Cytokine and T cell responses in post-chikungunya viral arthritis: A cross-sectional study.

OBJECTIVE: To define the relationship between chronic chikungunya post-viral arthritis disease severity, cytokine response and T cell subsets in order to identify potential targets for therapy. METHODS: Participants with chikungunya arthritis were recruited from Colombia from 2019-2021. Arthritis disease severity was quantified using the Disease Activity Score-28 and an Arthritis-Flare Questionnaire adapted for chikungunya arthritis. Plasma cytokine concentrations (interleukin (IL)-1ß, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12p70, IL-13, interferon-γ and tumor necrosis factor (TNF)) were measured using a Meso Scale Diagnostics assay. Peripheral blood T cell subsets were measured using flow cytometry. RESULTS: Among participants with chikungunya arthritis (N = 158), IL-2 levels and frequency of regulatory T cells (Tregs) were low. Increased arthritis disease activity was associated with higher levels of inflammatory cytokines (IL-6, TNF and CRP) and immunoregulatory cytokine IL-10 (p<0.05). Increased arthritis flare activity was associated with higher Treg frequencies (p<0.05) without affecting T effector (Teff) frequencies, Treg/Teff ratios and Treg subsets. Finally, elevated levels of IL-2 were correlated with increased Treg frequency, percent Tregs out of CD4+ T cells, and Treg subsets expressing immunosuppressive markers, while also correlating with an increased percent Teff out of live lymphocytes (p<0.05). CONCLUSION: Chikungunya arthritis is characterized by increased inflammatory cytokines and deficient IL-2 and Treg responses. Greater levels of IL-2 were associated with improved Treg numbers and immunosuppressive markers. Future research may consider targeting these pathways for therapy.

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.

Clinical profile of children incidentally found to have advanced kidney failure.

INTRODUCTION: No data exist on the epidemiology of children incidentally diagnosed with advanced kidney failure (KF) during evaluation for non-specific symptoms. This is likely related to unrecognized symptoms and signs of CKD. The objective of our study was to evaluate incidentally diagnosed patients with advanced KF requiring long-term kidney replacement therapy (KRT). METHODS: An IRB-approved retrospective chart review of children who started KRT with dialysis (hemo- or peritoneal) was conducted. Included were children with no prior knowledge or diagnosis of underlying kidney disease with chronic kidney disease (CKD) disease stage 4 (GFR 15-29 mL/min/1.73 m2) or 5 (GFR < 15 mL/min/1.73 m2) at initial presentation and started on chronic KRT within 2 months of presentation. RESULTS: Of 177 patients initiating KRT during the study period, 26 (15%) were categorized as incidental advanced KF. This cohort with mean age 12.25 years consisted of 42% males, 54% African Americans included 46% with glomerular, and 54% with non-glomerular etiology for kidney failure. Vomiting (42%) and fatigue (39%) were most common, while growth failure (19%) and hyperkalemia (7%) were less frequent on initial presentation. Anemia (100%), hypertension (96%), hyperparathyroidism (96%), and hyperphosphatemia (92%) were the most frequently seen CKD comorbidities. Chronic KRT was started within 24 h in 62% and within 2 weeks in 88% of the cohort. CONCLUSION: Under-diagnosis of patients with advanced KF is most likely related to milder non-specific clinical symptoms and normal growth in the majority of patients. A higher resolution version of the Graphical abstract is available as Supplementary information.

Manual Palpation vs. Femoral Arterial Doppler Ultrasound for Comparison of Pulse Check Time During Cardiopulmonary Resuscitation in the Emergency Department: A Pilot Study.

BACKGROUND: Manual palpation (MP) is frequently employed for pulse checks, but studies have shown that trained medical personnel have difficulty accurately identifying pulselessness or return of spontaneous circulation (ROSC) using MP. Any delays in identifying pulselessness can lead to significant delays in starting or resuming high-quality chest compressions. OBJECTIVES: This study explored whether femoral arterial Doppler ultrasound (FADU) decreases pulse check duration during cardiopulmonary resuscitation (CPR) compared with MP among patients in the emergency department (ED) receiving CPR directed by emergency medicine physicians who had received minimal additional didactic ultrasound training. METHODS: We performed a prospective observational cohort study from October 2018 to May 2019 at an urban community ED. Using convenience sampling, we enrolled patients arriving at our ED or who decompensated during their ED stay and received CPR. For continuous data, median (interquartile range [IQR]) were calculated, and medians were compared using Kruskal-Wallis test. RESULTS: Fifty-two eligible patients were enrolled and 135 pulse checks via MP and 35 via FADU were recorded. MP observations had a median (IQR) of 11.00 (7.36-15.48) s, whereas FADU had a median (IQR) of 8.98 (5.45-13.85) s. There was a difference between the two medians of 2.02 s (p = 0.05). CONCLUSIONS: In this study, the use of FADU was superior to MP in achieving shorter pulse check times. Further research is needed to confirm the accuracy of FADU for identifying ROSC as well as to determine whether FADU can improve clinical outcomes.

Acute Kidney Injury in Pediatric Acute SARS-CoV-2 Infection and Multisystem Inflammatory Syndrome in Children (MIS-C): Is There a Difference?

