The effect of anabolic-androgenic hormones on postprandial triglyceridaemia and lipoprotein profiles in man

It has been hypothesised that endogenous testosterone and AAS may predispose humans to premature CHD. However, there is no direct evidence to link these hormones with a greater prevalence of premature CHD. The aim of this thesis was to better describe atherosclerotic risk associated with these hormones by clarifying their effect on additional risk factors for premature atherosclerosis. Little is known about the effect of testosterone and AAS on 'atherogenic dyslipidaemia', a phenotype characterised by elevated postprandial triglyceridaemia, small dense LDL and a low HDLC concentration, which confers a high risk of CHD. Accordingly, the magnitude of postprandial triglyceridaemia, LDL and HDL particle size, and LDLC, HDLC and Lp(a) concentration were compared in male (n=9) and female (n=3) bodybuilders after self administration of AAS for 5-6 weeks (ON cycle) and again after a 4-6 week 'washout' period (OFF cycle), and in normal males (T) (n=10) before and during a reversible suppression of endogenous testosterone, induced using a GnRH agonist (triptorelin), and in a control group (C) (n=8). Lipoprotein size was assessed by gradient gel electrophoresis (GGE), lipoprotein concentrations by immuno and enzymatic assay, and postprandial triglyceridaemia by a standardised oral fat tolerance test (65g/m² ). HDLC decreased in male bodybuilders (0.94±0.30 vs 0.70±0.27 mmol/L, p=0.004; x ± SD) and female bodybuilders (1.3±0.5 vs 0.8±0.2 mmol/L) ON cycle. GGE studies suggested that mostly HDL₂ was reduced. There were no significant reductions in LDL particle size ON cycle. Two males had larger LDL species ON cycle. Lp(a) decreased in male bodybuilders (124.7±128.0 to 69.3±73.3 U/L, p=0.008). ON cycle postprandial triglyceride excursion was unchanged in female bodybuilders and reduced (11.6±10.0 vs 7.5±5.4 mmol/L.hr; p=0.027) in male bodybuilders. In the triptorelin study, HDLC was increased in T (1.07±0.18 vs 1.41±0.28 mmol/L, p=0.002) and not in C. GGE studies indicated an increase of HDL₂ in five T subjects and no increase in C. Total cholesterol increased in T (4.77±0.80 vs 5.24±1.04 mmol/L, p=0.039) but not in C. LDL size increased in four T subjects, and not in C. Lp(a) increased in T (277.9±149.l vs 376.5±222.2 U/L, p=0.004), but not in C. Postprandial triglyceridaemia was unchanged in both T and C. The results of these studies did not show any additional atherogenic effects of endogenous testosterone or AAS in humans. Rather, a suppression of Lp(a) may be an antiatherogenic effect of these hormones. A reduced postprandial triglyceridaemia and increased LDL size in individuals who are predisposed to 'atherogenic dyslipidaemia', may be further antiatherogenic effects of AAS use

Combined immunodeficiency and Epstein-Barr virus-induced B cell malignancy in humans with inherited CD70 deficiency

In this study, we describe four patients from two unrelated families of different ethnicities with a primary immunodeficiency, predominantly manifesting as susceptibility to Epstein-Barr virus (EBV)–related diseases. Three patients presented with EBV-associated Hodgkin’s lymphoma and hypogammaglobulinemia; one also had severe varicella infection. The fourth had viral encephalitis during infancy. Homozygous frameshift or in-frame deletions in CD70 in these patients abolished either CD70 surface expression or binding to its cognate receptor CD27. Blood lymphocyte numbers were normal, but the proportions of memory B cells and EBV-specific effector memory CD8+ T cells were reduced. Furthermore, although T cell proliferation was normal, in vitro–generated EBV-specific cytotoxic T cell activity was reduced because of CD70 deficiency. This reflected impaired activation by, rather than effects during killing of, EBV-transformed B cells. Notably, expression of 2B4 and NKG2D, receptors implicated in controlling EBV infection, on memory CD8+ T cells from CD70-deficient individuals was reduced, consistent with their impaired killing of EBV-infected cells. Thus, autosomal recessive CD70 deficiency is a novel cause of combined immunodeficiency and EBV-associated diseases, reminiscent of inherited CD27 deficiency. Overall, human CD70–CD27 interactions therefore play a nonredundant role in T and B cell–mediated immunity, especially for protection against EBV and humoral immunity

