Cerebral organoids (COs) are valuable for studying neurodegenerative diseases and pathogens, but their limited microglia pose challenges. Here, we present a protocol to generate organoids with physiologically relevant microglia (COiMs). We describe steps for differentiating induced pluripotent stem cells (iPSCs) into mesoderm, seeding embryoid bodies (EBs), and harvesting hematopoietic progenitor cells (HPCs). We detail procedures for co-culturing HPCs and iPSCs to generate CO-iMs, followed by their infection and treatment. The model provides a platform to study viral infections and neuroinflammation. For complete details on the use and execution of this protocol, please refer to Narasipura et al.1

Astrocytes are essential regulators of central nervous system (CNS) homeostasis, and their dysfunction can amplify neuroinflammation and neurodegeneration. HIV and methamphetamine (Meth) are known to downregulate β-catenin signaling and induce astrocyte senescence, but whether this senescent state is functionally pathogenic and contributes directly to neuroinflammatory processes remains unclear. Here, we investigated how senescent astrocytes respond across prototypical astrocytic functions. In vitro, human iPSC-derived astrocytes infected with HIV and/or exposed to Meth developed a senescence phenotype (increased p16INK4A) accompanied by reduced expression of the phagocytic receptor MEGF10 and impaired engulfment of apoptotic neurons. HIV/Meth exposure also shifted astrocyte secretomes toward proinflammatory profiles (IL-6, CCL2, CXCL1, and ICAM-1), and astrocyte-conditioned media decreased neuronal PSD95 and NFL and disrupted endothelial adherens and tight junction proteins (VE-cadherin, occludin, claudin-5), resulting in increased monocyte transmigration. Mechanistically, pharmacologic activation or lentiviral expression of active β-catenin protected astrocytes from senescence, preserving MEGF10 expression and phagocytic capacity under HIV/Meth exposure, while MEGF10 overexpression independently restored phagocytosis. In a human–mouse chimera model (NSG mice xenotransplanted with human iPSC-derived astrocytes), HIV infection and/or Meth administration increased p16INK4A and reduced MEGF10 expression in engrafted human astrocytes, recapitulating in vitro findings. Together, these studies demonstrate that HIV and Meth suppress β-catenin signaling to drive a functionally disruptive astrocyte senescence program linked to impaired MEGF10-dependent phagocytosis, diminished neuronal support, and compromised blood–brain barrier integrity. Restoring β-catenin signaling and preserving MEGF10 function emerge as rational strategies to prevent astrocyte-driven neuroinflammation and neuropathogenesis in HIV/Meth co-morbidity.

HIV-associated neurocognitive disorders (HAND) persist despite combination antiretroviral therapy (cART) and can be exacerbated by repeated cocaine (COC) exposure. Because COC, HAND, and cART independently disrupt medial prefrontal cortex (mPFC) function, their combined neurotoxic impact is a critical clinical concern. Using patch-clamp electrophysiology in HIV-1 transgenic (Tg) and non-Tg rats, we examined mPFC pyramidal neuron activity following repeated exposure to COC and/or cART. In non-Tg rats, COC and cART independently increased neuronal firing, trending toward an additive hyperactive effect when combined. Conversely, HIV-1 Tg rat neurons exhibited plateaued excitability, with no further firing elevations induced by COC or cART. Under intense depolarizing stimuli, treated neurons displayed overactivation-induced firing declines. These findings indicate that while COC and cART additively disrupt mPFC function in non-Tg rats, excitability mechanisms appear saturated in the HIV-1 Tg model. This restricted experimental context highlights the overlapping neurobiological impacts of cART and stimulant use, providing foundational insights into the comorbidity of COC use disorder and HAND.

