Highly conserved, non-human-like, and cross-reactive SARS-CoV-2 T cell epitopes for COVID-19 vaccine design and validation.

Natural and vaccine-induced SARS-CoV-2 immunity in humans has been described but correlates of protection are not yet defined. T cells support the SARS-CoV-2 antibody response, clear virus-infected cells, and may be required to block transmission. In this study, we identified peptide epitopes associated with SARS-CoV-2 T-cell immunity. Using immunoinformatic methods, T-cell epitopes from spike, membrane, and envelope were selected for maximal HLA-binding potential, coverage of HLA diversity, coverage of circulating virus, and minimal potential cross-reactivity with self. Direct restimulation of PBMCs collected from SARS-CoV-2 convalescents confirmed 66% of predicted epitopes, whereas only 9% were confirmed in naive individuals. However, following a brief period of epitope-specific T-cell expansion, both cohorts demonstrated robust T-cell responses to 97% of epitopes. HLA-DR3 transgenic mouse immunization with peptides co-formulated with poly-ICLC generated a potent Th1-skewed, epitope-specific memory response, alleviating safety concerns of enhanced respiratory disease associated with Th2 induction. Taken together, these epitopes may be used to improve our understanding of natural and vaccine-induced immunity, and to facilitate the development of T-cell-targeted vaccines that harness pre-existing SARS-CoV-2 immunity.

High ambient glucose induces angiotensin-independent AT-1 receptor activation, leading to increases in proliferation and extracellular matrix accumulation in MES-13 mesangial cells.

Diabetic nephropathy is associated with mesangial ECM (extracellular matrix) accumulation. We have shown that AT-1R [Ang II (angiotensin II) type I receptor] signalling induces ECM proteins via transactivation of PI3K (phosphoinositide 3-kinase) in mesangial cells. In the present study, we examined the mechanisms underlying the effect of high ambient glucose on cell proliferation and ECM expansion in a mesangial context. High glucose induced increases in PI3K activity, proliferation and ECM accumulation in mesangial cells. These effects were abrogated by losartan, an AT-1R antagonist, but not by [Sar1,Thr8]-Ang II (Sar is sarcosine), an inactive analogue of Ang II, or by a neutralizing antibody against Ang I/II. Overexpression of a constitutively active PI3Kalpha or AT-1R alone was sufficient to induce similar changes by high glucose. In contrast, overexpression of an inactive AT-1R lowered the basal levels and rendered the cells non-responsive to high glucose. Moreover, cells overexpressing wild-type AT-1R had enhanced sensitivity to acute Ang II stimulation. These cells, however, did not respond to conditioned medium obtained from mesangial cells cultured in high glucose. We further demonstrated that iAng (intracellular Ang II) can be induced by high glucose but only under certain conditions. Efficient suppression of iAng by short hairpin RNA against angiotensinogen, however, did not affect high glucose-induced effects on MES-13 cells. These results suggest that high ambient glucose induces activation of AT-1R in an Ang II-independent manner to transactivate PI3K, resulting in proliferation and ECM accumulation in mesangial cells.

Beta-adrenergic receptor mediated protection against doxorubicin-induced apoptosis in cardiomyocytes: the impact of high ambient glucose.

Recent studies have demonstrated that the beta2-adrenergic receptor (beta2AR)-Galphai signaling pathway exerts a cardiac antiapoptotic effect. The goals of this study were to determine the intracellular signaling factors involved in beta2AR-mediated protection against doxorubicin-induced apoptosis in H9c2 cardiomyocyte and explore the impact of high ambient glucose on the antiapoptotic effect. Under physiological glucose environment (100 mg/dl), beta2AR stimulation prevented doxorubicin-induced apoptosis, which was attenuated by cotreatment with wortmannin, a phosphoinositide 3-kinase (PI3K) inhibitor, or transfection of a dominant-negative Akt. Inhibition of Src kinase with 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine or cSrc small interfering RNA 32 also attenuated the antiapoptotic effect. Inhibition of platelet-derived growth factor receptor (PDGFR) with AG1296 reversed the beta2AR-induced antiapoptotic effect. Transfection of an active Src cDNA (Y529F) alone was sufficient to render the cells resistant to apoptosis, and the resistance was blocked by wortmannin. Transfection of an active PI3K minigene (iSH2-p110) alone also induced resistance to apoptosis, and the resistance was reversed by an Akt-inhibitor but not by AG1296. High ambient glucose (450 mg/dl) caused two major effects: 1) it significantly reduced betaAR-induced PDGFR phosphorylation, Src kinase activity, and activation of PI3K signaling pathway; and 2) it partially attenuated beta2AR-induced antiapoptotic effect. These data provide in vitro evidence supporting a signaling cascade by which beta2AR exerts a protective effect against doxorubicin-induced apoptosis via sequential involvement of Galphai, Gbetagamma, Src, PDGFR, PI3K, and Akt. High ambient glucose significantly attenuates beta2AR-mediated cardioprotection by suppressing factors involved in this cascade including PDGFR, Src, and PI3K/Akt.

Ontogeny and the effects of corticosteroid pretreatment on aquaporin water channels in the ovine cerebral cortex.

