An automated approach to determine antibody endpoint titers for COVID-19 by an enzyme-linked immunosorbent assay.

While a variety of therapeutic options continue to emerge for COVID-19 treatment, convalescent plasma (CP) has been used as a possible treatment option early in the pandemic. One of the most significant challenges with CP therapy, however, both when defining its efficacy and implementing its approach clinically, is accurately and efficiently characterizing an otherwise heterogenous therapeutic treatment. Given current limitations, our goal is to leverage a SARS antibody testing platform with a newly developed automated endpoint titer analysis program to rapidly define SARS-CoV-2 antibody levels in CP donors and hospitalized patients. A newly developed antibody detection platform was used to perform a serial dilution enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (Ig)G, IgM, and IgA SARS-CoV-2 antibodies. Data were then analyzed using commercially available software, GraphPad Prism, or a newly developed program developed in Python called TiterScape, to analyze endpoint titers. Endpoint titer calculations and analysis times were then compared between the two analysis approaches. Serial dilution analysis of SARS-CoV-2 antibody levels revealed a high level of heterogeneity between individuals. Commercial platform analysis required significant time for manual data input and extrapolated endpoint titer values when the last serial dilution was above the endpoint cutoff, occasionally producing erroneously high results. By contrast, TiterScape processed 1008 samples for endpoint titer results in roughly 14 minutes compared with the 8 hours required for the commercial software program analysis. Equally important, results generated by TiterScape and Prism were highly similar, with differences averaging 1.26 ± 0.2 percent (mean ± SD). The pandemic has created unprecedented challenges when seeking to accurately test large numbers of individuals for SARS-CoV-2 antibody levels with a rapid turnaround time. ELISA platforms capable of serial dilution analysis coupled with a highly flexible software interface may provide a useful tool when seeking to define endpoint titers in a high-throughput manner. Immunohematology 2021;37:33-43.While a variety of therapeutic options continue to emerge for COVID-19 treatment, convalescent plasma (CP) has been used as a possible treatment option early in the pandemic. One of the most significant challenges with CP therapy, however, both when defining its efficacy and implementing its approach clinically, is accurately and efficiently characterizing an otherwise heterogenous therapeutic treatment. Given current limitations, our goal is to leverage a SARS antibody testing platform with a newly developed automated endpoint titer analysis program to rapidly define SARS-CoV-2 antibody levels in CP donors and hospitalized patients. A newly developed antibody detection platform was used to perform a serial dilution enzyme-linked immunosorbent assay (ELISA) for immunoglobulin (Ig)G, IgM, and IgA SARS-CoV-2 antibodies. Data were then analyzed using commercially available software, GraphPad Prism, or a newly developed program developed in Python called TiterScape, to analyze endpoint titers. Endpoint titer calculations and analysis times were then compared between the two analysis approaches. Serial dilution analysis of SARS-CoV-2 antibody levels revealed a high level of heterogeneity between individuals. Commercial platform analysis required significant time for manual data input and extrapolated endpoint titer values when the last serial dilution was above the endpoint cutoff, occasionally producing erroneously high results. By contrast, TiterScape processed 1008 samples for endpoint titer results in roughly 14 minutes compared with the 8 hours required for the commercial software program analysis. Equally important, results generated by TiterScape and Prism were highly similar, with differences averaging 1.26 ± 0.2 percent (mean ± SD). The pandemic has created unprecedented challenges when seeking to accurately test large numbers of individuals for SARS-CoV-2 antibody levels with a rapid turnaround time. ELISA platforms capable of serial dilution analysis coupled with a highly flexible software interface may provide a useful tool when seeking to define endpoint titers in a high-throughput manner. Immunohematology 2021;37:33­43.

Activation of sphingosine kinase by muscarinic receptors enhances NO-mediated and attenuates EDHF-mediated vasorelaxation.

Local formation of the sphingomyelin metabolite sphingosine-1-phosphate (S1P) within the vascular wall has been shown to modulate vascular reactivity. In this study we investigated whether sphingosine kinase, the enzyme responsible for S1P synthesis, plays a role in muscarinic receptor-mediated NO production and vascular relaxation in different blood vessel types. For this purpose, sphingosine kinase translocation and sphingolipid-dependent NO-production after muscarinic receptor stimulation were assessed in an endothelial cell line. Furthermore, we used the sphingosine kinase inhibitor N,N-dimethylsphingosine (DMS) to investigate the role of sphingosine kinase in the relaxant responses to the muscarinic agonist methacholine (MCh) in isolated rat aorta and mesenteric arteries. Activation of M(3)-receptors in an endothelial cell line induced a fast translocation of YFP-tagged sphingosine kinase-1 from the cytosol to the plasma membrane. Concomitant NO-production in this cell line was partially inhibited by DMS. Accordingly, in rat aorta the relaxant responses to MCh were attenuated in the presence of DMS, while the responses to the NO-donor sodium nitroprusside were unaltered. In contrast, DMS enhanced the relaxant responses to MCh in mesenteric artery preparations. This effect could also be observed in the presence of NO synthase and cyclooxygenase inhibitors, indicating that sphingosine kinase inhibition specifically enhanced endothelium-derived hyperpolarizing factor-mediated (i.e. non-NO and non-prostacyclin-dependent) relaxation. We conclude that sphingosine kinase differentially regulates vascular tone in different vessel types, enhancing NO-dependent vasorelaxation but counteracting EDHF-dependent vasorelaxation. This observation enhances our understanding of the complex mechanisms by which sphingolipids regulate vascular homeostasis. Moreover, a disturbed regulation of sphingolipid metabolism in the vascular wall may therefore play a role in the aetiology/pathology of disease states characterized by endothelial dysfunction.