Objective: To evaluate the prevalence and factors associated with the risk of acute kidney injury (AKI) in pediatric patients with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and multisystem inflammatory syndrome in children (MIS-C). Study Design: We performed a retrospective chart review of 113 patients with SARS-CoV-2 infection with or without MIS-C admitted at Children's Hospital of Michigan (CHM) from March to August 2020. Patient demographic details, laboratory data, imaging studies, echocardiography reports, and treatment data were collected. Results: Of the 92 patients included in the final analysis, 22 (24%) developed AKI with 8/22 (36%) developing stage 3 AKI. The prevalence of AKI was much higher in patients with MIS-C 15/28 (54%) vs. those with acute SARS-CoV-2 infection 7/64 (11%), (p < 0.001). Overall, when compared to patients without AKI, patients with AKI were older in age (11 vs. 6.5 years, p = 0.007), African American (86 vs. 58%, p = 0.028), had MIS-C diagnosis (68 vs. 19%, p < 0.001), required ICU admission (91 vs. 20%, p < 0.001), had cardiac dysfunction (63 vs. 16%, p < 0.001), required inotropic support (59 vs. 6%, p < 0.001) and had a greater elevation in inflammatory markers. In a multivariate analysis, requirement of inotropes [Odds Ratio (OR)-22.8, p < 0.001], African American race (OR-8.8, p = 0.023) and MIS-C diagnosis (OR-5.3, p = 0.013) were the most significant predictors for AKI. All patients had recovery of kidney function, and none required kidney replacement therapy. Conclusion: Children with acute SARS-CoV-2 infection and MIS-C are at risk for AKI, with the risk being significantly greater with MIS-C. The pathogenesis of AKI in acute SARS-CoV-2 infection appears to be a combination of both renal hypo-perfusion and direct renal parenchymal damage whereas in MIS-C, the renal injury appears to be predominantly pre-renal from cardiac dysfunction and capillary leak from a hyperinflammatory state. These factors should be considered by clinicians caring for these children with a special focus on renal protective strategies to aid in recovery and prevent additional injury to this high-risk subgroup.

Intraperitoneal vancomycin treatment of multifocal methicillin-resistant Staphylococcus aureus osteomyelitis in a patient on peritoneal dialysis.

PURPOSE: We report the case of a 2-year-old girl with end-stage renal disease managed by peritoneal dialysis (PD) who developed methicillin-resistant staphylococcal osteomyelitis of the left shoulder and was successfully treated with intraperitoneal (IP) administration of vancomycin for 2 weeks followed by oral clindamycin therapy. SUMMARY: The patient was hospitalized with tactile fever and a 3-day history of worsening fussiness. Radiography of the left shoulder showed findings indicative of osteomyelitis. Vancomycin was administered via central venous line for 3 days, during which time the patient underwent PD 24 hours a day. After magnetic resonance imaging revealed proximal humeral osteomyelitis, septic arthritis of the shoulder joint, and osteomyelitis of the scapula, the patient underwent incision and drainage of the left shoulder joint. Both blood and joint drainage cultures grew methicillin-resistant Staphylococcus aureus that was sensitive to vancomycin. The patient's central venous catheter was removed on hospital day 4; due to difficulties with peripheral i.v. access and a desire to avoid placing a peripherally inserted central venous catheter, vancomycin administration was changed to the IP route, with vancomycin added to the PD fluid. During IP treatment, serum vancomycin levels were maintained at 13.5 to 18.5 mg/L, and the calculated ratio of vancomycin area under the curve to minimum inhibitory concentration was maintained above 400. After completing a 14-day course of IP vancomycin therapy, the patient was switched to oral clindamycin, with subsequent complete resolution of osteomyelitis. CONCLUSION: IP vancomycin was effective for treatment of invasive S. aureus infection in this case. This approach should be considered in patients undergoing PD for whom peripheral i.v. access options are limited and/or not preferred.

Case Report: Acute Thrombotic Microangiopathy in a Patient with STING-Associated Vasculopathy with Onset in Infancy (SAVI).

Stimulator of interferon genes (STING)-associated vasculopathy with onset in infancy (SAVI) is a rare disorder that is associated with extensive inflammation throughout the body due to a high interferon state. Common clinical manifestations of this disorder include chronic lung disease, digital necrosis, recurrent low-grade fevers, and inflammatory skin lesions. However, renal involvement in patients with SAVI has been sparsely documented. We describe a unique case of pediatric SAVI associated with thrombotic microangiopathy (TMA), collapsing focal segmental glomerulosclerosis, interstitial lung disease (from SAVI involvement), and chronic kidney disease. This patient had a substantial hospital course where he developed renal failure. Extensive studies were conducted to exclude all other causes, including infection and possible drug side effects. Ultimately, immunologic evaluation demonstrated normal complement studies, a low ADAMTS13, and presence of ADAMTS13 inhibitor. There was also evidence of thrombocytopenia and schistocytes on peripheral blood smear. Subsequently, the patient was diagnosed with TMA and he was treated with fresh frozen plasma. Repeat immunologic studies confirmed that the TMA had resolved. In addition to describing a novel association between TMA and SAVI, this case also illustrates the challenges associated with optimizing treatment regimens and the importance of clinical vigilance for atypical complications that may arise in patients with SAVI.