Compartmentalization of total and virus-specific tissue-resident memory CD8+ T Cells in human lymphoid organs

Disruption of T cell memory during severe immune suppression results in reactivation of chronic viral infections, such as Epstein Barr virus (EBV) and Cytomegalovirus (CMV). How different subsets of memory T cells contribute to the protective immunity against these viruses remains poorly defined. In this study we examined the compartmentalization of virus-specific, tissue resident memory CD8+ T cells in human lymphoid organs. This revealed two distinct populations of memory CD8+ T cells, that were CD69+CD103+ and CD69+CD103-, and were retained within the spleen and tonsils in the absence of recent T cell stimulation. These two types of memory cells were distinct not only in their phenotype and transcriptional profile, but also in their anatomical localization within tonsils and spleen. The EBV-specific, but not CMV-specific, CD8+ memory T cells preferentially accumulated in the tonsils and acquired a phenotype that ensured their retention at the epithelial sites where EBV replicates. In vitro studies revealed that the cytokine IL-15 can potentiate the retention of circulating effector memory CD8+ T cells by down-regulating the expression of sphingosine-1-phosphate receptor, required for T cell exit from tissues, and its transcriptional activator, Kruppel-like factor 2 (KLF2). Within the tonsils the expression of IL-15 was detected in regions where CD8+ T cells localized, further supporting a role for this cytokine in T cell retention. Together this study provides evidence for the compartmentalization of distinct types of resident memory T cells that could contribute to the long-term protection against persisting viral infections

Global wealth disparities drive adherence to COVID-safe pathways in head and neck cancer surgery

Peer reviewe

IL-15 and TGF-β co-operate to extinguish expression of <i>KLF2</i> and <i>S1PR1</i>.

<p>(A) Representative flow cytometry plots show the expression of CD69 and CD103 by CD8⁺ T cells following 7 day culture under different conditions: unstimulated (US), IL-15 or IL-15 + TGF-β and polyclonal stimulation (TAE). (B) Plot shows the proportion of CD69⁺CD103^- (left panel) and CD69⁺CD103⁺ (right panel) CD8⁺ T cells following 7 day culture with different cytokines. Data are represented as the mean and SEM of 5–11 donors. (C) Representative flow cytometry plots show the expression of CD69, CD137 and dilution of cell trace violet (CTV) dye following stimulation of circulating CD8⁺ T cells for 7 days with TAE beads (upper panels) or IL-15 (lower panels). (D) Representative flow cytometry plot and graph show the expression of CD69 and the dilution of cell trace violet dye following stimulation of purified circulating naïve (n = 4), TCM (n = 2), TEM (n = 8) and TEMRA (n = 5) CD8⁺ T cells for 7 days with IL-15. (E-F) Plots show the relative expression of <i>S1PR1</i> (E) and <i>KLF2</i> (F) in CD69⁺ or CD69^—CD8⁺ T cells following culture for 7 days with no stimulation or stimulation with IL-15 with and without TGF-β. Purified circulating CD8⁺ T cells were cultured for 7 days and the resulting CD69⁺ and CD69^—populations were purified by cell sorting. The expression levels of KLF2 and S1pr1 were quantified by RT-PCR. Individual dots represent different samples and the data is represented as the mean ± SEM. Statistical analysis was performed using one-way ANOVA and Tukey test. P<0.05 is noted with * and P<0.005 is noted with **. (G-H) The ability of IL-15 induced CD69⁺CD8⁺ T cells to migrate to S1P and CCL5 (20 nM) was tested in trans-well migration assays. Cultured cells were sorted as stated above (for F) and their ability to migrate towards different concentrations of S1P (G) or CCL5 (20 nM) (H) was determined. Data represent the mean and SEM of three independent experiments using three different donors. Statistical analysis was performed using two-way ANOVA and the p value was < 0.05.</p

CD69⁺CD103⁺CD8⁺ T cells localize near the epithelial barrier in tonsils.