Methamphetamine (Meth) is one of the most widely used illicit drugs worldwide, exerting potent psychostimulant effects that fuels its highly addictive nature. Chronic Meth use is associated with severe cognitive impairments, particularly in executive functions, decision-making, and working memory, which persist long even after cessation of Meth use. These cognitive deficits are associated with dysfunction of glutamatergic pyramidal neurons in the medial prefrontal cortex (mPFC), which regulates addiction and cognition. Both human and animal studies highlight Meth-induced mPFC dysfunction that contributes to compulsive behaviors and relapse. Emerging evidence also highlights significant sex differences in Meth use disorder (MUD). Previous studies suggest that Meth-induced behavioral and neuronal dysfunctions are different between males and females, but the cellular and molecular mechanism are not fully understood. Using behavioral and electrophysiological approaches (whole-cell patching), this study determined certain sex differences in neuronal dysfunction in the mPFC of rats that self-administered Meth (Meth-SA) followed by a short (2–5 d) or long-term (≥30 d) withdrawal. We found that both male and female rats self-administered methamphetamine in a similar pattern; however, the resulting hypoactivity, hyperactivity, and calcium dysregulation in mPFC neurons differed between the sexes. Such sex-specific neuronal dysfunction was associated with, and depended on, short or long-term withdrawal, respectively. By understanding these sex-specific behavioral/neuronal differences following different Meth withdrawal period, our novel findings demonstrate the role of sex as a biological variable in Meth-use and relapse, and reveal the effects of drug-using environment on mPFC neuronal dysfunction during withdrawal, providing insights for gender-specific treatment strategies.

BACKGROUND AND OBJECTIVES:A spectrum of neurologic complications associated with COVID-19 are well documented. While neuroinflammation in the brain of COVID-19 patients likely contributes to these complications, the mechanisms of neuroinflammation and correlates of neurologic complications remain elusive, especially since the etiologic pathogen of COVID-19, SARS-CoV-2, minimally invades the CNS. This study aimed to evaluate markers of neuroinflammation, IgG glycosylation patterns indicative of pro- or anti-inflammatory state, and prevalence of brain auto-reactive antibodies in the CSF of COVID-19 patients and their relationship to brain neuropathology. METHODS:We evaluated the CSF of 11 deceased unvaccinated COVID-19 donors and 13 matched non-COVID-19 controls. Markers of neuroinflammation, IgG glycosylation patterns, and brain auto-reactive antibodies were assessed, along with their correlation to brain neuropathology. Statistical analyses were performed to compare groups and assess relationships between variables, using non-parametric tests and bootstrap analysis. RESULTS:COVID-19 CSF showed higher levels of neopterin and ANNA-1, markers of neuroinflammation and autoimmunity, respectively, and lower IFN response compared to non-COVID-19 donors. In brain regions of high microglial activation, IL4 and RANTES were significantly increased. SARS-CoV-2 was undetectable in the CSF and brain of COVID-19 donors, yet anti-SARS-CoV-2 CSF antibodies were detected. Fucosylated IgG were associated with Spike IgG, CSF protein, and soluble CD14, whereas afucosylated bisecting IgG were inversely correlated with Spike IgG. Sialic acid containing IgG were positively correlated with IL1β and TNFα. These associations were not found in non-COVID-19 donors. Inflammatory agalactosylated fucosylated IgG (G0F) were associated with infiltrating CD4 + T cells in the brains of COVID-19 donors. COVID-19 donor CSF displayed higher levels of auto-reactive antibodies to human brain antigens compared to non-COVID-19 donors and donors with positive autoantibodies showed higher levels of neopterin. DISCUSSION:These data describe increased neuroinflammation and autoreactive antibody markers in the CSF of COVID-19 donors and suggest that IgG glycosylation and autoimmunity may contribute to COVID-19 pathology, highlighting potential mechanisms underlying the neurologic complications associated with COVID-19.

