In vitro and in vivo effects of kisspeptin antagonists p234, p271, p354, and p356 on GPR54 activation.

Kisspeptins (KPs) and their receptor (GPR54 or KiSS1R) play a key-role in regulation of the hypothalamic-pituitary-gonadal axis and are therefore interesting targets for therapeutic interventions in the field of reproductive endocrinology. As dogs show a rapid and robust LH response after the administration of KP10, they can serve as a good animal model for research concerning KP signaling. The aims of the present study were to test the antagonistic properties of KP analogs p234, p271, p354, and p356 in vitro, by determining the intracellular Ca2+ response of CHEM1 cells that stably express human GPR54, and to study the in vivo effects of these peptides on basal plasma LH concentration and the KP10-induced LH response in female dogs. Exposure of the CHEM1 cells to KP-10 resulted in a clear Ca2+ response. P234, p271, p354, and p356 did not prevent or lower the KP10-induced Ca2+ response. Moreover, the in vivo studies in the dogs showed that none of these supposed antagonists lowered the basal plasma LH concentration and none of the peptides lowered the KP10-induced LH response. In conclusion, p234, p271, p354, and p356 had no antagonistic effects in vitro nor any effect on basal and kisspeptin-stimulated plasma LH concentration in female dogs.

The effects of kisspeptin agonist canine KP-10 and kisspeptin antagonist p271 on plasma LH concentrations during different stages of the estrous cycle and anestrus in the bitch.

Kisspeptin (KP) plays a key role in the regulation of the hypothalamic-pituitary-gonadal axis via the release of GnRH. As normal KP signaling is essential for reproductive function, it could be an interesting new target for therapeutic interventions, e.g., nonsurgical contraception in dogs. The aims of the present study were to investigate the effect of KP-10 administration on plasma LH concentration in different stages of the reproductive cycle and to investigate the suitability of p271 as KP antagonist in the bitch. Two groups of six adult Beagle bitches were used. In one group, plasma LH concentration was determined before (40 and 0 minutes) and 10, 20, 40, and 60 minutes after the intravenous administration of 0.5-µg/kg body weight (BW) canine KP-10. In the other group, the bitches received a continuous intravenous infusion with p271 (50 µg/kg BW/h) for 3 hours, and 0.5-µg/kg BW canine KP-10 was administered intravenously 2 hours after the start of the p271 infusion. Their plasma LH concentration was determined before (-40 and 0 minutes) and 30, 60, 90, 120, 130, 140, 160, and 180 minutes after the start of the p271 infusion. In both groups, the experiments were performed during the follicular phase, the first and second half of the luteal phase, and during anestrus. Canine KP-10 induced an increase of plasma LH concentration during all estrous cycle stages and anestrus. There was no difference in LH response between the two groups. The lowest LH response was seen during the follicular phase and the highest response during anestrus. The area under the curve (AUC) for LH and LH increment in the follicular phase were lower than those in anestrus. The AUC LH and LH increment in the first half of the luteal phase were lower than those in the second half of the luteal phase and anestrus. The AUC LH and LH increment in the second half of the luteal phase were not different from those in anestrus. Continuous administration of the antagonist p271 did not alter basal plasma LH concentration and could not prevent or lower the LH response to KP-10 in any of the cycle stages and anestrus. It can be concluded that the LH response to KP-10 is dependent on estrous cycle stage and that peripheral administrated p271 cannot be used as KP antagonist in the dog. This provides new insight in reproductive endocrinology of the bitch, which is important when KP signaling is considered for therapeutic interventions, such as for estrus induction or nonsurgical contraception in the bitch.

Activation of nucleotide oligomerization domain 2 exacerbates a murine model of proteoglycan-induced arthritis.

In addition to its role in innate immunity, nucleotide oligomerization domain 2 (NOD2) has been shown to play a suppressive role in models of colitis. Notably, mutations in NOD2 cause the inherited granulomatous disease of the joints called Blau syndrome, thereby linking NOD2 with joint disease as well. However, the role of NOD2 in joint inflammation has not been clarified. We demonstrate here that NOD2 is functional within the mouse joint and promotes inflammation, as locally or systemically administered muramyl dipeptide (MDP; the NOD2 agonist) resulted in significant joint inflammation that was abolished in NOD2-deficient mice. We then sought to investigate the role of NOD2 in a mouse model of inflammatory arthritis dependent on adaptive immunity using TCR-transgenic mice whose T cells recognized the dominant epitope of proteoglycan (PG). Mice immunized with PG in the presence of MDP developed a more severe inflammatory arthritis and histopathology within the joints. Antigen-specific activation of splenocytes was enhanced by MDP with respect to IFN-gamma production, which would be consistent with the Th1-mediated disease in vivo. Intriguingly, NOD2 deficiency did not alter the PG-induced arthritis, indicating that NOD2 does not play an essential role in this model of joint disease when it is not activated by MDP. In conclusion, we demonstrate that in a model of inflammatory arthritis dependent on T and B cell priming, NOD2 activation potentiates disease. However, the absence of NOD2 does not alter the course of inflammatory arthritis, in contrast to models of intestinal inflammation.