Analytical Approaches for the Quantitation of Redox-active Pyridine Dinucleotides in Biological Matrices

Some of the main electron carriers in the metabolism are mono- or dinucleotides and they play crucial roles in maintaining a balanced redox homeostasis of cells, and in coupling many anabolic and catabolic reactions. Altered cellular redox status can be an indicator of various metabolic disorders such as obesity, the metabolic syndrome, or type 2 diabetes and of other pathological conditions, which involve oxidative stress, such as cardiovascular diseases. Adequate NAD+/NADH and NADP+/NADPH ratios are fundamental for normal cellular functions, thus accurate measurement of these pyridine dinucleotides is essential in biochemical research. Liquid chromatography coupled to tandem mass spectrometry has become the leading analytical technology in (targeted) state-of-the-art metabolic profiling. Main difficulties that hamper quantification of metabolites are chemical similarities, high polarity, and chemical and biological instability of the molecules to be measured. In this review, some critical steps of studying cellular redox status are described, in particular, different techniques of sample preparation and challenges in chromatographic separation

Simultaneous Quantitative Determination of Different Ceramide and Diacylglycerol Species in Cultured Cells by Using Liquid Chromatography–Electrospray Tandem Mass Spectrometry

A sensitive, reproducible reverse-phased high performance liquid chromatography electrospray tandem mass spectrometry (HPLC-ESI-MS/MS) method with simple sample preparation was developed for the simultaneous determination of a wide range of ceramides, diacylglycerols (DAGs) in cultured cells. Chromatographic separation of the compounds was achieved in a 14-minute run using a C8 column with a gradient elution by methanol and 10 mM ammonium acetate buffer as mobile phase at a flow rate of 0.5 ml/min. Various ceramides, DAGs were detected with a triple quadrupol system in multiple reaction monitoring mode, which is based on a soft positive electrospray ionization. The usual sample preparation process was shortened by the application of pure methanol for the extraction instead of the widely used methanol/chloroform mixture. C17:0 ceramide which does not occur in the cell samples, was used as an internal standard. The sample preparation process was optimized and the methodology was tested on a human hepatocarcinoma cell culture. Our results clearly showed accumulation of some ceramides and DAGs in the cells treated with BSA-conjugated palmitate for 8 hours. Since both ceramides and DAGs are important lipid intermediates and signal messengers, alteration in their cellular levels have major impact on cell functions, and thus our novel analytic method can be widely used in lipotoxicity research. The presented technique can be further developed to measure other intermediates of ceramide synthesis and other derivatives of DAGs as well

Application of Gas Chromatography – Flame Ionization Detection to Study Cellular Incorporation of Dietary Trans Fatty Acids of Medical Importance

Putative health effects of dietary trans fatty acids (TFAs) receive a growing attention; while very little is known about the metabolism of these special food components. In vitro studies carried out in cultured cells provide an efficient and standardizable approach to follow the metabolic fate of TFAs, but it requires suitable techniques for the quantitative measurement of FAs in cell samples. Here, the development and validation of a simple and reliable method for the quantification of a group of relevant FAs by gas chromatography and flame ionization detection is presented. Sample preparation used a fast one-step and chloroform-free process for simultaneous extraction and esterification, and chromatographic separation was achieved in 25 min using a Zebron ZB-88 capillary column. A linear calibration (of R2 >0.99) was obtained in the concentration range of 1-200 µg/mL for each FA. Recovery rate was 82 % for samples of non-esterified FAs and >95 % for complex lipids, such as ceramides, diglycerides and triglycerides. The LOD and LOQ were below 0.5 µg/mL, and a robust method precision was achieved (RSD % was below 6 % for each lipid classes). The present method was also tested on a cultured cell line with or without FA treatment at close to physiological concentration, and the observed changes in the metabolite concentration levels revealed characteristic differences between the metabolism of cis and trans unsaturated FAs

Cellular toxicity of dietary trans fatty acids and its correlation with ceramide and diglyceride accumulation

