Perivascular adipose tissue as regulator of the force of artery contractions in health and disease

During the last two decades, perivascular adipose tissue (PVAT) has been revealed as an important regulator of vascular processes such as proliferation of smooth muscle cells, pro- and anti-oxidant reactions in the vascular wall, angiogenesis, inflammation, apoptosis of neutrophils, migration of monocytes and others. PVAT derived mediators either increase or decrease the amplitudes of the force of artery contraction measured using isometric small vessel myography. In healthy animals and humans predominates the relaxing effect while in diseases the contractile influence of PVAT is common. In aging and pathological conditions like atherosclerosis and diabetes, or with environmental factors like tobacco smoke and high-fat diet, the phenotype of perivascular adipocytes is changed from anti-inflammatory to pro-inflammatory. This change is accompanied by a significant rearrangement of mediators released from PVAT

Serum IGF-1 levels measured by different ELISA assays and IRMA - a comparability study

Background: While techniques for measuring IGF-1 have evolved over the decades (Blum 1998), immunoassays are still the primary tool used in routine laboratories. ELISA, IRMA and Chemiluminiscence IGF-1 kits are today on the market and their calibrators, specifity, sensitivity, cross-reactivity and prices differ substantially. Comparability studies are therefore valuable, especially if IGF-1 results obtained by one method should be interpreted retrospectively after transfer of patients from the pediatric to the adult endocrinologists.Aim: To determine which of the used ELISA assays is closest to the IRMA IGF-1 assay and responds best to the Consensus Statement IGF-1 requirements from 2011.Material and methods: 24 patients aged 37.9±14.4 years, median 35; 12 females (42.25± 16.6, median 38.5); 12 males (33.58±10.9, median 34.5) years.The IGF-1 serum levels were measured by three different assays: Immunoradiometric (IRMA), IGFBP-blocked Immunoenzymetric (ELISA1) and Immunoenzymetric with acid-ethanol extraction (ELISA2). The results were interpreted according to the kit referent values adjusted for age and gender.Results: A very high positive correlation between the three assays (r=0.986 to 0.990) (p<0.01) was found. A significant difference was established only regarding the mean IGF-1 values measured by IRMA vs ELISA2 (Ñ€<0.05). The lowest IGF-1 mean values were measured by IRMA.Conclusions: Closest to the IRMA method were IGF1 levels measured by the indirect `sandwich` ELISA1 (IGFBP blocked) assay. Due to the significant difference between IRMA and the direct ELISA2 method, diagnostic pitfalls and therapeutic misinterpretations are possible. Priority has to be given to IGF-1 assays which best correspond to the Consensus Statement requirements from 2011. In this study this is fulfilled for the ELISA1, IGF blocked immunoassay. Future developments lie in fully automated assay systems, adapted to pediatric endocrine laboratories

IMMUNE RESPONSE TO SARS-COV-2 IN PATIENTS WITH CHRONIC HIV INFECTION

Introduction. Data for the long-term effects of SARS-CoV-2/HIV co-infection on immune restoration, as well as the level of post-exposure and post-vaccination immunity at the current stage of SARS-CoV-2 pandemic in HIV+ individuals is still scarce. We assessed SARS-CoV-2-specific immune responses, and the effects of SARS-CoV-2 infection on the immune recovery in HIV+cART+ patients with different exposure history.  Materials and methods. HIV+cART+ patients 9 (2-18) months after mild/moderate COVID-19 and completed immunization with anti-SARS-CoV-2 vaccine (n=13, group A), convalescent, not immunized (n=11, group B), or with no history of exposure to SARS-CoV-2 (n=11, group C) were included in the study. CD4AC and CD4/CD8 ratio were determined before and after the documented/probable contact with SARS-CoV-2 by 4-color flow cytometry (TRUCount, MultiTest, FACSCanto II). Virus-specific immunity was characterized by the SARS-CoV-2 specific IFNγ production (SARS-CoV-2 IGRA, Euroimmun) and the levels of RBD-IgG ((Euroimmun ELISA).  Results. SARS-CoV-2 specific T-cell and IgG responses were highly correlated and present, respectively, in 92% and 100%; 64% and 54%, 36% and 50% from group A, B and C patients. SARS-CoV-2 specific IFNy+T cells and RDB-IgG were significantly higher in the group with hybrid exposure (A) as compared to convalescent (B) and asymptomatic (C) patients. No significant difference existed between background and actual CD4AC (mean 836 vs 799 cells/µl, p>0.05, Mann-Whitney), and the CD4/CD8 ratio significantly increased in the group with hybrid exposure (0.92 vs 1.07, p<0.01, paired T-test).  Conclusion. Over 80% of tested HIV+ individuals have mounted a SARS-CoV-2 specific immune response. Immunization and hybrid exposure provide a durable and significantly stronger SARS-CoV-2-specific immune response as compared to mild/ asymptomatic infection, without affecting the long-term immune recovery

