Inappropriate sinus tachycardia (IST) is a common observation in patients with post-COVID-19 syndrome (PCS) but has not yet been fully described to date. To investigate the prevalence and the ...mechanisms underlying IST in a prospective population of PCS patients. Consecutive patients admitted to the PCS Unit between June and December 2020 with a resting sinus rhythm rate ≥ 100 bpm were prospectively enrolled in this study and further examined by an orthostatic test, 2D echocardiography, 24-h ECG monitoring (heart rate variability was a surrogate for cardiac autonomic activity), quality-of-life and exercise capacity testing, and blood sampling. To assess cardiac autonomic function, a 2:1:1 comparative sub-analysis was conducted against both fully recovered patients with previous SARS-CoV-2 infection and individuals without prior SARS-CoV-2 infection. Among 200 PCS patients, 40 (20%) fulfilled the diagnostic criteria for IST (average age of 40.1 ± 10 years, 85% women, 83% mild COVID-19). No underlying structural heart disease, pro-inflammatory state, myocyte injury, or hypoxia were identified. IST was accompanied by a decrease in most heart rate variability parameters, especially those related to cardiovagal tone: pNN50 (cases 3.2 ± 3 vs. recovered 10.5 ± 8 vs. non-infected 17.3 ± 10; p < 0.001) and HF band (246 ± 179 vs. 463 ± 295 vs. 1048 ± 570, respectively; p < 0.001). IST is prevalent condition among PCS patients. Cardiac autonomic nervous system imbalance with decreased parasympathetic activity may explain this phenomenon.
The phenotypic characterization of the cells in which HIV persists during antiretroviral therapy (ART) remains technically challenging. We developed a simple flow cytometry-based assay to quantify ...and characterize infected cells producing HIV proteins during untreated and treated HIV infection. By combining two antibodies targeting the HIV capsid in a standard intracellular staining protocol, we demonstrate that p24-producing cells can be detected with high specificity and sensitivity in the blood from people living with HIV. In untreated individuals, the frequency of productively infected cells strongly correlated with plasma viral load. Infected cells preferentially displayed a transitional memory phenotype and were enriched in Th17, peripheral Tfh and regulatory T cells subsets. These cells also preferentially expressed activation markers (CD25, HLA-DR, Ki67), immune checkpoint molecules (PD-1, LAG-3, TIGIT, Tim-3) as well as the integrins α4β7 and α4β1. In virally suppressed individuals on ART, p24-producing cells were only detected upon stimulation (median frequency of 4.3 p24+ cells/106 cells). These measures correlated with other assays assessing the size of the persistent reservoir including total and integrated HIV DNA, Tat/rev Induced Limiting Dilution Assay (TILDA) and quantitative viral outgrowth assay (QVOA). In ART-suppressed individuals, p24-producing cells preferentially displayed a transitional and effector memory phenotype, and expressed immune checkpoint molecules (PD-1, TIGIT) as well as the integrin α4β1. Remarkably, α4β1 was expressed by more than 70% of infected cells both in untreated and ART-suppressed individuals. Altogether, these results highlight a broad diversity in the phenotypes of HIV-infected cells in treated and untreated infection and suggest that strategies targeting multiple and phenotypically distinct cellular reservoirs will be needed to exert a significant impact on the size of the reservoir.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The phenotype of the rare HIV-infected cells persisting during antiretroviral therapies (ART) remains elusive. We developed a single-cell approach that combines the phenotypic analysis of ...HIV-infected cells with near full-length sequencing of their associated proviruses to characterize the viral reservoir in 6 male individuals on suppressive ART. We show that individual cells carrying clonally expanded identical proviruses display very diverse phenotypes, indicating that cellular proliferation contributes to the phenotypic diversification of the HIV reservoir. Unlike most viral genomes persisting on ART, inducible and translation-competent proviruses rarely present large deletions but are enriched in defects in the Ψ locus. Interestingly, the few cells harboring genetically intact and inducible viral genomes express higher levels of the integrin VLA-4 compared to uninfected cells or cells with defective proviruses. Viral outgrowth assay confirmed that memory CD4+ T cells expressing high levels of VLA-4 are highly enriched in replication-competent HIV (27-fold enrichment). We conclude that although clonal expansions diversify the phenotype of HIV reservoir cells, CD4+ T cells harboring replication-competent HIV retain VLA-4 expression.
