Leonid Margolis’s research while affiliated with Ilia State University and other places

Introduction The mechanisms of the SARS-CoV-2-triggered complex alterations in immune cell activation and production of cytokines in lung tissue remain poorly understood, in part because of the limited use of adequate tissue models that simulate the structure and cell composition of the lung in vivo. We developed a novel ex vivo model of SARS-CoV-2 infection of lung explants, that maintains the intact tissue composition and the viral load for up to 7–10 days. Using this model, we studied cytokine production during SARS-CoV-2 infection. Materials and methods Lung tissue was monitored for viability and cell composition using flow cytometry and histological analysis. SARS-CoV-2 infection was verified immunohistochemically, viral loads in tissue and culture medium were monitored by qPCR. A panel of 41 cytokines was measured in culture medium using xMAP technology. Results The explant lung tissue was viable and maintained viral infection that influenced the cytokine production. Elevated concentrations of G-CSF, GM-CSF, GRO-a, IFN-g, IL-6, IL-8, IP-10, MCP-3, MIP-1a, PDGF-AA, and VEGF, and decreased IL-1RA concentration were observed in infected tissue compared to non-infected tissue. Discussion Our results generally reflect the data obtained in COVID-19 patients. GRO-a, IFN-g, IL-6, IL-8, MCP-1, MCP-3, and RANTES correlated with the viral load, forming a distinct pro-inflammatory cluster. Thus, our lung ex vivo model faithfully reproduces some aspects of cytokine alterations in COVID-19 patients at an early disease stage, making the investigation of SARS-CoV-2 infection mechanisms more accessible and providing a potential platform for antiviral drug testing.

Problem Preeclampsia is a heterogeneous syndrome of diverse etiologies and molecular pathways leading to distinct clinical subtypes. Herein, we aimed to characterize the extracellular vesicle (EV)‐associated and soluble fractions of the maternal plasma proteome in patients with preeclampsia and to assess their value for disease prediction. Method of Study This case–control study included 24 women with term preeclampsia, 23 women with preterm preeclampsia, and 94 healthy pregnant controls. Blood samples were collected from cases on average 7 weeks before the diagnosis of preeclampsia and were matched to control samples. Soluble and EV fractions were separated from maternal plasma; EVs were confirmed by cryo‐EM, NanoSight, and flow cytometry; and 82 proteins were analyzed with bead‐based, multiplexed immunoassays. Quantile regression analysis and random forest models were implemented to evaluate protein concentration differences and their predictive accuracy. Preeclampsia subgroups defined by molecular profiles were identified by hierarchical cluster analysis. Significance was set at p < 0.05 or false discovery rate‐adjusted q < 0.1. Results In preterm preeclampsia, PlGF, PTX3, and VEGFR‐1 displayed differential abundance in both soluble and EV fractions, whereas angiogenin, CD40L, endoglin, galectin‐1, IL‐27, CCL19, and TIMP1 were changed only in the soluble fraction ( q < 0.1). The direction of changes in the EV fraction was consistent with that in the soluble fraction for nine proteins. In term preeclampsia, CCL3 had increased abundance in both fractions ( q < 0.1). The combined EV and soluble fraction proteomic profiles predicted preterm and term preeclampsia with an AUC of 78% (95% CI, 66%–90%) and 68% (95% CI, 56%–80%), respectively. Three clusters of preeclampsia featuring distinct clinical characteristics and placental pathology were identified based on combined protein data. Conclusions Our findings reveal distinct alterations of the maternal EV‐associated and soluble plasma proteome in preterm and term preeclampsia and identify molecular subgroups of patients with distinct clinical and placental histopathologic features.

Plasma cytokine levels were quantified among 30 persons with HIV (PWH) identified as elite controllers (15 transient controllers [studied a median of 1.38 years before losing viral control] and 15 persistent controllers). Thirty antiretroviral therapy (ART)-naive PWH, 30 ART-treated PWH with undetectable viremia, and 30 HIV-uninfected controls also were studied. Higher levels of cytokines were recognized among PWH than among controls, with EC displaying the highest levels. Elevated levels of IP-10 and MIG were identified among transient controllers as predictors of the loss of viral control. These findings offer feasible biomarkers for predicting virologic outcome and loss of control in EC.

