Production and utilization of monoclonal antibodies to human/rat corticotrophin-releasing factor-41

Self

Hillhouse

1993

British J Pharmacology

The actions of the following pyrogens: lipopolysaccharide (LPS), polyinosinic:polycytidylic acid (Poly‐I:C), human interleukin (IL)‐1α and IL‐1β, human IL‐6 and rat interferon (INF) on corticotrophin‐releasing factor‐41 (CRF‐41) and prostanglandin E2 (PGE2) release from the intact rat hypothalamus in vitro have been studied. Rat hypothalami were incubated in vitro in an artificial cerebrospinal fluid. Immunoreactive (ir)‐CFR‐41 and PGE2 released into the medium were measured by two‐site enzyme amplified immunometric assay (EAIA) and radioimmunoassay (RIA) respectively. Human IL‐6 (1 to 10,000 iu ml−1) caused a dose‐dependent release of irCRF‐41, rising to a maximal 3–4 fold increase over basal at the highest dose tested. Human IL‐1α (1 to 1000 iu ml−1), human IL‐1β (1 to 1000 iu ml−1), poly‐I:C (10 pg ml−1 to 100 μg ml−1) and rat INF (1 to 10,000 IRu ml−1) all failed to alter irCRF‐41 release. LPS (1 mg ml−1) caused a 35% decrease in irCRF‐41 release; however, over the dose‐range of 0.1 μg ml−1 to 100 μg ml−1, LPS failed to alter irCRF‐41 release. The decreased irCRF‐41 release in response to LPS (1 mg ml−1) was accompanied by a decrease in the subsequent 56 mm KCl stimulation of irCRF‐41. Human IL‐1α and IL‐1β (1000 iu ml−1) were able to stimulate the release of irPGE2 from intact hypothalami, causing a 2 fold increase over basal release. Poly‐I:C (100 μg ml−1), LPS (0.1 μg ml−1 to 1 mg ml−1), rat INF (10,000 IRu ml−1) and human IL‐6 (1 to 10,000 iu ml−1) all failed to alter irPGE2 release. In conclusion, these results suggest that the in vitro release of CRF‐41 and PGE2, in response to pyrogens, are mediated via different cytokines. In view of this it is possible that different cytokines may mediate the temperature, prostaglandin and hypothalamo‐pituitary‐adrenocortical axis activation seen during pyrogenic stimulation in vivo.

Section: Rat Hypothalamus In Vitromentioning

confidence: 99%

Effects of pyrogenic immunomodulators on the release of corticotrophin‐releasing factor‐41 and prostaglandin E₂ from the intact rat hypothalamus in vitro

Self

Hillhouse

1993

British J Pharmacology

“…It has ensued from this study that, as different antisera recognize different substances, most studies using a single antibody to determine irCRF in brain tissue may fail to detect the various molecular forms of the pep tide. The use of more than one antibody to determine irCRF may lead to the identification of differential molec ular forms of the peptide and, as a result, expand the existing knowledge on the anatomical distribution of CRF in the CNS [20][21][22][23]47]. The importance of the 34-41 C-terminal sequence within CRF-1-41, has been demonstrated by Milton et al [47] in their study on the characterization of monoclonal antibodies to rat/human CRF-1-41.…”

Section: Discussionmentioning

confidence: 99%

“…The use of more than one antibody to determine irCRF may lead to the identification of differential molec ular forms of the peptide and, as a result, expand the existing knowledge on the anatomical distribution of CRF in the CNS [20][21][22][23]47]. The importance of the 34-41 C-terminal sequence within CRF-1-41, has been demonstrated by Milton et al [47] in their study on the characterization of monoclonal antibodies to rat/human CRF-1-41. The highly purified monoclonal antibodies were found to bind CRF-21-41 but not CRF-1-33 in vitro, suggesting that the epitope, recognized by the antibodies, contains residues 34-41.…”

Section: Discussionmentioning

confidence: 99%

Structure-Related Effects of CRF and CRF-Derived Peptides: Dissociation of Behavioral, Endocrine and Autonomic Activity

