Cytokine production by memory T cells is a key mechanism of T cell mediated protection. However, we have limited understanding of the persistence of cytokine producing T cells during memory cell maintenance and secondary responses. We interrogated antigen-specific CD4 T cells using a mouse influenza A virus infection model. Although CD4 T cells detected using MHCII tetramers declined in lymphoid and non-lymphoid organs, we found similar numbers of cytokine
Monocytes play an important role in hypertension. Circulating monocytes in humans exist as classical, intermediate, and non-classical forms. Monocyte differentiation can be influenced by the endothelium, which in turn is activated in hypertension by mechanical stretch. We sought to examine the role of increased endothelial stretch and hypertension on monocyte phenotype and function. Human monocytes were cultured with confluent human aortic endothelial cells undergoing either 5% or 10% cyclical stretch. We also characterized circulating monocytes in normotensive and hypertensive humans. In addition, we quantified accumulation of activated monocytes and monocyte-derived cells in aortas and kidneys of mice with Angiotensin II-induced hypertension. Increased endothelial stretch enhanced monocyte conversion to CD14++CD16+ intermediate monocytes and monocytes bearing the CD209 marker and markedly stimulated monocyte mRNA expression of interleukin (IL)-6, IL-1β, IL-23, chemokine (C-C motif) ligand 4, and tumour necrosis factor α. STAT3 in monocytes was activated by increased endothelial stretch. Inhibition of STAT3, neutralization of IL-6 and scavenging of hydrogen peroxide prevented formation of intermediate monocytes in response to increased endothelial stretch. We also found evidence that nitric oxide (NO) inhibits formation of intermediate monocytes and STAT3 activation. In vivo studies demonstrated that humans with hypertension have increased intermediate and non-classical monocytes and that intermediate monocytes demonstrate evidence of STAT3 activation. Mice with experimental hypertension exhibit increased aortic and renal infiltration of monocytes, dendritic cells, and macrophages with activated STAT3. These findings provide insight into how monocytes are activated by the vascular endothelium during hypertension. This is likely in part due to a loss of NO signalling and increased release of IL-6 and hydrogen peroxide by the dysfunctional endothelium and a parallel increase in STAT activation in adjacent monocytes. Interventions to enhance bioavailable NO, reduce IL-6 or hydrogen peroxide production or to inhibit STAT3 may have anti-inflammatory roles in hypertension and related conditions.
Aortic adaptive immunity plays a role in atherosclerosis; however, the precise mechanisms leading to T-cell activation in the arterial wall remain poorly understood.Here, we have identified naïve T cells in the aorta of wild-type and T-cell receptor transgenic mice and we demonstrate that naïve T cells can be primed directly in the vessel wall with both kinetics and frequency of T-cell activation found to be similar to splenic and lymphoid T cells. Aortic homing of naïve T cells is regulated at least in part by the P-selectin glycosylated ligand-1 receptor. In experimental atherosclerosis the aorta supports CD4+ T-cell activation selectively driving Th1 polarization. By contrast, secondary lymphoid organs display Treg expansion.Our results demonstrate that the aorta can support T-cell priming and that naïve T cells traffic between the circulation and vessel wall. These data underpin the paradigm that local priming of T cells specific for plaque antigens contributes to atherosclerosis progression.
Diabetic retinopathy (DR) may evolve from retinal neurovascular dysfunction and oxidative stress.We therefore investigated the effects of euglycaemic (6 mmol/l) and hyperglycaemic clamps (15 mmol/l) with insulin infusions (6 pmol/kg/min), with or without vitamin C (2 g intravenous), on pattern electroretinogram (PERG) and flicker-induced vasodilation responses in 12 adults with type 1 diabetes.The PERG ratio (0.8° vs. 7° checks) increased by (mean [95% C.I.]) 0.31 (0.03 -0.59) after hyperglycaemia with vitamin C. Venule maximum dilations were absolutely increased by 1.7 (0.5 -3.0) % after euglycaemic clamps.Arteriole maximum dilations were non-significantly larger with hyperglycaemia and vitamin C (P = 0.07 by ANOVA).Insulin, and possibly vitamin C, may improve retinal neurovascular function in type 1 diabetes.Diabetic retinopathy (DR) is a potentially blinding complication of diabetes mellitus.Among people with a 20-year history of type 1 diabetes, roughly 92% will have some level of DR and 18% will have vision-threatening DR (1).DR may evolve in part as a consequence of sustained retinal neurovascular dysfunction, which in turn may be exacerbated by poor glycaemic control and oxidative stress.Retinal flicker-induced vasodilation (2-7) and PERG responses (6-9) are potential sensitive markers of DR.Based on previous work (2-20), we hypothesised that flicker-induced vasodilation would improve and be impaired by euglycaemic and hyperglycaemic clamps, respectively, in people with type 1 diabetes.We simultaneously predicted that PERG responses, markers of ganglion cell function, would be stable across glucose conditions.Twelve people with type 1 diabetes aged ≥ 18 years and no significant history of ocular or systemic disease were recruited.Smokers and those with known mild non-
Purpose.: To investigate the role of epoxyeicosatrienoic acids (EETs) and prostaglandins (PGs) in retinal blood vessel calibers and vasodilation during flicker light stimulation in humans. Methods.: Twelve healthy nonsmokers participated in a balanced crossover study. Oral fluconazole 400 mg and dispersible aspirin 600 mg were used to inhibit production of EETs and PGs, respectively. Retinal imaging was performed 1 hour after drug ingestion with the Dynamic Vessel Analyzer. Resting calibers of selected vessel segments were recorded in measurement units (MU). Maximum percentage dilations during flicker stimulation were calculated from baseline calibers. We then studied six participants each after fluconazole and aspirin ingestions at 30-minute intervals for 2 hours. Within-subject differences were assessed by ANOVA and Dunnett-adjusted pairwise comparisons with significance taken at P < 0.05. Results.: In crossover study participants, mean (SD) arteriole and venule dilations without drug administration were 4.4% (2.0%) and 4.6% (1.7%), respectively. Neither drug affected vasodilation during flicker stimulation. Mean (SD) resting arteriole and venule calibers on no-drug visits were 119.6 (10.6) MU and 145.7 (17.0) MU, respectively. Fluconazole reduced mean (±95% CI) resting venule calibers by 5.1 (4.3) MU. In repeated measures participants, neither drug affected vasodilations, but fluconazole reduced resting venule calibers over 2 hours (P < 0.001). Conclusions.: Epoxyeicosatrienoic acids and prostaglandins are unlikely to be primary mediators of flicker light–induced retinal vasodilation in humans. However, EETs may play a role in the regulation of retinal vascular tone and blood flow under resting physiological conditions.