FERROPTOSIS IN CD4+ AND CD8+ T-CELLS IN THE SETTINGS OF HIV INFECTION

Introduction: Elevation of intracellular iron concentration triggers ferroptosis. Understanding the regulation and pathophysiological mechanisms of this process in HIV infection may contribute to antiretroviral therapy (cART) monitoring. Aim: To perform a correlation analysis of the intracellular labile-bound iron pool (LIP) in CD4+ and CD8+ T cells in association with CD4+, CD8+ T cells absolute count (AC) and CD4/CD8 index in HIV+ individuals on continuous cART with sustained viral suppression. Material and methods: Peripheral blood samples (Li heparin, n=34) were collected in the course of the routine immune monitoring of HIV+ individuals at four time points during 24 months. Plasma HIV viral load (VL) was determined with the Abbott Real-Time HIV-1 test (sensitivity 40 copies/ml). AC and percentage of CD4+ and CD8+ T cells were determined by direct flow cytometry (Multitest, BD Trucount, FACS Canto II). The intracellular content of LIP in CD4 and CD8 T cells (LIP CD4, LIP CD8) was measured at the beginning of the study, using acetoxymethyl ester and subsequent incubation with a chelator (Deferiprone). LIP was quantified according to the mean fluorescence intensity (MFI) (FACSCanto II, Diva 6.1.2). Results: In the settings of a higher LIP CD4 , high LIP CD8 correlated with increased CD8AC (Rho=0.70, p<0.05) up to 11 (min. 6, max. 15) months after LIP measurement., and decreased CD4/CD8 ratio correlated inversely with LIP CD8 in all consecutive measurements (Rho= -0.71, p<0.01 for all), Importantly, high LIP CD8 correlated with a lower CD4AC (Rho=-0.65, p<0.05) up to five (min.1, max.8) months after LIP measurement. Conclusion: The increased concentration of intracellular LIP in CD8 cells in HIV+cART individuals could indicate viral activity in the settings of undetectable HIV VL, directly associated with ongoing cell ferroptosis

Expression of membrane-bound β-Klotho on peripheral blood CD4+ and CD8+ T lymphocytes of healthy subjects

AbstractImmunosenescence is driven by repeated and continuous immune activation. Sensitive markers are warranted to detect and monitor the process of premature immune ageing. We analysed the β-Klotho expression on T-cell subsets from healthy donors: young adults, n = 12 (20–30 y); middle-aged, n = 62 (30–50 y) and elderly, n = 18 (>70 y). The absolute counts (AC) and percentages of total, CD4+, CD8+ and CD8 + CD27-CD57+ T-cells, CD4/CD8 ratio, and the share of β-Klotho + T-cell subsets were analysed by multicolour flow cytometry. AC of T-cell subsets showed non-significant differences among the groups. The percentages of β-Klotho(+) total, CD4+ and CD8+ T-cells in young adults exceeded significantly the expression in the elderly and middle-aged subjects: (mean) 2.13 vs. 0.48; 3.23 vs. 0.48; 6.87 vs. 2.28, and 0.75 vs. 2.21; 0.93 vs. 3.32; 6.62 vs. 6.62, respectively (p < 0.05 all). The β-Klotho expression on total, CD4+ and CD8+ T-cells among middle-aged participants was heterogeneous and significantly higher from the elderly only in CD8 + T-cells (mean): 0.75 vs. 0.47 (p = 0.1); 0.93 vs. 0.72 (p = 1.0) and 3.62 vs 1.75 (p = 0.01), respectively. The proportion of β-Klotho(+) CD8+ cells did not correlate with age, but correlated inversely and significantly with the share of CD27-CD57 + CD8+ T-cells in middle-aged subjects (R = −0.4, p < 0.01). We hypothesise that CD8 + T-cells showed higher sensitivity to the effects of immune-senescence, at least in part, due to the higher expression and dependence on the effects of KLOTHO gene products. We propose that a reduced share of β-Klotho + CD8+ T-cells indicates exhaustion due to immune activation, and may serve as an early marker of premature immunosenescence