Extracellular vesicles (EVs) serve as pivotal mediators of intercellular communication in both health and disease, delivering biologically active molecules from vesicle‐producing cells to recipient cells. In the context of HIV infection, EVs have been shown to carry the viral protein Nef, a key pathogenic factor associated with HIV‐related co‐morbidities. Despite this recognition, the specific localisation of Nef within the vesicles has remained elusive. This study addresses this critical knowledge gap by investigating Nef‐containing EVs. Less than 1% of the total released Nef was associated with EVs; most Nef existed as free protein released by damaged cells. Nevertheless, activity of EV‐associated Nef in downregulating the major cholesterol transporter ABCA1, a critical aspect linked to the pathogenic effects of Nef, was comparable to that of free Nef present in the supernatant. Through a series of biochemical and microscopic assays, we demonstrate that the majority of EV‐associated Nef molecules are localised on the external surface of the vesicles. This distinctive distribution prompts the consideration of Nef‐containing EVs as potential targets for immunotherapeutic interventions aimed at preventing or treating HIV‐associated co‐morbidities. In conclusion, our results shed light on the localisation and functional activity of Nef within EVs, providing valuable insights for the development of targeted immunotherapies to mitigate the impact of HIV‐associated co‐morbidities.

CD8 T cells are emerging as important mediators in atherosclerosis and cardiovascular disease (CVD). Immune activation may play a particular role in people with HIV (PWH) who are at an increased risk of CVD, even after controlling for known CVD risk factors. Latent CMV infection is associated with increased CVD risk for both PWH and people without HIV, and human CMV-specific CD4 and CD8 T cells are enriched for an immunosenescent phenotype. We previously showed that CMV coinfection in PWH promotes vascular homing and activation of inflammatory CD4 T cells through the CD2–LFA-3 axis. However, the role of CD2/LFA3 costimulation of CD8 T cells in PWH with CMV has yet to be described. In the present study, we demonstrate that CD2 expression on CX3CR1+CD57+CD28− inflammescent CD8 T cells is increased on cells from CMV-seropositive PWH. In vitro CD2/LFA-3 costimulation enhances TCR-mediated activation of these inflammatory CD8 memory T cells. Finally, we show that LFA-3 is highly expressed in aortas of SIV-infected rhesus macaques and in atherosclerotic plaques of people without HIV. Our findings are consistent with a model in which CMV infection enhances CD2 expression on highly proinflammatory CD8 T cells that can then be stimulated by LFA-3 expressed in the vasculature, even in the absence of CD28 costimulation. This model, in which CMV infection exacerbates toxic cytokine and granzyme production by CD8 T cells within the vasculature, highlights a potential therapeutic target in atherosclerosis development and progression, especially for PWH.

Biomaterials with antimicrobial activity are gaining attention due to their biodegradability and efficacy in interacting with a wide range of microorganisms. A new cellulose nano-biomaterial, endospermic nanocellulose crystals (ENC) obtained from parenchymal tissue of ivory nut endosperm, has a natural capacity as a universal binder. This feature is enhanced when it is chemically functionalized, and can be exploited in the fight against microbes. We tested the ability of sulfated ENC in aqueous suspension to encapsulate viruses through a crosslinking reaction mediated by cations. 0.25% w/v ENC suspensions efficiently encapsulated spike (S) protein, preventing its interaction with ACE2 receptor. ENC was further able to encapsulate SARS-CoV-2 pseudoviruses and prevent infection of 293T-hsACE2 cells. ENC also suppressed infection of MT-4 cells with HIV-1LAI.04. This antiviral activity of sulfated ENC is due to the irreversible interaction of ENC with viral particles mediated by crosslinking, as antiviral activity was less effective in the absence of cations. Additionally, ENC was used as a matrix to immobilize recombinant ACE2 receptors and anti-S IgG, creating molecular lures that efficiently inhibited SARS-CoV-2 infections in vitro. These results show that sulfated ENC from ivory nuts can be used as an efficient antiviral material.