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.

Glucopyranosyl lipid adjuvant enhances immune response to Ebola virus-like particle vaccine in mice.

The identification of adjuvants that promote lasting antigen-specific immunity and augment vaccine efficacy are integral to the development of new protein-based vaccines. The Ebola virus-like particle (VLP) vaccine expressing Ebola virus glycoprotein (GP) and matrix protein (VP40) was used in this study to evaluate the ability of TLR4 agonist glucopyranosyl lipid adjuvant (GLA) formulated in a stable emulsion (SE) to enhance immunogenicity and promote durable protection against mouse-adapted Ebola virus (ma-EBOV). Antibody responses and Ebola-specific T cell responses were evaluated post vaccination. Survival analysis after lethal ma-EBOV challenge was performed 4 weeks and 22 weeks following final vaccination. GLA-SE enhanced EBOV-specific immunity and resulted in long-term protection against challenge with ma-EBOV infection in a mouse model. Specifically, GLA-SE elicited Th1-skewed antibodies and promoted the generation of EBOV GP-specific polyfunctional T cells. These results provide further support for the utility of TLR4 activating GLA-SE-adjuvanted vaccines.

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.

Chikungunya Arthritis Mechanisms in the Americas: A Cross-Sectional Analysis of Chikungunya Arthritis Patients Twenty-Two Months After Infection Demonstrating No Detectable Viral Persistence in Synovial Fluid.

OBJECTIVE: To determine if chikungunya virus persists in synovial fluid after infection, potentially acting as a causative mechanism of persistent arthritis. METHODS: We conducted a cross-sectional study of 38 Colombian participants with clinical chikungunya virus infection during the 2014-2015 epidemic who reported chronic arthritis and 10 location-matched controls without chikungunya virus or arthritis. Prior chikungunya virus infection status was serologically confirmed, and the presence of synovial fluid chikungunya virus, viral RNA, and viral proteins was determined by viral culture, quantitative reverse transcription-polymerase chain reaction (qRT-PCR), and mass spectrometry, respectively. Biomarkers were assessed by multiplex analysis. RESULTS: Patients with serologically confirmed chikungunya arthritis (33 of 38 [87%]) were predominantly female (82%) and African Colombian (55%) or white Colombian (33%), with moderate disease activity (mean ± SD Disease Activity Score in 28 joints 4.52 ± 0.77) a median of 22 months after infection (interquartile range 21-23 months). Initial symptoms of chikungunya virus infection included joint pain (97%), swelling (97%), stiffness (91%), and fever (91%). The most commonly affected joints were the knees (87%), elbows (76%), wrists (75%), ankles (56%), fingers (56%), and toes (56%). Synovial fluid samples from all patients with chikungunya arthritis were negative for chikungunya virus on qRT-PCR, showed no viral proteins on mass spectrometry, and cultures were negative. Case and control plasma cytokine and chemokine concentrations did not differ significantly. CONCLUSION: This is one of the largest observational studies involving analysis of the synovial fluid of chikungunya arthritis patients. Synovial fluid analysis revealed no detectable chikungunya virus. This finding suggests that chikungunya virus may cause arthritis through induction of potential host autoimmunity, suggesting a role for immunomodulating agents in the treatment of chikungunya arthritis, or that low-level viral persistence exists in synovial tissue only and is undetectable in synovial fluid.

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.

Prevalence and correlates of a lifetime cannabis use disorder among pregnant former tobacco smokers.

BACKGROUND: Following tobacco and alcohol, cannabis is the most commonly used substance during pregnancy. Given the high prevalence of concurrent cannabis and tobacco use as well as the health consequences associated with prenatal substance use, we sought to document the relative contributions of psychosocial and psychiatric factors commonly associated with cannabis use in predicting a lifetime cannabis use disorder (CUD) among women who had quit smoking tobacco as a result of pregnancy. METHODS: Pregnant former tobacco smokers (n=273) enrolled in a larger randomized controlled trial for postpartum tobacco relapse prevention completed semi-structured psychiatric interviews and self-reported demographic, pregnancy, health, psychosocial, and tobacco use factors during their third trimester of pregnancy. RESULTS: In total, 14% (n=38) of women met criteria for a lifetime CUD. The strongest predictors of a lifetime CUD were a history of having multiple psychiatric disorders (OR=36.44; 95% CI=5.03-264.27; p<0.001) followed by a lifetime alcohol use disorder (OR=3.54; 95% CI=1.27-9.87; p<0.05). In addition, more frequent attempts to quit smoking tobacco (OR=1.12; 95% CI=1.01-1.25; p<0.05) and lower self-efficacy about weight management after quitting smoking tobacco (OR=0.78; 95% CI=0.62-0.97; p<0.05) also were significantly associated with a lifetime CUD. CONCLUSIONS: Women with a history of both cannabis and tobacco dependence may represent a subset of women who need more specialized treatment during the perinatal period to improve substance use outcomes.

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.