The aim of the present study was to determine the ontogeny and effects of corticosteroid pretreatment on aquaporin 4 (AQP4) channel mRNA and protein expression in the cerebral cortex of sheep during development. A portion of the cerebral cortex was snap-frozen from fetuses of dexamethasone- and placebo-treated ewes at 60%, 80% and 90% of gestation, dexamethasone- and placebo-treated newborn lambs and adult sheep. Cerebral cortical samples were obtained 18 h after the last of four 6 mg dexamethasone or placebo injections were given over 48 h to the ewes and adult sheep. Lambs were treated with 0.01 mg kg(-1) dexamethasone or placebo in the same schedule as the ewes and adult sheep. Amplification of an ovine AQP4 cDNA fragment was accomplished by reverse transcription-polymerase chain reaction using primers based on a homologous bovine sequence. The resulting cDNA was used to determine AQP4 channel mRNA expression by Northern hybridisation using phosphorimaging. The relative abundance of AQP4 mRNA was normalised to the ovine ribosomal gene L32. A portion of the frontal cortex was also analysed for AQP4 protein expression by Western immunoblot. Densitometry was performed and the results expressed as a ratio to an adult brain pool. Aquaporin 4 channel mRNA and protein were detectable as early as at 60% gestation. There were no changes in AQP4 mRNA expression among the fetal, newborn and adult groups or after dexamethasone pretreatment in any age group. The expression of the AQP4 protein was higher (P < 0.05) in fetuses at 80% and 90% of gestation (2.9- and 3.3-fold, respectively), in lambs (3.2-fold) and in adult sheep (3.8-fold) compared with fetuses at 60% of gestation, as well as in adult sheep (1.3-fold) compared with fetuses at 80% of gestation. Dexamethasone pretreatment resulted in decreases (P < 0.05) in AQP4 protein expression in the lambs and adult sheep, but not in the fetal groups. We conclude that: (1) AQP4 mRNA and protein were expressed early in fetal and throughout ovine development; (2) protein, but not mRNA, expression increased between 60% and 80% of gestation and did not differ from adult levels by 90% of gestation; and (3) dexamethasone pretreatment resulted in decreases in AQP4 protein expression in lambs and adult sheep, but not in fetuses. The maturational increases in AQP4 protein expression and dexamethasone-related decreases in expression were post-transcriptional, because changes in AQP4 mRNA expression were not observed.

Ontogeny of phosphoinositide 3-kinase signaling in developing heart: effect of acute beta-adrenergic stimulation.

Signaling pathways underlying transition of cardiomyocyte growth from hyperplasia in fetal/newborn to hypertrophy in postnatal/adult hearts are not well understood. We have shown that beta-adrenergic receptor (beta-AR)-mediated regulation of neonatal cardiomyocyte proliferation involves p70 ribosomal protein S6 kinase (p70S6K). Here we examined the ontogeny of phosphoinositide 3-kinase (PI3K)/p70S6K signaling pathway in rat hearts and investigated the influence of beta-AR on this pathway during development. Cardiac PI3K and p70S6K1 activities were high in the embryonic day 20 fetus, decreased gradually postnatally, and were low in the adult. In contrast, p70S6K2 was barely detectable. Phosphorylation of p70S6K1, Akt, and phosphoinositide-dependent protein kinase 1 were markedly increased in late gestation and early postnatal life but not in adult hearts. Phosphatase and tensin homolog on chromosome 10 (PTEN), a negative regulator of PI3K, was highly expressed in adult hearts but only at low levels and mostly in the phosphorylated (inactivated) form in the fetus. Beta-AR stimulation resulted in increased cardiac p70S6K1 activity only in animals > or = 2 wk old, whereas Akt level was increased in all developmental stages tested. These increases were accompanied by increased Bcl-2 associated death promoter (Ser136) phosphorylation without changes in PTEN level. Thus there is globally high input of cardiac PI3K signaling during the fetal-neonatal transition period. Inactivation of PTEN may in part contribute to the high activity of PI3K signaling, which coincides with the period of high cardiomyocyte proliferation. Beta-AR stimulation activates cardiac p70S6K1 and Akt in postnatal animals and may activate cardiac survival signals. These data provide further evidence for the importance of beta-AR and PI3K signaling in the regulation of cardiac growth during development.

Molecular interactions between glucocorticoid and catecholamine signaling pathways.

To study the mechanism underlying glucocorticoid regulation of the beta(1)-adrenergic receptor (beta(1)AR), we identified a 43-bp region (-1274 to -1232 from the translation start site) that contains a novel glucocorticoid regulatory unit (GRU) that confers glucocorticoid responsiveness. The sequence encompassing the GRU is (5')TAATTA(3'), which is a core-binding motif for the homeodomain proteins; an E-box ((5')CACGTG(3')) binding site for the Myc/Max family proteins, and an overlapping glucocorticoid response element half-site ((5')TGTTCT(3')). We showed that the half-site is critical for GRU-protein interactions, which also require binding of proteins to the E-box and the homeodomain region. Expression of proteins binding to the GRU was shown to be developmentally regulated, being high in embryonic hearts, reduced in newborn hearts, and undetectable in adult hearts. Overexpression of c-myc antisense significantly reduced glucocorticoid responsiveness of the beta(1)AR gene. We further demonstrated that transcriptional regulation of the beta(1)AR gene is closely related to that of the c-myc gene and that the beta(1)AR may be a potential target of c-myc. We conclude that the ovine beta(1)AR gene contains a novel, functional GRU and that the nuclear factors that transactivate through this element may have important developmental implications.