High fatty acid (FA) levels are deleterious to pancreatic β-cells, largely due to the accumulation of biosynthetic lipid intermediates, such as ceramides and diglycerides, which induce ER stress and apoptosis. Toxicity of palmitate (16:0) and oleate (18:1 cis-Δ9) has been widely investigated, while very little data is available on the cell damages caused by elaidate (18:1 trans-Δ9) and vaccenate (18:1 trans-Δ11), although the potential health effects of these dietary trans fatty acids (TFAs) received great publicity. We compared the effects of these four FAs on cell viability, apoptosis, ER stress, JNK phosphorylation and autophagy as well as on ceramide and diglyceride contents in RINm5F insulinoma cells. Similarly to oleate and unlike palmitate, TFAs reduced cell viability only at higher concentration, and they had mild effects on ER stress, apoptosis and autophagy. Palmitate increased ceramide and diglyceride levels far more than any of the unsaturated fatty acids; however, incorporation of TFAs in ceramides and diglycerides was strikingly more pronounced than that of oleate. This indicates a correlation between the accumulation of lipid intermediates and the severity of cell damage. Our findings reveal important metabolic characteristics of TFAs that might underlie a long term toxicity and hence deserve further investigation

Gonadal androgens are associated with decreased type I interferon production by plasmacytoid dendritic cells and increased IgG titres to BNT162b2 following co-vaccination with live attenuated influenza vaccine in adolescents

mRNA vaccine technologies introduced following the SARS-CoV-2 pandemic have highlighted the need to better understand the interaction of adjuvants and the early innate immune response. Type I interferon (IFN-I) is an integral part of this early innate response that primes several components of the adaptive immune response. Women are widely reported to respond better than men to tri- and quadrivalent influenza vaccines. Plasmacytoid dendritic cells (pDCs) are the primary cell type responsible for IFN-I production, and female pDCs produce more IFN-I than male pDCs since the upstream pattern recognition receptor Toll-like receptor 7 (TLR7) is encoded by X chromosome and is biallelically expressed by up to 30% of female immune cells. Additionally, the TLR7 promoter contains several putative androgen response elements, and androgens have been reported to suppress pDC IFN-I in vitro. Unexpectedly, therefore, we recently observed that male adolescents mount stronger antibody responses to the Pfizer BNT162b2 mRNA vaccine than female adolescents after controlling for natural SARS-CoV-2 infection. We here examined pDC behaviour in this same cohort to determine the impact of IFN-I on anti-spike and anti-receptor-binding domain IgG titres to BNT162b2. Through flow cytometry and least absolute shrinkage and selection operator (LASSO) modelling, we determined that serum-free testosterone was associated with reduced pDC IFN-I, but contrary to the well-described immunosuppressive role for androgens, the most bioactive androgen dihydrotestosterone was associated with increased IgG titres to BNT162b2. Also unexpectedly, we observed that co-vaccination with live attenuated influenza vaccine boosted the magnitude of IgG responses to BNT162b2. Together, these data support a model where systemic IFN-I increases vaccine-mediated immune responses, yet for vaccines with intracellular stages, modulation of the local IFN-I response may alter antigen longevity and consequently improve vaccine-driven immunity

Cellular immunity to SARS-CoV-2 following intrafamilial exposure in seronegative family members

Introduction: Family studies of antiviral immunity provide an opportunity to assess virus-specific immunity in infected and highly exposed individuals, as well as to examine the dynamics of viral infection within families. Transmission of SARS-CoV-2 between family members represented a major route for viral spread during the early stages of the pandemic, due to the nature of SARS-CoV-2 transmission through close contacts. Methods: Here, humoral and cellular immunity is explored in 264 SARS-CoV-2 infected, exposed or unexposed individuals from 81 families in the United Kingdom sampled in the winter of 2020 before widespread vaccination and infection. Results: We describe robust cellular and humoral immunity into COVID-19 convalescence, albeit with marked heterogeneity between families and between individuals. T-cell response magnitude is associated with male sex and older age by multiple linear regression. SARS-CoV-2-specific T-cell responses in seronegative individuals are widespread, particularly in adults and in individuals exposed to SARS-CoV-2 through an infected family member. The magnitude of this response is associated with the number of seropositive family members, with a greater number of seropositive individuals within a family leading to stronger T-cell immunity in seronegative individuals. Discussion: These results support a model whereby exposure to SARS-CoV-2 promotes T-cell immunity in the absence of an antibody response. The source of these seronegative T-cell responses to SARS-CoV-2 has been suggested as cross-reactive immunity to endemic coronaviruses that is expanded upon SARS-CoV-2 exposure. However, in this study, no association between HCoV-specific immunity and seronegative T-cell immunity to SARS-CoV-2 is identified, suggesting that de novo T-cell immunity may be generated in seronegative SARS-CoV-2 exposed individuals