Cerebral organoids (COs) are valuable tools for studying the intricate interplay between glial cells and neurons in brain development and disease, including HIV-associated neuroinflammation. We developed a novel approach to generate microglia containing COs (CO-iMs) by co-culturing hematopoietic progenitors and inducing pluripotent stem cells. This approach allowed for the differentiation of microglia within the organoids concomitantly with the neuronal progenitors. Compared with conventional COs, CO-iMs were more efficient at generating CD45+/CD11b+/Iba-1+ microglia and presented a physiologically relevant proportion of microglia ( 7

Background:Native pyogenic spinal infections (PSIs), including spondylodiscitis and vertebral osteomyelitis, are challenging to diagnose due to low culture sensitivity and delayed results. Metagenomic next-generation sequencing (mNGS) has emerged as a promising diagnostic tool, but its comparative clinical utility remains uncertain. The purpose of this study is to systematically compare the diagnostic performance and clinical impact of mNGS versus conventional microbial culture in detecting pathogens responsible for native PSIs. Methods:The current systematic review and meta-analysis was conducted in accordance with Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines. A comprehensive literature search was performed across 6 major databases. Eligible studies directly compared mNGS with standard culture for native PSIs and reported diagnostic performance metrics. Data were extracted and analyzed using a random-effects model to produce pooled estimates. Study quality was assessed using the Newcastle-Ottawa Scale. Primary outcomes included pooled sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). Secondary outcomes assessed diagnostic yield, time to diagnosis, treatment modification, and false-positive or contamination events. Results:A total of 1,227 patients from 12 studies were included, encompassing those with suspected or confirmed native PSIs. Pooled sensitivity and specificity of mNGS were 89.7% (95% CI: 85.6-93.1%) and 86.2% (95% CI: 80.5-91.0%), respectively. mNGS demonstrated a significantly higher diagnostic yield (69-90%) compared to culture (27.2-44.7%) and enabled faster diagnosis (range, 17.7-48 hours). mNGS informed antimicrobial selection in up to 70.3% of cases and detected a broader pathogen spectrum. The incidence of false positives was low (range, 1-5) but non-negligible, emphasizing the need for careful interpretation. Conclusions:mNGS outperforms conventional culture in sensitivity, speed, and breadth of pathogen detection in native PSIs and supports more tailored antimicrobial therapy. However, careful interpretation is necessary due to potential false positives. These findings support the integration of mNGS into clinical workflows, particularly in complex or culture-negative infections.

IntroductionMethamphetamine (Meth) is a highly addictive psychostimulant that disrupts neuronal function in the medial prefrontal cortex (mPFC), inducing Meth use disorders (MUD). MUD is often complicated by HIV-associated neurocognitive disorders (HAND, a.k.a. neuroAIDS/neuroHIV), and vice versa. MUD and neuroHIV also disrupt astrocytes, altering their role in supporting normal neuron function. The mechanism(s) underlying Meth and neuroHIV’s impact on astrocytes and astrocyte-neuron interplay remains unknown.MethodsTo define that, we assessed the activity of cortical astrocyte K+ channels that regulate extracellular K+ homeostasis ([K+]e), and substantially neuronal excitability in the brain. HIV-1 transgenic (Tg) rats, a rodent model of neuroHIV with combined antiretroviral therapy (cART) that have no active HIV-1 replication but expression of viral proteins, were given daily repeated Meth administrations. Saline-pretreated non-Tg rats served as control. We then conducted electrophysiological assessment in mPFC astrocytes after acute Meth (20, 100 μM in bath) or daily repeated Meth administrations (5 mg/kg/day s.c. for 5 days) followed by a 3-day withdrawal.ResultsWe found that both Meth and neuroHIV suppressed the activity of astrocytic K+ channels, regardless of subtypes. The maximum reduction occurred in response to combined Meth/neuroHIV, showing exacerbated astrocyte dysfunction. Blocking the trace amine-associated receptor 1 (TAAR1)/G protein-coupled signaling pathway abolished Meth-induced, but not neuroHIV-induced, suppression of K2P, Kv, and Kir channel activity.DiscussionCollectively, these findings demonstrate that Meth and neuroHIV inhibit astrocyte function, which could exacerbate mPFC neuronal dysfunction in MUD and/or neuroHIV. They also suggest that Meth- and neuroHIV-induced astrocytic K+ channel function was mediated by TAAR1-and/or chemokine receptor-coupled signaling pathways.