Diamant¹,

Wied²

1993

Neuroendocrinology

Two C-terminal and one N-terminal peptide fragments derived from corticotropin-releasing factor (CRF-1–41), CRF-28–41, CRF-34–41, and CRF-1–8, and the CRF receptor antagonist α-helical CRF-9–41 (αhCRF) were evaluated for their behavioral, endocrine and autonomic nervous effects in rats. To this purpose, three different approaches were used. First, rats were tested in a passive avoidance behavioral (PAB) task after intracerebroventricular (i.c.v.) injection the peptides at five different doses. I.c.v. CRF-1–41 was found to attenuate PAB. The effects of CRF-1–41 on PAB were completely antagonized by i.c.v. pretreatment with αhCRF. When given as a sole treatment, the antagonist produced a bimodal effect on PAB. At low doses, αhCRF tended to facilitate PAB, while high doses of the antagonist significantly attenuated PAB. Both CRF fragments showed behavioral effects similar to CRF-1–41. Of all peptides tested, CRF-34–41 was found to most attenuate PAB at both retention tests. In a second experiment, the behaviorally most potent fragment CRF-34–41 was compared to CRF-1–41 for its adrenocorticotropic activity after iv injection in pentobarbital-anesthetized rats. Treatment with CRF-1–41, in a dose-related fashion, produced a significant rise in plasma ACTH, whereas CRF-34–41 was without effect. Finally, the effect of i.c.v. injected CRF-1–41 and CRF fragments on heart rate (HR) and gross activity was measured in conscious rats in their home cages during a 60-min period, using a wireless telemetry system. Concomitantly, the occurrence of grooming behavior was recorded. During the first 10 min after i.c.v. treatment, of all peptides tested, CRF-34–41 produced the most marked increase in HR, which remained significant only during the first 30 min of recording. CRF-28–41 induced a non significant transient tachycardia. The parent molecule CRF-1–41 also induced an immediate, significant tachycardia but this effect lasted longer than 60 min. The N-terminal CRF-1–8 remained without effect. No significant effects on gross activity were observed with the two short C-terminal peptides, whereas i.c.v. injected CRF-1–41 induced excessive grooming behavior. A significant grooming response was also recorded in rats given CRF-34–41 fragment, but not in those treated with CRF-28–41 or CRF-1–8. The observed structure-related dissociation of endocrine, autonomic and behavioral effects suggests the possible existence of peptide fragments, derived from CRF-1–41 in the central nervous system, with distinct biological activity, acting on different receptors.

“…Immunoglobulin (IgG) fractions were isolated from monoclonal anti-CRF-41 antibody ascites (Milton et al 1990) or control normal mouse serum using caprylic acid (Harlow & Lane, 1988). The IgG was precipitated using ammonium sulphate and after dialysis against phosphate-buffered saline (PBS) the final antibody solutions were concentrated to 10 mg/ml IgG, aliquoted and lyophilized.…”

Section: Caprylic Acid Purification Of Antibodiesmentioning

confidence: 99%

“…Poly(I) poly(C) stimulates a biphasic fever, like endotoxin, and activates the HPA axis in a CRF-41-dependent manner in rabbits (Milton, Hillhouse & Milton, 1992). We have studied the effects of peripherally administered monoclonal anti-CRF-41 antibodies (Milton, Hillhouse, Nicholson, Self & McGregor, 1990) and a CRF-41 antagonist (Rivier, Rivier & Vale, 1984;Fisher. Rivier, Rivier & Brown, 1991) on febrile responses to a submaximal dose of poly(I) poly(C).…”

Section: Introductionmentioning

confidence: 99%

A possible role for endogenous peripheral corticotrophin‐releasing factor‐41 in the febrile response of conscious rabbits.

Hillhouse

The Journal of Physiology

1993

SUMMARY1. The actions of peripheral corticotrophin-releasing factor-41 (CRF-41) on the febrile responses of conscious rabbits induced by peripherally administered polyinosinic . polycytidylic acid (poly(J) . poly(C)) have been studied using a CRF-41 receptor antagonist (a-helical CRF(9-41) and anti-CRF-41 monoclonal antibodies.2. Temperature responses were monitored continuously using rectal thermistor probes. Test substances were administered intravenously (i.v.), or for central CRF-41 antagonism experiments, via an indwelling third ventricle cannula (i.c.v.). Blood samples were taken at time intervals from a marginal ear vein and plasma cortisol levels determined by radioimmunoassay. 6. CRF-41 immunoneutralization after the onset of temperature rises caused an immediate and significant defervescence.7. In conclusion, these results suggest a modulatory pro-pyretic role for endogenous peripheral CRF-41 in the febrile responses to poly(J) poly(C).