HIV cure efforts are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in patients receiving ...antiretroviral therapy. We combine fluorescent in situ RNA hybridization with detection of HIV protein and flow cytometry, enabling detection of 0.5–1 gag-pol mRNA+/Gag protein+-infected cells per million. In the peripheral blood of untreated persons, active HIV replication correlated with viremia and occurred in CD4 T cells expressing T follicular helper cell markers and inhibitory co-receptors. In virally suppressed subjects, the approach identified latently infected cells capable of producing HIV mRNA and protein after stimulation with PMA/ionomycin and latency-reversing agents (LRAs). While ingenol-induced reactivation mirrored the effector and central/transitional memory CD4 T cell contribution to the pool of integrated HIV DNA, bryostatin-induced reactivation occurred predominantly in cells expressing effector memory markers. This indicates that CD4 T cell differentiation status differentially affects LRA effectiveness.
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•HIV RNA and protein co-expression allows ex vivo characterization of patient CD4 T cells•HIV-infected CD4s show markers of exhaustion and peripheral follicular helper cells•Translation-competent latent reservoir can be detected in most ART-treated patients•PKC agonist Bryostatin preferentially reactivates HIV from effector memory CD4s
Technological limitations hamper characterization of CD4 T cells supporting ongoing HIV infection and quantification of the latent reservoir. Baxter et al. (2016) use simultaneous detection of viral protein and mRNA to quantify and phenotype both the ongoing infection during viremia and the translation-competent inducible reservoir in virally supressed, treated patients.
Highly active antiretroviral therapy (HAART) results in potent and durable suppression of HIV-1 viremia. However, HIV-1 replication resumes if therapy is interrupted. Although it is generally ...believed that active replication has been halted in individuals on HAART, immune activation and inflammation continue at abnormal levels, suggesting continued, low-level viral replication. To assess whether active replication might be driving immune activation in HAART, we examined the impact of treatment intensification with the integrase inhibitor raltegravir on viral complementary DNA and immune activation parameters. In the presence of raltegravir, linear HIV-1 cDNA is prevented from integrating into chromatin and is subsequently converted to episomal cDNAs. Raltegravir intensification of a three-drug suppressive HAART regimen resulted in a specific and transient increase in episomal DNAs in a large percentage of HAART-suppressed subjects. Furthermore, in subjects with these episomal DNAs, immune activation was higher at baseline and was subsequently normalized after raltegravir intensification. These results suggest that, despite suppressive HAART, active replication persists in some infected individuals and drives immune activation. The ability of raltegravir intensification to perturb the reservoir that supports active replication has implications for therapeutic strategies aimed at achieving viral eradication.
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Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
With the increasing number of therapeutic strategies tested in humans to reduce the size of the latent reservoir, the development of a robust, precise and clinical trial scalable assay that measures ...the frequency of infected cells carrying inducible replication-competent HIV is urgently needed. The size of the pool of cells carrying replication-competent HIV is largely overestimated by DNA assays, as a result of a large proportion of defective viruses, and underestimated by co-culture outgrowth assays. New culture methods that measure the inducible HIV reservoir have been developed during the past few years. In these induction assays, CD4
T cells from virally suppressed individuals are activated and HIV RNA is measured in cell extracts or cell supernatants. In this review, we summarize the principle and outcomes of these assays and discuss the potential of these methods in the evaluation of HIV eradication strategies.
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DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Efforts to cure HIV are hampered by limited characterization of the cells supporting HIV replication in vivo and inadequate methods for quantifying the latent viral reservoir in individuals receiving ...antiretroviral therapy (ART). We describe a protocol for flow cytometric identification of viral reservoirs, based on concurrent detection of cellular HIV Gagpol mRNA by in situ RNA hybridization combined with antibody staining for the HIV Gag protein. By simultaneously detecting both HIV RNA and protein, the CD4 T cells harboring translation-competent virus can be identified. The HIV
method is 1,000-fold more sensitive than Gag protein staining alone, with a detection limit of 0.5-1 Gagpol mRNA
/Gag protein
cells per million CD4 T cells. Uniquely, the HIV
assay also allows parallel phenotyping of viral reservoirs, including reactivated latent reservoirs in clinical samples. The assay takes 2 d, and requires antibody labeling for surface and intracellular markers, followed by mRNA labeling and multiple signal amplification steps.