Cerebral organoids (COs) are a valuable tool to study the intricate interplay between glial cells and neurons in brain development and disease, including HIV-associated neuroinflammation. We developed a novel approach to generate microglia containing COs (CO-iMs) by co-culturing hematopoietic progenitors and induced pluripotent stem cells. This approach allowed for the differentiation of microglia within the organoids concomitantly to the neuronal progenitors. CO- iMs exhibited higher efficiency in generation of CD45 + /CD11b + /Iba-1 + microglia cells compared to conventional COs with physiologically relevant proportion of microglia (∼7%). CO-iMs exhibited substantially higher expression of microglial homeostatic and sensome markers as well as markers for the complement cascade. CO-iMs showed susceptibility to HIV infection resulting in a significant increase in several pro-inflammatory cytokines/chemokines and compromised neuronal function, which were abrogated by addition of antiretrovirals. Thus, CO-iM is a robust model to decipher neuropathogenesis, neurological disorders, and viral infections of brain cells in a 3D culture system.

Bone mineral density (BMD) loss in people living with HIV occurs with the initiation of combined antiretroviral therapy (cART), particularly with tenofovir disoproxil fumarate (TDF) containing cART. Switching from TDF to abacavir (ABC) or dolutegravir (DTG) leads to increased BMD. Whether BMD gains are due to cessation of TDF or anabolic effects of ABC or DTG is unclear. We investigated the effects of ABC and DTG on osteoblast lineage cells in vitro and in vivo. Primary human osteoblasts and male C57BL/6 mice were treated with individual antiretrovirals (ARVs) or a combination of ABC/DTG/lamivudine (3TC). Nearly all ARVs and cART inhibited osteogenic activity in vitro. Due to the importance of Wnt/beta-catenin in bone formation, we further investigated ARV effects on the Wnt/beta-catenin pathway. ABC, alone and as part of ABC/DTG/3TC, increased osteoblastic beta-catenin activity as indicated by increased TOPFlash activity, hypo-phosphorylated (active) beta-catenin staining, and beta-catenin targeted gene expression. Mice treated with TDF had decreased lumbar spine BMD and trabecular connectivity density in the vertebrae, while those treated with ABC/DTG/3TC reduced cortical area and thickness in the femur. Mice treated with ABC alone had no bone structural changes, increased circulating levels of the bone formation marker, P1NP, and elevated expression of the Wnt/beta-catenin target gene, Lef1, in osteocyte enriched samples. Further, bones from ARV-treated mice were isolated to evaluate ARV distribution. All ARVs were detected in the bone tissue, which was inclusive of bone marrow, but when bone marrow was removed, only TDF, ABC, and DTG were detected at similar to 0.1% of the circulating levels. Overall, our findings demonstrate that ABC activates Wnt/beta-catenin signaling, but whether this leads to increased bone formation requires further study. Assessing the impact of ARVs on bone is critical to informing ARV selection and/or discovery of regimens that do not negatively impact the skeleton.

The following study aimed to determine the existence of blood biomarkers in symptomatic patients with or without lumbar Modic changes (MC). A cross-sectional sub-analyses of a prospective cohort was performed. Fasting blood samples were collected from patients with and without lumbar MC who had undergone spinal fusion or microdiscectomy. An 80-plex panel and CCL5/RANTES were used to assess preoperative plasma cytokine concentrations. Patient demographics and imaging phenotypes were also assessed. Thirty-one subjects were analysed (n = 18 no MC; n = 13 MC). No significant differences were found in age, sex, body mass index, smoking and alcohol history, and surgical procedure (i.e. fusion, decompression) between the two groups (p > 0.05). Several statistically significant blood biomarkers in MC patients were identified, including elevated levels of C–C Motif Chemokine Ligand 5 (CCL5, p = 0.0006), while Macrophage Migration Inhibitory Factor (MIF) was significantly lower (p = 0.009). Additionally, C-X-C Motif Chemokine Ligand 5 (CXCL5, p = 0.052), Pentraxin 3 (PTX3, p = 0.06) and Galectin-3 (Gal-3, p = 0.07) showed potential relevance. Moreover, MC patients exhibited significantly higher levels of disc degeneration (p = 0.0001) and displacement severity (p = 0.020). Based on multivariate analyses and controlling for disc degeneration/displacement, CCL5 (OR 1.02; 95

