Point-of-care biomarker assay for rapid multiplexed detection of CRP and IP-10.

Rapid and accurate measurements of immune protein markers are essential for diagnosis and treatment in all clinical settings. The recent pandemic has revealed a stark need for developing new tools and assays that could be rapidly used in diverse settings and provide useful information to clinicians. Here, we describe the development and test application of a novel one-step CRP/IP-10 duplex assay for the LightDeck platform capable of delivering reproducible and accurate measurements in under eight minutes. We used the optimized assay to measure CRP and IP-10 levels in human blood and serum samples from healthy, SARS-CoV-2 (COVID-19) positive, and influenza-like illness (ILI) presenting patients. Our results agreed with previously published analyte levels and enabled us to make statistically significant comparisons relevant to multiple clinical parameters. Our duplex assay is a simple and powerful tool for aiding prognostic decision-making in diverse settings.

Assessing the use of a micro-sampling device for measuring blood protein levels in healthy subjects and COVID-19 patients.

BACKGROUND: Venous phlebotomy performed by trained personnel is critical for patient diagnosis and monitoring of chronic disease, but has limitations in resource-constrained settings, and represents an infection control challenge during outbreaks. Self-collection devices have the potential to shift phlebotomy closer to the point of care, supporting telemedicine strategies and virtual clinical trials. Here we assess a capillary blood micro-sampling device, the Tasso Serum Separator Tube (SST), for measuring blood protein levels in healthy subjects and non-hospitalized COVID-19 patients. METHODS: 57 healthy controls and 56 participants with mild/moderate COVID-19 were recruited at two U.S. military healthcare facilities. Healthy controls donated Tasso SST capillary serum, venous plasma and venous serum samples at multiple time points, while COVID-19 patients donated a single Tasso SST serum sample at enrolment. Concentrations of 17 protein inflammatory biomarkers were measured in all biospecimens by Ella multi-analyte immune-assay. RESULTS: Tasso SST serum protein measurements in healthy control subjects were highly reproducible, but their agreements with matched venous samples varied. Most of the selected proteins, including CRP, Ferritin, IL-6 and PCT, were well-correlated between Tasso SST and venous serum with little sample type bias, but concentrations of D-dimer, IL-1B and IL-1Ra were not. Self-collection at home with delayed sample processing was associated with significant concentrations differences for several analytes compared to supervised, in-clinic collection with rapid processing. Finally, Tasso SST serum protein concentrations were significantly elevated in in non-hospitalized COVID-19 patients compared with healthy controls. CONCLUSIONS: Self-collection of capillary blood with micro-sampling devices provides an attractive alternative to routine phlebotomy. However, concentrations of certain analytes may differ significantly from those in venous samples, and factors including user proficiency, temperature control and time lags between specimen collection and processing need to be considered for their effect on sample quality and reproducibility.

Natural History of Aerosol Induced Lassa Fever in Non­Human Primates.

Lassa virus (LASV), an arenavirus causing Lassa fever, is endemic to West Africa with up to 300,000 cases and between 5000 and 10,000 deaths per year. Rarely seen in the United States, Lassa virus is a CDC category A biological agent inasmuch deliberate aerosol exposure can have high mortality rates compared to naturally acquired infection. With the need for an animal model, specific countermeasures remain elusive as there is no FDA-approved vaccine. This natural history of aerosolized Lassa virus exposure in Macaca fascicularis was studied under continuous telemetric surveillance. The macaque response to challenge was largely analogous to severe human disease with fever, tachycardia, hypotension, and tachypnea. During initial observations, an increase trend of activated monocytes positive for viral glycoprotein was accompanied by lymphocytopenia. Disease uniformly progressed to high viremia followed by low anion gap, alkalosis, anemia, and thrombocytopenia. Hypoproteinemia occurred late in infection followed by increased levels of white blood cells, cytokines, chemokines, and biochemical markers of liver injury. Viral nucleic acids were detected in tissues of three non­survivors at endpoint, but not in the lone survivor. This study provides useful details to benchmark a pivotal model of Lassa fever in support of medical countermeasure development for both endemic disease and traditional biodefense purposes.

Neutralizing Antibodies from Convalescent Chikungunya Virus Patients Can Cross-Neutralize Mayaro and Una Viruses.