Eicosanoid and cytokine levels differentiate between stages of MTB infection

Abstract Introduction: The need for biomarkers predicting the course of MTB infection and the necessity of specific therapy are well recognized. Recent data point to the role of cytokines and lipid mediators in protective immunity against tuberculosis. Aim: We evaluated the balance between cytokines, and eikosanoids as a possible prognostic indicator in MTB infection. Material and methods: The induced expression of effector and regulatory cytokines IFN-γ, TNF-α, IL-2, IL-17, IL-6, and IL-10 was measured in relation to the lipid mediators PGE2 and LXA4 in active TB infection (ATB, n=15) before and after therapy (ATB-T, n=6), established latent infection (LTBI, n=22), recent contacts of ATB (RC, n=12), and healthy controls (n=11) A flow cytometry microarray (CBA, BD Biosciences) and quantitative ELISA (SunRed Tech) were employed. Results: The regulatory cytokines (RC) were characterized by a high potential for IL-17 and Th1 cytokine secretion, combined with low IL-6 expression, while ATB donors had a partially preserved TNF-α potential, and higher IL-6 expression. The PGE2-to-LXA4 ratio discriminated between situations with high bacterial load (ATB), and contained infection (LTBI, ATB-T), and defined clearly cut subgroups among RC and ATB donors. Conclusions: Our results suggest that increased PGE2/LXA4 ratio coupled with high induced IL-10 level indicates infection after a recent contact. In the settings of ATB, increased ratio and low TNF-α level point to inefficient granuloma formation in the settings of ATB

Virus-Specific Stem Cell Memory CD8+ T Cells May Indicate a Long-Term Protection against Evolving SARS-CoV-2

Immune memory to SARS-CoV-2 is key for establishing herd immunity and limiting the spread of the virus. The duration and qualities of T-cell-mediated protection in the settings of constantly evolving pathogens remain an open question. We conducted a cross-sectional study of SARS-CoV-2-specific CD4+ and CD8+ T-cell responses at several time points over 18 months (30–750 days) post mild/moderate infection with the aim to identify suitable methods and biomarkers for evaluation of long-term T-cell memory in peripheral blood. Included were 107 samples from 95 donors infected during the periods 03/2020–07/2021 and 09/2021–03/2022, coinciding with the prevalence of B.1.1.7 (alpha) and B.1.617.2 (delta) variants in Bulgaria. SARS-CoV-2-specific IFNγ+ T cells were measured in ELISpot in parallel with flow cytometry detection of AIM+ total and stem cell-like memory (TSCM) CD4+ and CD8+ T cells after in vitro stimulation with peptide pools corresponding to the original and delta variants. We show that, unlike IFNγ+ T cells, AIM+ virus-specific CD4+ and CD8+ TSCM are more adequate markers of T cell memory, even beyond 18 months post-infection. In the settings of circulating and evolving viruses, CD8+ TSCM is remarkably stable, back-differentiated into effectors, and delivers immediate protection, regardless of the initial priming strain

Transmitted HIV Drug Resistance in Bulgaria Occurs in Clusters of Individuals from Different Transmission Groups and Various Subtypes (2012–2020)

Transmitted HIV drug resistance in Bulgaria was first reported in 2015 using data from 1988–2011. We determined the prevalence of surveillance drug resistance mutations (SDRMs) and HIV-1 genetic diversity in Bulgaria during 2012–2020 using polymerase sequences from 1053 of 2010 (52.4%) antiretroviral therapy (ART)-naive individuals. Sequences were analyzed for DRM using the WHO HIV SDRM list implemented in the calculated population resistance tool at Stanford University. Genetic diversity was inferred using automated subtyping tools and phylogenetics. Cluster detection and characterization was performed using MicrobeTrace. The overall rate of SDRMs was 5.7% (60/1053), with 2.2% having resistance to nucleoside reverse transcriptase inhibitors (NRTIs), 1.8% to non-nucleoside reverse transcriptase inhibitors (NNRTIs), 2.1% to protease inhibitors (PIs), and 0.4% with dual-class SDRMs. We found high HIV-1 diversity, with the majority being subtype B (60.4%), followed by F1 (6.9%), CRF02_AG (5.2%), A1 (3.7%), CRF12_BF (0.8%), and other subtypes and recombinant forms (23%). Most (34/60, 56.7%) of the SDRMs were present in transmission clusters of different subtypes composed mostly of male-to-male sexual contact (MMSC), including a 14-member cluster of subtype B sequences from 12 MMSC and two males reporting heterosexual contact; 13 had the L90M PI mutation and one had the T215S NRTI SDRM. We found a low SDRM prevalence amid high HIV-1 diversity among ART-naive patients in Bulgaria during 2012–2020. The majority of SDRMs were found in transmission clusters containing MMSC, indicative of onward spread of SDRM in drug-naive individuals. Our study provides valuable information on the transmission dynamics of HIV drug resistance in the context of high genetic diversity in Bulgaria, for the development of enhanced prevention strategies to end the epidemic