Objective: HIV-associated neuroinflammation persists in the brain despite suppressive combination antiretroviral therapy (cART). We evaluated associations between a subset of CD8 + T cells, termed CD4 dim CD8 bright T cells, and soluble markers of immune activation and/or neuroinflammation in the cerebrospinal fluid (CSF) and plasma of people with HIV (PWH). Design: Fifteen cART-naive PWH were enrolled and underwent blood draw, lumbar puncture for CSF collection, and neuropsychological tests at week 0 (pre-cART) and 24 weeks after cART initiation. Methods: CSF and peripheral blood T cells were evaluated with flow cytometry and soluble markers of immune activation were measured by multiplex and singleplex assays. Spearman bootstrap correlation coefficients with 10 000 resamples were computed and reported with corresponding 95% confidence intervals (CIs) for each marker of interest and T-cell type. Results: The frequency of CSF CD4 dim CD8 bright T cells at week 0 was inversely related with CSF neopterin. In contrast, at week 24, CSF CD4 − CD8 + T cells were positively correlated with CSF s100β, a marker of brain injury. In the blood, at week 0, CD4 dim CD8 bright T cells were inversely correlated with MCP-1, IP-10, IL-8, IL-6, G-CSF, and APRIL and positively correlated with plasma RANTES and MMP1. At week 0, the frequency of blood CD4 − CD8 + were positively correlated with CRP and BAFF. Conclusion: CD4 dim CD8 bright T cells are associated with some anti-inflammatory properties, whereas CD4 − CD8 + T cells may contribute to inflammation and injury. Assessing the contrast between these two cell populations in neuroHIV may inform targeted therapeutic intervention to reduce neuroinflammation and associated neurocognitive impairment.

Human inducible pluripotent stem cell (hiPSC)-derived astrocytes (iAs) are critical to study astrocytes in health and disease. They provide several advantages over human fetal astrocytes in research, which include consistency, availability, disease modeling, customization, and ethical considerations. The generation of iAs is hampered by the requirement of Matrigel matrix coating for survival and proliferation. We provide a protocol demonstrating that human iAs cultured in the absence of Matrigel are viable and proliferative. Further, through a side-by-side comparison of cultures with and without Matrigel, we show significant similarities in astrocyte-specific profiling, including morphology (shape and structure), phenotype (cell-specific markers), genotype (transcriptional expression), metabolic (respiration), and functional aspects (glutamate uptake and cytokine response). In addition, we report that, unlike other CNS cell types, such as neuronal progenitor cells and neurons, iAs can withstand the absence of Matrigel coating. Our study demonstrates that Matrigel is dispensable for the culture of human iPSC-derived astrocytes, facilitating an easy, streamlined, and cost-effective method of generating these cells.

Musculoskeletal diseases (MSDs) are characterized as injuries and illnesses that affect the musculoskeletal system. MSDs affect every population worldwide and are associated with substantial global burden. Variations in the makeup of the gut microbiota may be related to chronic MSDs. There is growing interest in exploring potential connections between chronic MSDs and variations in the composition of gut microbiota. The human microbiota is a complex community consisting of viruses, archaea, bacteria, and eukaryotes, both inside and outside of the human body. These microorganisms play crucial roles in influencing human physiology, impacting metabolic and immunological systems in health and disease. Different body areas host specific types of microorganisms, with facultative anaerobes dominating the gastrointestinal tract (able to thrive with or without oxygen), while strict aerobes prevail in the nasal cavity, respiratory tract, and skin surfaces (requiring oxygen for development). Together with the immune system, these bacteria have coevolved throughout time, forming complex biological relationships. Changes in the microbial ecology of the gut may have a big impact on health and can help illnesses develop. These changes are frequently impacted by lifestyle choices and underlying medical disorders. The potential for safety, expenses, and efficacy of microbiota-based medicines, even with occasional delivery, has attracted interest. They are, therefore, a desirable candidate for treating MSDs that are chronic and that may have variable progression patterns. As such, the following is a narrative review to address the role of the human microbiome as it relates to MSDs.