Most alphaviruses are mosquito-borne and can cause severe disease in domesticated animals and humans. The most notable recent outbreak in the Americas was the 2014 chikungunya virus (CHIKV) outbreak affecting millions and producing disease highlighted by rash and arthralgia. Chikungunya virus is a member of the Semliki Forest (SF) serocomplex, and before its arrival in the Americas, two other member of the SF complex, Una (UNAV) and Mayaro (MAYV) viruses, were circulating in Central and South America. This study examined whether antibodies from convalescent CHIKV patients could cross-neutralize UNAV and MAYV. Considerable cross-neutralization of both viruses was observed, suggesting that exposure to CHIKV can produce antibodies that may mitigate infection with UNAV or MAYV. Understanding the impact of CHIKV exposure on population susceptibility to other emerging viruses may help predict outbreaks; moreover, identification of cross-reactive immune responses among alphaviruses may lead to the development of vaccines targeting multiple viruses.

Arachidonic Acid Stress Impacts Pneumococcal Fatty Acid Homeostasis.

Free fatty acids hold dual roles during infection, serving to modulate the host immune response while also functioning directly as antimicrobials. Of particular importance are the long chain polyunsaturated fatty acids, which are not commonly found in bacterial organisms, that have been proposed to have antibacterial roles. Arachidonic acid (AA) is a highly abundant long chain polyunsaturated fatty acid and we examined its effect upon Streptococcus pneumoniae. Here, we observed that in a murine model of S. pneumoniae infection the concentration of AA significantly increases in the blood. The impact of AA stress upon the pathogen was then assessed by a combination of biochemical, biophysical and microbiological assays. In vitro bacterial growth and intra-macrophage survival assays revealed that AA has detrimental effects on pneumococcal fitness. Subsequent analyses demonstrated that AA exerts antimicrobial activity via insertion into the pneumococcal membrane, although this did not increase the susceptibility of the bacterium to antibiotic, oxidative or metal ion stress. Transcriptomic profiling showed that AA treatment also resulted in a dramatic down-regulation of the genes involved in fatty acid biosynthesis, in addition to impacts on other metabolic processes, such as carbon-source utilization. Hence, these data reveal that AA has two distinct mechanisms of perturbing the pneumococcal membrane composition. Collectively, this work provides a molecular basis for the antimicrobial contribution of AA to combat pneumococcal infections.

Nutritional regulation of long-chain PUFA biosynthetic genes in rainbow trout (Oncorhynchus mykiss).

Most studies on dietary vegetable oil in rainbow trout (Oncorhynchus mykiss) have been conducted on a background of dietary EPA (20 : 5n-3) and DHA (22 : 6n-3) contained in the fishmeal used as a protein source in aquaculture feed. If dietary EPA and DHA repress their endogenous synthesis from α-linolenic acid (ALA, 18 : 3n-3), then the potential of ALA-containing vegetable oils to maintain tissue EPA and DHA has been underestimated. We examined the effect of individual dietary n-3 PUFA on the expression of the biosynthetic genes required for metabolism of ALA to DHA in rainbow trout. A total of 720 juvenile rainbow trout were allocated to twenty-four experimental tanks and assigned one of eight diets. The effect of dietary ALA, EPA or DHA, in isolation or in combination, on hepatic expression of fatty acyl desaturase (FADS)2a(Δ6), FADS2b(Δ5), elongation of very long-chain fatty acid (ELOVL)5 and ELOVL2 was examined after 3 weeks of dietary intervention. The effect of these diets on liver and muscle phospholipid PUFA composition was also examined. The expression levels of FADS2a(Δ6), ELOVL5 and ELOVL2 were highest when diets were high in ALA, with no added EPA or DHA. Under these conditions ALA was readily converted to tissue DHA. Dietary DHA had the largest and most consistent effect in down-regulating the gene expression of all four genes. The ELOVL5 expression was the least responsive of the four genes to dietary n-3 PUFA changes. These findings should be considered when optimising aquaculture feeds containing vegetable oils and/or fish oil or fishmeal to achieve maximum DHA synthesis.

Rainbow trout (Oncorhynchus mykiss) Elov15 and Elov12 differ in selectivity for elongation of omega-3 docosapentaenoic acid.

The synthesis of the omega-3 long-chain polyunsaturated fatty acids (LCPUFA) eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n--3) from dietary α-linolenic acid (ALA; 18:3n--3) requires three desaturation and three elongation steps in vertebrates. The elongation of EPA to docosapentaenoic acid (DPA; 22:5n-3) can be catalysed by the elongase enzymes Elov15 or Elov12, but further elongation of DPA to 24:5n-3, the penultimate precursor of DHA, is limited to Elov12, at least in mammals. Elov15 enzymes have been characterised from seventeen fish species but Elov12 enzymes have only been characterised in two of these fish. The essentiality of Elov12 for DHA synthesis is unknown in fish. This study is the first to identify an Elov12 in rainbow trout (Oncorhynchus mykiss) and functionally chafacterise the Elovl5 and Elovl2 using a yeast expression system. Elovl5 was active with C18-20 PUFA substrates and not C22 PUFA. In contrast, Elovl2 was active with C20-22 PUFA substrates and not C18 PUFA. Thus, rainbow trout is dependent on Elovl2 for DPA to 24:5n--3 synthesis and ultimately DHA synthesis. The expression of elov15 was significantly higher than elov12 in liver. Elucidating this dependence on Elov12 to elongate DPA and the low elov12 gene expression compared with elovl5 are critical findings in understanding the potential for rainbow trout to synthesize DHA.

