Expression and purification of recombinant ovine interleukin-1β from Escherichia coli

Seow, Heng Fong; Rothel, J.S.; Wood, P.R.

doi:10.1016/0165-2427(94)90099-x

Cited by 24 publications

(7 citation statements)

References 18 publications

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“…Recombinant ovine IL‐6 protein encoded by the pQE30 vector was generated and purified as previously described (47) and further purified on diethylaminoethyl‐convective interaction media anion‐exchange monolithic resins (BIASeparations, Villach, Austria). The contaminants were bound tightly to the DEAE column, and the IL‐6 proteins were collected in the unbound fraction.…”

Section: Methodsmentioning

confidence: 99%

Anti‐IL‐6 neutralizing antibody modulates blood‐brain barrier function in the ovine fetus

et al. 2015

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Impaired blood-brain barrier function represents an important component of hypoxic-ischemic brain injury in the perinatal period. Proinflammatory cytokines could contribute to ischemia-related blood-brain barrier dysfunction. IL-6 increases vascular endothelial cell monolayer permeability in vitro. However, contributions of IL-6 to blood-brain barrier abnormalities have not been examined in the immature brain in vivo. We generated pharmacologic quantities of ovine-specific neutralizing anti-IL-6 mAbs and systemically infused mAbs into fetal sheep at 126 days of gestation after exposure to brain ischemia. Anti-IL-6 mAbs were measured by ELISA in fetal plasma, cerebral cortex, and cerebrospinal fluid, bloodbrain barrier permeability was quantified using the bloodto-brain transfer constant in brain regions, and IL-6, tight junction proteins, and plasmalemma vesicle protein (PLVAP) were detected by Western immunoblot. Anti-IL-6 mAb infusions resulted in increases in mAb (P < 0.05) in plasma, brain parenchyma, and cerebrospinal fluid and decreases in brain IL-6 protein. Twenty-four hours after ischemia, anti-IL-6 mAb infusions attenuated ischemiarelated increases in blood-brain barrier permeability and modulated tight junction and PLVAP protein expression in fetal brain. We conclude that inhibiting the effects of IL-6 protein with systemic infusions of neutralizing antibodies attenuates ischemia-related increases in blood-brain barrier permeability by inhibiting IL-6 and modulates tight junction proteins after

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Section: Methodsmentioning

confidence: 99%

Anti‐IL‐6 neutralizing antibody modulates blood‐brain barrier function in the ovine fetus

et al. 2015

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“…The bacterial samples were lysed, sonicated, the proteins column-purified, and then sterile filtered to remove any possibility of bacterial contamination as previously described (for IL-1 ␤ [36,37] and IL-6 [38] ). The purity of ovine IL-6 was 95% and that of IL-1 ␤ was 90% as evidenced by SDS-PAGE and Western immunoblot analysis, consistent with that described by McWaters et al [38] and Rothel et al [36] , re-spectively, using the same methods.…”

Section: Purification and Iodination Of Cytokinesmentioning

confidence: 99%

Ovine Proinflammatory Cytokines Cross the Murine Blood-Brain Barrier by a Common Saturable Transport Mechanism

Threlkeld

Lynch

et al. 2010

Neuroimmunomodulation

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port of ovine 131 I-IL-1 ␤ or 131 I-IL-6, respectively, across the BBB. Transport of both ovine 131 I-IL-1 ␤ and 131 I-IL-6 was significantly inhibited by 1 g/mouse of murine IL-1 ␤ or IL-6, respectively. In contrast, 1 g/mouse of unlabeled ovine IL-1 ␤ or IL-6 did not inhibit the transport of murine 131 I-IL-1 ␤ or 131 I-IL-6. Conclusions: Ovine IL-1 ␤ and IL-6 cross the mouse BBB by saturable transport. Inhibition of transport by murine homologs indicates that both species use the same transport mechanisms. Conversely, an inability of ovine cytokines to significantly inhibit the transport of murine cytokines indicates that mouse BBB has a lower affinity for ovine than murine cytokines. Knowledge of species-conserved BBB transport mechanisms may facilitate the development of novel animal models of central nervous system pathogenesis.

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“…Recombinant ovine IL-1b (1.2´10 7 units/mg) and rOvTNF-a (1´10 6 units/mg) were prepared as described by Seow et al 13,14 Before use in vivo, the puri®ed cytokines were passed through a Detoxi-Gel column (Pierce, Rockford, IL, USA) to avoid the possibility of gross endotoxin contamination. Endotoxin levels were below the detection level of a Limulus Amebocyte Lysate assay (Endotoxin Kit; Sigma Chemical Co., St Louis, MO, USA).…”

Section: Recombinant Ovine Cytokinesmentioning

confidence: 99%

The use of recombinant ovine IL‐1β and TNF‐α as natural adjuvants and their physiological effects in vivo

Seow

Lightowlers

et al. 1998

Immunol Cell Biol

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In the present study we have investigated the use of recombinant ovine IL-1beta and TNF-alpha both alone and in combination, as natural adjuvants in vaccination trials in sheep. Initial experiments were conducted to investigate the physiological effects of the cytokines in vivo and determine what dose could be administered without adverse pyrogenic effects. Even at the maximum dose tested (100 microg) the only significant physiological effect was a transient increase in body temperature of approximately 2 degrees C in sheep injected with TNF-alpha. Administration of either cytokine had profound effects on the levels of circulating leucocytes for up to 5 days postinjection. The incorporation of either IL-1beta or TNF-alpha in aqueous or Al(OH)3 vaccine formulations enhanced antibody responses to a recombinant antigen from the cestode parasite Taenia ovis. The addition of IL-1beta to aqueous vaccine formulations increased antibody responses 15-20-fold and in Al(OH)3 formulations by three to six fold. TNF-alpha stimulated 1.5 to six-fold and 2.5 to seven-fold increases in antibody levels in aqueous and Al(OH)3-based formulations, respectively, in a dose-dependent manner. The addition of either cytokine to Quil A or IFA vaccines did not enhance the antibody levels elicited. When 10 microg of both IL-1beta and TNF-alpha were incorporated in the aqueous or Al(OH)3 vaccine formulations, increases of 21-fold and 25-fold, respectively, were observed in antibody levels. The adjuvant activity of IL-1beta and TNF-alpha in combination in the Al(OH)3-based vaccine resulted in antibody levels commensurate with those obtained using Quil A or IFA.

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Expression and purification of recombinant ovine interleukin-1β from Escherichia coli

Cited by 24 publications

References 18 publications

Anti‐IL‐6 neutralizing antibody modulates blood‐brain barrier function in the ovine fetus

Anti‐IL‐6 neutralizing antibody modulates blood‐brain barrier function in the ovine fetus

Ovine Proinflammatory Cytokines Cross the Murine Blood-Brain Barrier by a Common Saturable Transport Mechanism

The use of recombinant ovine IL‐1β and TNF‐α as natural adjuvants and their physiological effects in vivo

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