Enzyme Linked Immunosorbent Assay for Quantification of Bovine Interleukin‐8 to Study Infection and Immunity in the Female Genital Tract

Cronin, James G.; Hodges, R M; Pedersen, Sara R; Sheldon, I. Martin

doi:10.1111/aji.12344

Cited by 31 publications

(21 citation statements)

References 24 publications

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“…The concentrations of CXCL8, CXCL1, CXCL2, and CXCL3 were measured by ELISA as described previously [41,42]. The inter-and intraassay coefficients of variation were all less than 10%; the limits of detection were 13 pg/ml for IL1b, 75 pg/ml for IL6, 83 pg/ml for TNF, 200 pg/ml for interferon gamma, 75 pg/ml for CCL2, 65 pg/ml for CXCL8, 200 pg/ml for CXCL1, 100 pg/ml for CXCL2, and 150 pg/ml for CXCL3.…”

Section: Enzyme-linked Immunosorbent Assaymentioning

confidence: 99%

Polarized Epithelial Cells Secrete Interleukin 6 Apically in the Bovine Endometrium1

Healy

Cronin

Sheldon

2015

Biology of Reproduction

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Section: Enzyme-linked Immunosorbent Assaymentioning

confidence: 99%

Polarized Epithelial Cells Secrete Interleukin 6 Apically in the Bovine Endometrium1

Healy

Cronin

Sheldon

2015

Biology of Reproduction

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“…At the end of each experiment, supernatants were collected and stored at −20°C for subsequent analyses, and organ weights were recorded. Concentrations of IL-1β and IL-6 in supernatants were measured in duplicate by ELISA according to the manufacturer's instructions (Bovine IL-1β Screening Set ESS0027, Thermo Fisher Scientific; Bovine IL-6 DuoSet DY8190, R&D Systems, Abingdon, UK); bovine IL-8 was measured by ELISA as described previously (Cronin et al, 2015). The interand intraassay coefficients of variation were all <10%, and the limits of detection were 13 pg/mL for IL-1β, 75 pg/mL for IL-6, and 32 pg/mL for IL-8.…”

mentioning

confidence: 99%

Short communication: Glutamine modulates inflammatory responses to lipopolysaccharide in ex vivo bovine endometrium

Noleto

Saut

Sheldon

2017

Journal of Dairy Science

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Bacteria infect the endometrium lining the uterus of cattle after parturition, and clearance of these microbes depends on a robust innate immune response to bacterial molecules, such as the endotoxin lipopolysaccharide (LPS). Endometrial inflammation is characterized by secretion of the cytokines IL-1β and IL-6 and the chemokine IL-8. However, animals often fail to clear invading bacteria and develop uterine disease if they are in negative energy balance, with reduced abundance of glucose and glutamine, which are substrates for energy in tissues. Depletion of glucose blunts inflammatory responses in the endometrium, but the role of glutamine is not clear. The present study tested the hypothesis that depletion of glutamine compromises inflammatory responses to LPS in endometrial tissue. Ex vivo organ cultures of endometrium were challenged with LPS, and culture supernatants accumulated IL-1β, IL-6, and IL-8, as expected. However, reducing the availability of glutamine in culture medium containing glucose reduced the accumulation of IL-1β, IL-6, and IL-8 by >50%. Surprisingly, in the absence of glucose, supplying increasing amounts of glutamine was not sufficient to augment inflammatory responses to LPS, whereas, in the absence of glutamine, supplying more glucose increased inflammation. Furthermore, inhibiting glycolysis reduced the accumulation of IL-1β, IL-6, and IL-8 by >50%, even when glutamine and glucose were abundant. In conclusion, depletion of glutamine reduces inflammatory responses to LPS in the endometrium, and the activity of glutamine depends on glucose and glycolysis. These data provide mechanistic insights into how negative energy balance may be linked to postpartum uterine disease.

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“…CVM collected from 16 cows from one farm was used to optimize the technique and processed using two different protocols: the standard protocol using reducing agent (DTT) [ 9 , 15 ] and our modified protocol without reducing agent. In the modified protocol, CVM was centrifuged at 3000 x g for 15 min at 4 °C and 500 μl of the upper part was collected and mixed with 1000 μl of sterile PBS in 2 ml eppendorf tubes and vortexed.…”

Section: Methodsmentioning

confidence: 99%

“…These inflammatory biomarkers have been detected in uterine mucus and CVM [ 8 – 10 ]. Due to the physical properties of mucus, processing of CVM with reducing agents is routinely recommended before the analysis of soluble-phase biomarkers [ 4 , 9 , 11 ]. N-acetyl-L-cysteine (NAC) and Dithiothreitol (DTT) are commonly used to homogenize mucus by reducing the disulfide bonds of mucins [ 12 – 14 ].…”

Section: Introductionmentioning

confidence: 99%

Improved detection of biomarkers in cervico-vaginal mucus (CVM) from postpartum cattle

et al. 2018

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BackgroundIn the postpartum cow, early diagnosis of uterine disease is currently problematic due to the lack of reliable, non-invasive diagnostic methods. Cervico-vaginal mucus (CVM) is an easy to collect potentially informative source of biomarkers for the diagnosis and prognosis of uterine disease in cows. Here, we report an improved method for processing CVM from postpartum dairy cows for the measurement of immune biomarkers. CVM samples were collected from the vagina using gloved hand during the first two weeks postpartum and processed with buffer alone or buffer containing different concentrations of the reducing agents recommended in standard protocols: Dithiothriotol (DTT) or N-Acetyl-L-Cysteine (NAC). Total protein was measured using the bicinchoninic acid (BCA) assay; interleukin 6 (IL-6), IL-8 and α1-acid glycoprotein (AGP) were measured by ELISA.ResultsWe found that use of reducing agents to liquefy CVM affects protein yield and the accuracy of biomarker detection. Our improved protocol results in lower protein yields but improved detection of cytokines and chemokines. Using our modified method to measure AGP in CVM we found raised levels of AGP at seven days postpartum in CVM from cows that went on to develop endometritis.ConclusionWe conclude that processing CVM without reducing agents improves detection of biomarkers that reflect uterine health in cattle. We propose that measurement of AGP in CVM during the first week postpartum may identify cows at risk of developing clinical endometritis.

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Enzyme Linked Immunosorbent Assay for Quantification of Bovine Interleukin‐8 to Study Infection and Immunity in the Female Genital Tract

Abstract: A bovine-specific IL-8 ELISA was developed, which accurately measured IL-8 secretion from endometrial cells.

Cited by 31 publications

References 24 publications

Polarized Epithelial Cells Secrete Interleukin 6 Apically in the Bovine Endometrium1

Polarized Epithelial Cells Secrete Interleukin 6 Apically in the Bovine Endometrium1

Short communication: Glutamine modulates inflammatory responses to lipopolysaccharide in ex vivo bovine endometrium

Improved detection of biomarkers in cervico-vaginal mucus (CVM) from postpartum cattle

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