Functional characterization of the duck and turkey fatty acyl elongase enzymes ELOVL5 and ELOVL2.

In most Western countries, the consumption of fish is low and insufficient to provide the recommended daily intake of the n-3 (ω-3) long-chain polyunsaturated fatty acids (PUFAs) eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3). Poultry has the potential to be a sustainable source of EPA and DHA if poultry species are capable of synthesizing these n-3 PUFAs from dietary plant-derived α-linolenic acid (ALA; 18:3n-3). In most animals, the elongation of very long-chain fatty acids (ELOVL) enzyme ELOVL2 is essential for conversion of dietary ALA to DHA because only ELOVL2 and not ELOVL5 can elongate docosapentaenoic acid (DPA; 22:5n-3) to 24:5n-3, the precursor of DHA. The chicken is the only poultry species in which elongase enzymes have been functionally characterized, and chicken ELOVL5 had unique DPA-to-24:5n-3 activity, which may enable chickens to synthesize more DHA than other animals. By using a yeast expression system, we examined the duck and turkey elongases, ELOVL2 and ELOVL5, to understand if all poultry species have similar potential to synthesize EPA and DHA. The duck and turkey ELOVL5 enzymes were active with C18-20 PUFAs only. The duck ELOVL2 had a broad substrate specificity with C18-22 PUFAs, whereas the turkey ELOVL2 was active only with EPA and C22 PUFAs. Both duck and turkey ELOVL2 enzymes catalyzed 2 rounds of EPA elongation, with the products being DPA and its elongation product, 24:5n-3. With exogenous DPA, both duck and turkey ELOVL2 synthesized 24:5n-3, with the duck ELOVL2 being more active than the turkey ELOVL2. The reason for the lack of DPA elongation activity by the duck and turkey ELOVL5 enzymes compared with the chicken ELOVL5 could not be elucidated by protein sequence comparisons. By using the elongase enzyme activities only as a predictor of DHA synthesis, ducks may have a similar ability to chickens to convert increasing dietary ALA to DHA.

Molecular basis for differential elongation of omega-3 docosapentaenoic acid by the rat Elovl5 and Elovl2.

Functional characterization of the rat elongases, Elovl5 and Elovl2, has identified that Elovl2 is crucial for omega-3 docosahexaenoic acid (DHA) (22:6n-3) synthesis. While the substrate specificities of the rat elongases had some overlap, only Elovl2 can convert the C22 omega-3 PUFA docosapentaenoic acid (DPA) (22:5n-3) to 24:5n-3, which is the penultimate precursor of DHA. In order to better understand the potential for these elongases to be involved in DHA synthesis, we have examined the molecular reasons for the differences between Elovl5 and Elovl2 in their ability to elongate DPA to 24:5n-3. We identified a region of heterogeneity between Elovl5 and Elovl2 spanning transmembrane domains 6 and 7. Using a yeast expression system, we examined a series of Elovl2/Elovl5 chimeras and point mutations to identify Elovl2 residues within this region which are responsible for DPA substrate specificity. The results indicate that the cysteine at position 217 in Elovl2 and a tryptophan at the equivalent position in Elovl5 explain their differing abilities to elongate DPA to 24:5n-3. Further studies confirmed that Elovl2 C217 is a critical residue for elongation of DPA at the level observed in the native protein. Understanding the ability of elongases to synthesize 24:5n-3 may provide a basis for using sequence data to predict their ability to ultimately support DHA synthesis.

Functional characterization of the chicken fatty acid elongases.