Discordant Impact of HLA on Viral Replicative Capacity and Disease Progression in Pediatric and Adult HIV Infection

HLA class I polymorphism has a major influence on adult HIV disease progression. An important mechanism mediating this effect is the impact on viral replicative capacity (VRC) of the escape mutations selected in response to HLA-restricted CD8+ T-cell responses. Factors that contribute to slow progression in pediatric HIV infection are less well understood. We here investigate the relationship between VRC and disease progression in pediatric infection, and the effect of HLA on VRC and on disease outcome in adult and pediatric infection. Studying a South African cohort of >350 ART-naïve, HIV-infected children and their mothers, we first observed that pediatric disease progression is significantly correlated with VRC. As expected, VRCs in mother-child pairs were strongly correlated (p = 0.004). The impact of the protective HLA alleles, HLA-B*57, HLA-B*58:01 and HLA-B*81:01, resulted in significantly lower VRCs in adults (p<0.0001), but not in children. Similarly, in adults, but not in children, VRCs were significantly higher in subjects expressing the disease-susceptible alleles HLA-B*18:01/45:01/58:02 (p = 0.007). Irrespective of the subject, VRCs were strongly correlated with the number of Gag CD8+ T-cell escape mutants driven by HLA-B*57/58:01/81:01 present in each virus (p = 0.0002). In contrast to the impact of VRC common to progression in adults and children, the HLA effects on disease outcome, that are substantial in adults, are small and statistically insignificant in infected children. These data further highlight the important role that VRC plays both in adult and pediatric progression, and demonstrate that HLA-independent factors, yet to be fully defined, are predominantly responsible for pediatric non-progression

T cell assays differentiate clinical and subclinical SARS-CoV-2 infections from cross-reactive antiviral responses

Identification of protective T cell responses against SARS-CoV-2 requires distinguishing people infected with SARS-CoV-2 from those with cross-reactive immunity to other coronaviruses. Here we show a range of T cell assays that differentially capture immune function to characterise SARS-CoV-2 responses. Strong ex vivo ELISpot and proliferation responses to multiple antigens (including M, NP and ORF3) are found in 168 PCR-confirmed SARS-CoV-2 infected volunteers, but are rare in 119 uninfected volunteers. Highly exposed seronegative healthcare workers with recent COVID-19-compatible illness show T cell response patterns characteristic of infection. By contrast, >90% of convalescent or unexposed people show proliferation and cellular lactate responses to spike subunits S1/S2, indicating pre-existing cross-reactive T cell populations. The detection of T cell responses to SARS-CoV-2 is therefore critically dependent on assay and antigen selection. Memory responses to specific non-spike proteins provide a method to distinguish recent infection from pre-existing immunity in exposed populations

Two doses of SARS-CoV-2 vaccination induce robust immune responses to emerging SARS-CoV-2 variants of concern

The extent to which immune responses to natural infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and immunization with vaccines protect against variants of concern (VOC) is of increasing importance. Accordingly, here we analyse antibodies and T cells of a recently vaccinated, UK cohort, alongside those recovering from natural infection in early 2020. We show that neutralization of the VOC compared to a reference isolate of the original circulating lineage, B, is reduced: more profoundly against B.1.351 than for B.1.1.7, and in responses to infection or a single dose of vaccine than to a second dose of vaccine. Importantly, high magnitude T cell responses are generated after two vaccine doses, with the majority of the T cell response directed against epitopes that are conserved between the prototype isolate B and the VOC. Vaccination is required to generate high potency immune responses to protect against these and other emergent variants