Limited data exists on SARS-CoV-2 sustained-response to vaccine in patients with rheumatic diseases. This study aims to evaluate neutralizing antibodies (nAB) induced by SARS-CoV-2 vaccine after 3 to ...6 months from administration in Systemic Lupus Erythematosus (SLE) patients, as a surrogate of sustained-immunological response. This cross-sectional study compared nAB titre of 39 SLE patients and 37 Healthy individuals with no previous SARS-CoV-2 infection, who had all received a complete regimen of a mRNA SARS-CoV-2 vaccine within the last 3 to 6 months. We included four lines of SLE treatment including Not-treated, Hydroxychloroquine, immunosuppressive drugs and biological therapy. Glucocorticoids were allowed in all groups. Healthy and Not-treated individuals showed the highest levels of nAB. Treated patients presented lower nAB titres compared to Healthy: a 73% decrease for First-Line patients, 56% for Second-Line treatment and 72% for Third-Line. A multivariate analysis pointed to Glucocorticoids as the most associated factor with declining nAB levels (75% decrease) in treated SLE. Furthermore, a significant reduction in nAB titres was observed for Rituximab-users compared to Healthy subjects (89% decrease). Medium-term response of SLE patients to SARS-CoV-2 mRNA vaccines is negatively impacted in Glucocorticoids and Rituximab users. These findings might help to inform recommendations in vaccination protocols for SLE patients.
COVID-19 pathophysiology is currently not fully understood, reliable prognostic factors remain elusive, and few specific therapeutic strategies have been proposed. In this scenario, availability of ...biomarkers is a priority. MS-based Proteomics techniques were used to profile the proteome of 81 plasma samples extracted in four consecutive days from 23 hospitalized COVID-19 associated pneumonia patients. Samples from 10 subjects that reached a critical condition during their hospital stay and 10 matched non-severe controls were drawn before the administration of any COVID-19 specific treatment and used to identify potential biomarkers of COVID-19 prognosis. Additionally, we compared the proteome of five patients before and after glucocorticoids and tocilizumab treatment, to assess the changes induced by the therapy on our selected candidates. Forty-two proteins were differentially expressed between patients' evolution groups at 10% FDR. Twelve proteins showed lower levels in critical patients (fold-changes 1.20-3.58), of which OAS3 and COG5 found their expression increased after COVID-19 specific therapy. Most of the 30 proteins over-expressed in critical patients (fold-changes 1.17-4.43) were linked to inflammation, coagulation, lipids metabolism, complement or immunoglobulins, and a third of them decreased their expression after treatment. We propose a set of candidate proteins for biomarkers of COVID-19 prognosis at the time of hospital admission. The study design employed is distinctive from previous works and aimed to optimize the chances of the candidates to be validated in confirmatory studies and, eventually, to play a useful role in the clinical practice.
Some HIV-infected c-ART-suppressed individuals show incomplete CD4+ T-cell recovery, abnormal T-cell activation and higher mortality. One potential source of immune activation could be coinfection ...with cytomegalovirus (CMV). IgG and IgM levels, immune activation, inflammation and T-cell death in c-ART-suppressed individuals with CD4+ T-cell counts >350 cells/μL (immunoconcordant, n = 133) or <350 cells/μL (immunodiscordant, n = 95) were analyzed to evaluate the effect of CMV humoral response on immune recovery. In total, 27 HIV-uninfected individuals were included as controls. In addition, the presence of CMV IgM antibodies was retrospectively analyzed in 58 immunoconcordant individuals and 66 immunodiscordant individuals. Increased CMV IgG levels were observed in individuals with poor immune reconstitution (p = 0.0002). Increased CMV IgG responses were significantly correlated with lower nadir and absolute CD4+ T-cell counts. In contrast, CMV IgG responses were positively correlated with activation (HLA-DR+) and death markers in CD4+ T-cells and activated memory CD8+ T-cells (CD45RA-CD38+). Longitudinal subanalysis revealed an increased frequency of IgM+ samples in individuals with poor CD4+ T-cell recovery, and an association was observed between retrospective IgM positivity and the current level of IgG. The magnitude of the humoral immune response to CMV is associated with nadir CD4+ T-cell counts, inflammation, immune activation and CD4+ T-cell death, thus suggesting that CMV infection may be a relevant driving force in the increased morbidity/mortality observed in HIV+ individuals with poor CD4+ T-cell recovery.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
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