PEGylation of a glycosaminoglycan-binding, dominant-negative CXCL8 mutant retains bioactivity in vitro and in vivo.

We have recently shown that a dominant-negative mutant of CXCL8, dnCXCL8, with increased glycosaminoglycan (GAG) binding affinity and inactivated GPCR signaling function is able to efficiently prevent neutrophil infiltration into murine lungs (Adage et al., 2015). Here we present evidence that chemical PEGylation of dnCXCL8 with 20 kDa and 40 kDa PEG does not significantly interfere with GAG binding affinity, nor does it influence the mutant's disabled chemotaxis function, while it strongly improved bioavailability and serum half-life of the chemokine mutant. In a murine model of lung inflammation, only the 40 kDa PEGylated dnCXCL8 showed a significant reduction of neutrophils in bronchoalveolar lavage (BAL) fluid. In combination with an almost three-fold increase (compared to non-PEGylated dnCXCL8) in plasma half-life after intravenous administration, our results prove that PEGylation of chemokine-derived biologics is an amenable way for the treatment of chronic inflammatory conditions.

Hypothalamic, metabolic, and behavioral responses to pharmacological inhibition of CNS melanocortin signaling in rats.

The CNS melanocortin (MC) system is implicated as a mediator of the central effects of leptin, and reduced activity of the CNS MC system promotes obesity in both rodents and humans. Because activation of CNS MC receptors has direct effects on autonomic outflow and metabolism, we hypothesized that food intake-independent mechanisms contribute to development of obesity induced by pharmacological blockade of MC receptors in the brain and that changes in hypothalamic neuropeptidergic systems known to regulate weight gain [i.e., corticotropin-releasing hormone (CRH), cocaine-amphetamine-related transcript (CART), proopiomelanocortin (POMC), and neuropeptide Y (NPY)] would trigger this effect. Relative to vehicle-treated controls, third intracerebroventricular (i3vt) administration of the MC receptor antagonist SHU9119 to rats for 11 d doubled food and water intake (toward the end of treatment) and increased body weight ( approximately 14%) and fat content ( approximately 90%), hepatic glycogen content ( approximately 40%), and plasma levels of cholesterol ( approximately 48%), insulin ( approximately 259%), glucagon ( approximately 80%), and leptin ( approximately 490%), whereas spontaneous locomotor activity and body temperature were reduced. Pair-feeding of i3vt SHU9119-treated animals to i3vt vehicle-treated controls normalized plasma levels of insulin, glucagon, and hepatic glycogen content, but only partially reversed the elevations of plasma cholesterol ( approximately 31%) and leptin ( approximately 104%) and body fat content ( approximately 27%). Reductions in body temperature and locomotor activity induced by i3vt SHU9119 were not reversed by pair feeding, but rather were more pronounced. None of the effects found can be explained by peripheral action of the compound. The obesity effects occurred despite a lack in neuropeptide expression responses in the neuroanatomical range selected across the arcuate (i.e., CART, POMC, and NPY) and paraventricular (i.e., CRH) hypothalamus. The results indicate that reduced activity of the CNS MC pathway promotes fat deposition via both food intake-dependent and -independent mechanisms.

Hypothalamic histamine release in normal and stressed rats is affected by sex and aging.

Changes in the baseline and in the 40 mM K+-evoked release of histamine from hypothalamus slices were compared in male and female rats aged 2, 6, 12, 18, and 24 months. The baseline release declined in the 12-, 18-, and 24-month males. In contrast, the K+-evoked release remained constant in the males, but in the females it decreased in animals more than 2 months old. The efficiency of the H3 receptors was also determined by measuring the reduction of the K+-evoked release induced by the H3 receptor agonist (R)-alpha-methylhistamine. This substance significantly decreased the amount of HA released in all age groups, except the 24-month-old males. Histamine release was also measured after exposure to a weak electrical stress. In 2- and 6-month-old males, there was a marked reduction of both the baseline and the K+-evoked release, and also of the inhibitory effect of the H3 agonist. There were no changes in the 12- and 18-month age groups, but both releases were enhanced in the 24-month group. In females, electrical stress had no significant effect, except in the youngest age group. Stress-dependent release of plasma corticosterone was decreased in males older than 12 months and in females older than 6 months. These changes gave a good correlation with variation in the H3 receptors. This study, therefore, demonstrates that aging modifies, in a sex-dependent way, the basal and stress-stimulated functions of the hypothalamic presynaptic histaminergic neurons.

N-Acetyltransferase mechanism for alpha-melanocyte stimulating hormone regulation in rat ageing.

alpha-Melanocyte stimulating hormone (alpha-MSH) is a proopiomelanocortin-derived peptide involved in such behavioural activities as arousal, grooming, memory, learning and attention. Because of these effects, alpha-MSH can be considered the 'adaptation neuropeptide'. Two alpha-MSH major forms were described: acetyl alpha-MSH and des-acetyl alpha-MSH. Since the acetylated form of alpha-MSH is biologically significantly more effective than des-acetyl alpha-MSH, we studied the activity of N-acetyltransferase, the enzyme responsible for MSH acetylation, during ageing in rat hippocampus and pituitary. We observed a substantial decrease of enzyme activity during lifetime, suggesting that the lower synthesis of the more efficient acetylated alpha-MSH form can be related to the reduced adaptive capabilities of aged subjects.