The health benefits of the (n-3) PUFA, EPA, and DHA have created a demand for fish and fish oil, the main sources of these PUFA. Production animals, such as poultry, are potential alternate and sustainable sources of EPA and DHA, provided these fatty acids can be synthesized from plant-derived α-linolenic acid [ALA, 18:3(n-3)]. Because elongases are potential control points in the conversion of ALA to DHA in rats, we examined the chicken elongases, ELOVL2 and ELOVL5, which had not been characterized. ELOVL2 activity was limited to C20-22 PUFA substrates and the major product of ELOVL2 metabolism of EPA was 24:5(n-3). This indicates that ELOVL2 can sequentially elongate EPA to docosapentaenoic acid [DPA, 22:5(n-3)] and then onto 24:5(n-3). ELOVL5 selectivity was broader with elongation of C18-22 PUFA substrates. The ability of chicken ELOVL5 to efficiently synthesize 24:5(n-3) is unique compared with ELOVL5 enzymes from other species. The expression of ELOVL5 was higher than ELOVL2 in livers of broiler chickens and their expression did not change when dietary ALA was increased from 0.6 to 1.3% of dietary energy for 42 d. The expression of both genes was higher than previously seen in rats. The chicken elongase enzymes are unlike those of any species studied to date, because both ELOVL2 and ELOVL5 have the ability to efficiently elongate DPA. In addition, the relative abundance of ELOVL2 and ELOVL5 in the liver suggests that chickens may be able to metabolize more DPA through to 24:5(n-3), the precursor of DHA, compared with other species such as rats.

Fatty acid desaturase 2 promoter mutation is not responsible for Δ6-desaturase deficiency.

Dietary essential polyunsaturated fatty acids (PUFAs) require fatty acid desaturases (FADS) for conversion to long-chain PUFAs (LCPUFAs), which are critical for many aspects of human health. A Δ6-desaturase deficiency in a single patient was attributed to an insertion mutation in the FADS2 promoter. Later population studies have shown this thymidine nucleotide (T) insertion to be a common polymorphism (rs3834458). We examined correlations between rs3834458 variants and fatty acid evidence of FADS2 activity in a cohort of rheumatoid arthritis patients selected for low or nil consumption of n-3 LCPUFA as fish or fish oil. The presence of the T allele was associated with higher FADS2 activity, as indicated by higher conversion of plasma n-3 PUFA to LCPUFA. However, the T-insertion/deletion polymorphism did not affect FADS2 promoter activity in luciferase reporter assays in HepG2 or NIH/3T3 cells. Our results indicate that the polymorphism rs3834458 does not appear to directly affect FADS2 promoter activity and is not responsible for a previously reported Δ6-desaturase deficiency.

Development of a fish cell culture model to investigate the impact of fish oil replacement on lipid peroxidation.

Fish oils are rich in omega-3 long-chain polyunsaturated fatty acids (n-3 LC-PUFA), predominantly 20:5n-3 and 22:6n-3, whereas vegetable oils contain abundant C(18)-PUFA, predominantly 18:3n-3 or 18:2n-6. We hypothesized that replacement of fish oils with vegetable oils would increase the oxidative stability of fish lipids. Here we have used the long established and easily cultivated FHM cell line derived from the freshwater fish species fathead minnow (Pimephales promelas) to test this hypothesis. The FHM cells were readily able to synthesize 20:5n-3 and 24:6n-3 from 18:3n-3 but 22:6n-3 synthesis was negligible. Also, they were readily able to synthesize 20:3n-6 from 18:2n-6 but 20:4n-6 synthesis was negligible. Mitochondrial ß-oxidation was greatest for 18:3n-3 and 20:5n-3 and the rates for 16:0, 18:2n-6, 22:6n-3 and 18:1n-9 were significantly lower. Fatty acid incorporation was predominantly into phospholipids (79-97%) with very little incorporation into neutral lipids. Increasing the fatty acid concentration in the growth medium substantially increased the concentrations of 18:3n-3 and 18:2n-6 in the cell phospholipids but this was not the case for 20:5n-3 or 22:6n-3. When they were subjected to oxidative stress, the FHM cells supplemented with either 20:5n-3 or 22:6n-3 (as compared with 18:3n-3 or saturated fatty acids) exhibited significantly higher levels of thiobarbituric reactive substances (TBARS) indicating higher levels of lipid peroxidation. The results are discussed in relation to the effects of fatty acid unsaturation on the oxidative stability of cellular lipids and the implications for sustainable aquaculture.

Elongase reactions as control points in long-chain polyunsaturated fatty acid synthesis.

