into metabolic and molecular alterations in the brain. This review presents a comprehensive overview of CEST principles, methodological developments, and translational applications in neuroinflammation. It covers the basic mechanisms, pulse sequence designs, readout strategies, and various CEST contrasts used to probe molecular changes associated with inflammation. Recent advancements in fast CEST imaging, including optimized undersampling strategies and accelerated reconstruction methods are discussed. Improvements in post-processing and quantification techniques are also highlighted. The growing role of artificial intelligence (AI) in CEST imaging for image reconstruction, artifact correction, and biomarker extraction, is examined. Preclinical and clinical studies show CEST’s potential to detect neuroinflammation across neurological disorders. The impact of high-field MRI on enhancing CEST sensitivity and specificity are also discussed. Despite notable progress, several challenges remain. These include sensitivity to field inhomogeneities, lack of acquisition standardization, and limited clinical validation. We outline current limitations, translational barriers, and provide recommendations for improving reproducibility, facilitating clinical adoption, and integrating AI-based approaches for robust molecular characterization. Overall, CEST imaging shows great potential as a non-invasive biomarker for neuroinflammation. It can deepen understanding of the molecular and metabolic mechanisms underlying neurological diseases, while addressing technical and translational challenges remains key for its broader clinical implementation.