Sphingosine kinase-dependent activation of endothelial nitric oxide synthase by angiotensin II.

OBJECTIVE: In addition to their role in programmed cell death, cell survival, and cell growth, sphingolipid metabolites such as ceramide, sphingosine, and sphingosine-1-phosphate have vasoactive properties. Besides their occurrence in blood, they can also be formed locally in the vascular wall itself in response to external stimuli. This study was performed to investigate whether vasoactive compounds modulate sphingolipid metabolism in the vascular wall and how this might contribute to the vascular responses. METHODS AND RESULTS: In isolated rat carotid arteries, the contractile responses to angiotensin II are enhanced by the sphingosine kinase inhibitor dimethylsphingosine. Endothelium removal or NO synthase inhibition by N(omega)-nitro-L-arginine results in a similar enhancement. Angiotensin II concentration-dependently induces NO production in an endothelial cell line, which can be diminished by dimethylsphingosine. Using immunoblotting and intracellular calcium measurements, we demonstrate that this sphingosine kinase-dependent endothelial NO synthase activation is mediated via both phosphatidylinositol 3-kinase/Akt and calcium-dependent pathways. CONCLUSIONS: Angiotensin II induces a sphingosine kinase-dependent activation of endothelial NO synthase, which partially counteracts the contractile responses in isolated artery preparations. This pathway may be of importance under pathological circumstances with reduced NO bioavailability. Moreover, a disturbed sphingolipid metabolism in the vascular wall may lead to reduced NO bioavailability and endothelial dysfunction.

Vascular effects of sphingolipids.

UNLABELLED: The sphingomyelin metabolites ceramide, sphingosine 1-phosphate (S1P) and sphingosylphosphorylcholine (SPC) are emerging modulators of vascular tone. While ceramide appears to act primarily intracellularly, S1P and SPC appear to mainly work via specific receptors, although those for SPC have not yet been defined unequivocally. Each of the sphingomyelin metabolites can induce both vasoconstriction and vasodilatation and, in some cases--ceramide on the one hand, and S1P and SPC on the other hand--have opposite effects on vascular tone. The differences in effects between vessels may relate to the relative roles of endothelial and smooth muscle cells in mediating them, as well as to the distinct expression patterns of S1P receptors among vascular beds and among endothelial and smooth muscle cells. Recent evidence suggests that vascular tone is not only modulated by sphingomyelin metabolites which are exogenously added or reach the vessel wall via the bloodstream but also by those formed locally by cells in the vessel wall. Such local formation can be induced by known vasoactive agents such as angiotensin II and may serve a signalling function. CONCLUSION: We conclude that sphingomyelin metabolites are important endogenous modulators of vascular function, which may contribute to the pathophysiology of some diseases and be targets for therapeutic interventions.

Role of transforming growth factor beta in rat bladder smooth muscle cell proliferation.

Conditions associated with hypertrophy of the urinary bladder have repeatedly been associated with an increased urinary excretion of transforming growth factor (TGF) beta in both rats and patients. Because TGFbeta can have both growth-promoting and -inhibiting effects, we have studied its effects on cell growth and death in primary cultures of rat bladder smooth muscle cells. TGFbeta1, TGFbeta2, or TGFbeta3 did not cause apoptosis, but all three isoforms inhibited DNA synthesis with similar potency (EC(50) of approximately 0.1 ng/ml) and efficacy. Such inhibition was antagonized by a specific TGFbeta receptor antagonist and independent of the presence of serum. Mitogen-activated protein kinases (MAPKs) are involved in the control of cell growth, and all three TGFbeta isoforms inhibited activation of the extracellular signal-regulated kinase, c-Jun NH(2)-terminal kinase, and p38 MAPK subfamilies. Nevertheless, the inhibitory effects of the TGFbeta isoforms on DNA synthesis were not affected by presence of inhibitors of the three MAPK pathways. TGFbeta did not alter cell size as measured by flow cytometry or mitochondrial activity, an integrated measure of cell size and number. We conclude that our data do not support the hypothesis that TGFbeta is a mediator of rat bladder hypertrophy.