BACKGROUND: Δ6-Desaturase (Fads2) is widely regarded as rate-limiting in the conversion of dietary α-linolenic acid (18:3n-3; ALA) to the long-chain omega-3 polyunsaturated fatty acid docosahexaenoic acid (22:6n-3; DHA). However, increasing dietary ALA or the direct Fads2 product, stearidonic acid (18:4n-3; SDA), increases tissue levels of eicosapentaenoic acid (20:5n-3; EPA) and docosapentaenoic acid (22:5n-3; DPA), but not DHA. These observations suggest that one or more control points must exist beyond ALA metabolism by Fads2. One possible control point is a second reaction involving Fads2 itself, since this enzyme catalyses desaturation of 24:5n-3 to 24:6n-3, as well as ALA to SDA. However, metabolism of EPA and DPA both require elongation reactions. This study examined the activities of two elongase enzymes as well as the second reaction of Fads2 in order to concentrate on the metabolism of EPA to DHA. METHODOLOGY/PRINCIPAL FINDINGS: The substrate selectivities, competitive substrate interactions and dose response curves of the rat elongases, Elovl2 and Elovl5 were determined after expression of the enzymes in yeast. The competitive substrate interactions for rat Fads2 were also examined. Rat Elovl2 was active with C(20) and C(22) polyunsaturated fatty acids and this single enzyme catalysed the sequential elongation reactions of EPA→DPA→24:5n-3. The second reaction DPA→24:5n-3 appeared to be saturated at substrate concentrations not saturating for the first reaction EPA→DPA. ALA dose-dependently inhibited Fads2 conversion of 24:5n-3 to 24:6n-3. CONCLUSIONS: The competition between ALA and 24:5n-3 for Fads2 may explain the decrease in DHA levels observed after certain intakes of dietary ALA have been exceeded. In addition, the apparent saturation of the second Elovl2 reaction, DPA→24:5n-3, provides further explanations for the accumulation of DPA when ALA, SDA or EPA is provided in the diet. This study suggests that Elovl2 will be critical in understanding if DHA synthesis can be increased by dietary means.

Cloning and functional characterisation of a fatty acyl elongase from southern bluefin tuna (Thunnus maccoyii).

The synthesis of long chain polyunsaturated fatty acids (LCPUFA), such as eicosapentaenoic acid (EPA; 20:5n-3) and docosahexaenoic acid (DHA; 22:6n-3), involves fatty acyl desaturase and elongase enzymes. The marine fish species southern bluefin tuna (SBT) can accumulate large quantities of omega-3 (n-3) LCPUFA in its flesh but their capacity to synthesize EPA and DHA is uncertain. A cDNA, sbtElovl5, encoding a putative fatty acyl elongase was amplified from SBT liver tissue. The cDNA included an open reading frame (ORF) encoding 294 amino acids. Sequence comparisons and phylogenetic analyses revealed a high level of sequence conservation between sbtElovl5 and fatty acyl elongase sequences from other fish species. Heterologous expression of the sbtElovl5 ORF in Saccharomyces cerevisiae confirmed that it encoded a fatty acyl elongase capable of elongating C(18/20) polyunsaturated fatty acid (PUFA) substrates, but not C(22) PUFA substrates. For the first time in an Elovl5, the substrate competition occurring in nature was investigated. Higher activity towards n-3 PUFA substrates than omega-6 (n-6) PUFA substrates was exhibited, regardless of substrate chain length. The sbtElovl5 preferentially elongated 18:4n-3 and 18:3n-6 rather than 20:5n-3 and 20:4n-6. The sbtElovl5 enzyme also elongated saturated and monounsaturated fatty acids.

Application of a flow cytometric cytotoxicity assay for monitoring cancer vaccine trials.

In this study, we evaluated the applicability of a flow cytometry-based cytotoxicity (FC) assay previously developed by our laboratory, for monitoring cancer vaccine trials. The assay simultaneously measures effector cell degranulation and target cell death. Clinically relevant samples consisted of frozen peripheral blood mononuclear cells (PBMC) from vaccinated melanoma patients with known response to the melanoma peptide g209. Both PBMC and 7 day in vitro-stimulated lymphocyte from patient samples were used as effector cells in the FC assay. Activity against the relevant g209 and the control g280 peptide measured in the FC assay was directly compared with results obtained from the Granzyme B enzyme-linked immunosorbent spot assay and the standard 51Cr-release assay run in tandem. The FC assay detected low or no activity when PBMC were used as effector cells. Using cytotoxic T lymphocytes as effector cells, little or no effector cell degranulation or cytotoxicity was measured in the FC assay in prevaccination samples. After vaccination, an increase in both degranulation and target cell death could be determined when target cells were pulsed with g209. No or low reactivity was found against g280 at any time point. Our findings exhibited excellent correlation between CD107a expression and GrB secretion and also Annexin V binding to target cells and specific lysis measured in the 51Cr-release assay. Results obtained from the FC assay were highly reproducible. Therefore, the FC assay may be applied to vaccine trial monitoring and allows the measurement of effector cell degranulation and target cell death simultaneously in a single sample.