Surface-enhanced Raman spectroscopy (SERS) is an emerging molecular imaging technique suitable for the highly sensitive simultaneous detection of multiple analytes. We aimed to apply SERS for the multiplex detection of adhesion molecules in human umbilical vein endothelial cells and macrophages in vitro, as a step towards applying this technology for simultaneously detecting multiple biomarkers of vascular inflammation and atherosclerotic plaque instability in vivo. Immunofluorescent microscopy and flow cytometry identified the expression of intercellular adhesion molecule (ICAM)−1, vascular cell adhesion molecule (VCAM)−1 and p-Selectin on human umbilical vein endothelial cells (HUVECs) following stimulation with 10 ng/ml TNFα for 24 hours. Phorbol 12-myristate 13-acetate (PMA) differentiated THP-1 macrophage-like cells expressed only ICAM-1 in both unstimulated and 24 hours 10 ng/ml TNFα stimulated conditions. Gold nanoparticles were PEGylated and functionalized with Raman reporters, in order to endow a specific pre-determined SERS signal, alongside antibodies against the desired adhesion molecule targets. This provided ‘nanotags’ configured as follows (antibody/Raman reporter); anti-ICAM-1/BPE; anti-VCAM-1/PYOT, anti-p-Selectin/PPY, IgG Isotype Control/DP. TNFα stimulated HUVEC were exposed to a mixture of all nanotags simultaneously (final concentration of 20 ug/L) and were subsequently imaged using SERS microscopy. This rational allowed for the simultaneous detection and discrimination of ICAM-1, VCAM-1 and p-Selectin on activated HUVEC. Adhesion molecules were detectible as soon as 15 min following nanotag addition, while IgG control nanotags produced negligible signal at all concentrations and time points investigated. Following simultaneous exposure to all nanotags, SERS imaging identified the expression of ICAM-1 on PMA differentiated TNFα stimulated THP-1, with little-to-no contribution from anti-VCAM-1, anti-p-Selectin, or IgG control nanotags. Here we have developed a SERS-based molecular imaging methodology for the multiplex detection of adhesion molecules in vitro.
The high mortality rate of Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) infection is a critical concern of the coronavirus disease 2019 (COVID-19) pandemic. Strikingly, men account for the majority of COVID-19 deaths, with current figures ranging from 59% to 75% of total mortality. However, despite clear implications in relation to COVID-19 mortality, most research has not considered sex as a critical factor in data analysis. Here, we highlight fundamental biological differences that exist between males and females, and how these may make significant contributions to the male-biased COVID-19 mortality. We present preclinical evidence identifying the influence of biological sex on the expression and regulation of angiotensin-converting enzyme 2 (ACE2), which is the main receptor used by SARS-CoV-2 to enter cells. However, we note that there is a lack of reports showing that sexual dimorphism of ACE2 expression exists and is of functional relevance in humans. In contrast, there is strong evidence, especially in the context of viral infections, that sexual dimorphism plays a central role in the genetic and hormonal regulation of immune responses, both of the innate and the adaptive immune system. We review evidence supporting that ineffective anti-SARS-CoV-2 responses, coupled with a predisposition for inappropriate hyperinflammatory responses, could provide a biological explanation for the male bias in COVID-19 mortality. A prominent finding in COVID-19 is the increased risk of death with pre-existing cardiovascular comorbidities, such as hypertension, obesity, and age. We contextualize how important features of sexual dimorphism and inflammation in COVID-19 may exhibit a reciprocal relationship with comorbidities, and explain their increased mortality risk. Ultimately, we demonstrate that biological sex is a fundamental variable of critical relevance to our mechanistic understanding of SARS-CoV-2 infection and the pursuit of effective COVID-19 preventative and therapeutic strategies.
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