<p>The locations of CD69⁺CD8⁺ T cell subsets were determined by immunohistochemistry. (A) Immunofluorescence microscopy images of human tonsils show the localization of CD8 (green), CD69 (blue) and CD103 (red). Scale bar represents 100 μm. (B) Higher magnification of areas 1 & 2 show the localization of CD103⁺CD69⁺CD8⁺ and CD103CD69⁺CD8⁺ T cells within the tonsils. (C) White arrowheads point to CD103⁺CD69⁺CD8⁺ T cells in areas 1 and CD103^—CD69⁺CD8⁺ T cells in area 2. (C) Quantitative analysis of the distance of CD103⁺CD3⁺CD8⁺ T cells from the epithelium shows majority localizing near the epithelial surface (P = 0.0022 by two-tailed Mann Whitney U-test). (D) Immunofluorescence microscopy of human spleen sections shows the localization of CD8 (green), CD69 (blue) and CD103 (red). Scale bar represents 100 μm. Higher magnification of areas 1 and 2 show the distribution of CD103⁺CD69⁺CD8⁺ and CD103^—CD69⁺CD8⁺ T cells. White arrowheads in area 1 show the CD103⁺CD69⁺CD8⁺ T cells and in area 2 show the CD103^—CD69⁺CD8⁺ T cells.</p

Two subsets of CD8⁺ T cells are retained within human lymphoid tissues.

<p>The co-expression of CD69 and CD103 by CD8⁺ T cells isolated from spleen and tonsils were determined by flow cytometry. (A and B) Representative flow cytometry plots (left) and graphs (right; mean ± SEM; n = 10) show the proportion of CD69⁺CD8⁺ T cells expressing CD103 in human spleen (A) and tonsils (B). (C—E) Representative flow cytometry plots showing the expression levels of CCR7, CD45RA and CD11a between CD69^—CD103^—(blue), CD69⁺CD103^—(red) and CD69⁺CD103⁺ (green) CD8⁺ T cell subsets from the spleen (C) and tonsils (D) and the expression of PD-1, TIM3 and BTLA in tonsils (E). Data is representative of 3–5 independent experiments. (F) Relative expression of <i>KLF2</i> and <i>S1PR1</i> in purified CD8⁺ T subsets from the spleen (left panels) and tonsils (right panels). Individual dots represent different donor samples (n = 8 for spleen and n = 3 for tonsils). Statistical analysis was performed using one-way ANOVA and Tukey test. P<0.05 is noted with * and P<0.0005 is noted with ***.</p

Constitutive expression of IL-15 within tonsils.

<p>Immunofluorescence microscopy of frozen section of the tonsils show the presence of IL-15 expressing cells in the T cell areas and the epithelial lining of the tissue. Sections were stained with anti-IL-15 (red), anti-CD8 (green) and anti-IgM (blue). Yellow dashed line marks the epithelial barrier surface and the white dashed-lines show B cell follicles (B). ‘T” indicates the extra-follicular regions where T cells localize.</p

CD69 is expressed on CD8⁺ T cells in the absence of recent T cell activation.

<p>The phenotype of CD8⁺ T cells isolated from peripheral blood, spleen and tonsils were examined by flow cytometry. (A) The mean (± SD; n = 13 for blood and spleen and n = 7 for tonsils) percent of CD69⁺CD8⁺ T cells in these compartments. (B) CD69 expression on naïve (CCR7⁺CD45RA⁺), central memory (TCM, CCR7⁺CD45RA^—), effector memory (TEM, CCR7^—CD45RA^—) and TEMRA (CCR7^—CD45RA⁺) CD8⁺ T cells from blood (blue), spleen (red) and tonsils (green). Individual dots represent different donor samples. (C and D) Representative flow cytometry histogram plots show the expression levels of activation markers CD25, CD137, HLA-DR and KLRG1 between the CD69⁺CD8⁺ T cells (red) and CD69^—CD8⁺ T cells (blue) in the spleen (C) and tonsils (D). Data is representative of 5 independent experiments. (E) Graph shows the relative expression levels of <i>BCL2</i> between CD69⁺ TEM CD8⁺ T cells and CD69^—TEM CD8⁺ T cells from human spleen (n = 5). P = 0.0625 by one-way ANOVA.</p