Objectives Fetal death is a complication of pregnancy caused by multiple etiologies rather than being the end-result of a single disease process. Many soluble analytes in the maternal circulation, such as hormones and cytokines, have been implicated in its pathophysiology. However, changes in the protein content of extracellular vesicles (EVs), which could provide additional insight into the disease pathways of this obstetrical syndrome, have not been examined. This study aimed to characterize the proteomic profile of EVs in the plasma of pregnant women who experienced fetal death and to evaluate whether such a profile reflected the pathophysiological mechanisms of this obstetrical complication. Moreover, the proteomic results were compared to and integrated with those obtained from the soluble fraction of maternal plasma. Methods This retrospective case-control study included 47 women who experienced fetal death and 94 matched, healthy, pregnant controls. Proteomic analysis of 82 proteins in the EVs and the soluble fractions of maternal plasma samples was conducted by using a bead-based, multiplexed immunoassay platform. Quantile regression analysis and random forest models were implemented to assess differences in the concentration of proteins in the EV and soluble fractions and to evaluate their combined discriminatory power between clinical groups. Hierarchical cluster analysis was applied to identify subgroups of fetal death cases with similar proteomic profiles. A p-value of <.05 was used to infer significance, unless multiple testing was involved, with the false discovery rate controlled at the 10% level (q < 0.1). All statistical analyses were performed by using the R statistical language and environment-and specialized packages. Results Nineteen proteins (placental growth factor, macrophage migration inhibitory factor, endoglin, regulated upon activation normal T cell expressed and presumably secreted (RANTES), interleukin (IL)-6, macrophage inflammatory protein 1-alpha, urokinase plasminogen activator surface receptor, tissue factor pathway inhibitor, IL-8, E-Selectin, vascular endothelial growth factor receptor 2, pentraxin 3, IL-16, galectin-1, monocyte chemotactic protein 1, disintegrin and metalloproteinase domain-containing protein 12, insulin-like growth factor-binding protein 1, matrix metalloproteinase-1(MMP1), and CD163) were found to have different plasma concentrations (of an EV or a soluble fraction) in women with fetal death compared to controls. There was a similar pattern of change for the dysregulated proteins in the EV and soluble fractions and a positive correlation between the log2-fold changes of proteins significant in either the EV or the soluble fraction (ρ = 0.89, p < .001). The combination of EV and soluble fraction proteins resulted in a good discriminatory model (area under the ROC curve, 82%; sensitivity, 57.5% at a 10% false-positive rate). Unsupervised clustering based on the proteins differentially expressed in either the EV or the soluble fraction of patients with fetal death relative to controls revealed three major clusters of patients. Conclusion Pregnant women with fetal death have different concentrations of 19 proteins in the EV and soluble fractions compared to controls, and the direction of changes in concentration was similar between fractions. The combination of EV and soluble protein concentrations revealed three different clusters of fetal death cases with distinct clinical and placental histopathological characteristics.

Plasma extracellular vesicle (EV)-associated cytokines were quantified in people with HIV (PWH) with different virological control status, including elite controllers (EC) who maintain persistent control (PC) or not (TC). Cytokine signatures and pathways were determined for each group. Median EV-associated cytokine levels were higher among PWH than HIV-uninfected. EC showed the highest levels of EV-associated cytokines among PWH with PC levels higher than TC levels. IL-18 levels best distinguished PWH from uninfected controls, and EC from ART-treated, and IL-3 distinguished PC from TC. The role of EV-cytokines in intercellular communication and endogenous control of HIV expression should be investigated further.

The development of an effective vaccine to protect against HIV acquisition will be greatly bolstered by in-depth understanding of the innate and adaptive responses to vaccination. We report here that the efficacy of DNA/ALVAC/gp120/alum vaccines, based on V2-specific antibodies mediating apoptosis of infected cells (V2-ADCC), is complemented by efferocytosis, a cyclic AMP (cAMP)-dependent antiphlogistic engulfment of apoptotic cells by CD14⁺ monocytes. Central to vaccine efficacy is the engagement of the CCL2/CCR2 axis and tolerogenic dendritic cells producing IL-10 (DC-10). Epigenetic reprogramming in CD14⁺ cells of the cyclic AMP/CREB pathway and increased systemic levels of miRNA-139-5p, a negative regulator of expression of the cAMP-specific phosphodiesterase PDE4D, correlated with vaccine efficacy. These data posit that efferocytosis, through the prompt and effective removal of apoptotic infected cells, contributes to vaccine efficacy by decreasing inflammation and maintaining tissue homeostasis.

Pneumonia is an acute respiratory disease of varying aetiology, which drew much attention during the COVID-19 pandemic. Among many thoroughly studied aspects of pneumonia, lipid metabolism has been addressed insufficiently. Here, we report on abnormal lipid metabolism of both COVID-19- and non-COVID-19-associated pneumonias in human lungs. Morphometric analysis revealed extracellular and intracellular lipid depositions, most notably within vessels adjacent to inflamed regions, where they apparently interfere with the blood flow. Lipids were visualized on Sudan III- and Oil Red O-stained cryosections and on OsO4-contrasted semi-thin and ultrathin sections. Chromato-mass spectrometry revealed that unsaturated fatty acid content was elevated at inflammation sites compared with the intact sites of the same lung. The genes involved in lipid metabolism were downregulated in pneumonia, as shown by qPCR and in silico RNAseq analysis. Thus, pneumonias are associated with marked lipid abnormalities, and therefore lipid metabolism can be considered a target for new therapeutic strategies.