Log transformed bacterial counts (CFU/gram) of the right prescapular lymph nodes.

<p>The data was log transformed to accommodate for the large variance. As several lymph nodes had shown no growth, equal to bacterial count = 0, +1 was added to all values to allow for log transformation.</p

Experimental timeline for the study.

<p>Baseline values were established from T-4 until T0 with a sampling interval of 2–3 weeks. After vaccination (T0) until the end of the experiment (T12), the sampling interval was 1 week. Animals were skin tested at T9, challenged with BCG at T9 + 3 days and euthanized at T12. T(x) = time point (week number).</p

<i>In vitro</i> and <i>in vivo</i> effects of kisspeptin antagonists p234, p271, p354, and p356 on GPR54 activation

<div><p>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 <i>in vitro</i>, by determining the intracellular Ca²⁺ response of CHEM1 cells that stably express human GPR54, and to study the <i>in vivo</i> 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 <i>in vivo</i> 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 <i>in vitro</i> nor any effect on basal and kisspeptin-stimulated plasma LH concentration in female dogs.</p></div

Calcium responses of CHEM1-GPR54 cells to human KP10 after incubation with different concentrations of p234, p271, p354 and p356.

<p>The median (n = 3) Ca²⁺ response of CHEM1-GPR54 cells to the addition of 10⁻⁸ M human KP10 and 10⁻¹³–10⁻⁵ M of p234 (a), p271 (b), p354 (c), and p356 (d). The error bars represents the range (min and max). * indicates a significant difference from control (no antagonist).</p

Median (and range) plasma LH concentrations (μg/L).

<p>* = min. after cKP10 administration.</p

The plasma LH concentration during kisspeptin antagonist infusion and single canine KP10 administration.

<p>The median plasma LH concentration (μg/L) during continuous infusion of 50 μg/kg/h of different KP antagonists and the controls. n = 6 dogs per group. At t = 120 min 0.5 μg canine KP10/kg was administered intravenously. The antagonist infusion started at t = 0 min and lasted until t = 180 min (grey bar). The arrow indicates the time of cKP10 administration.</p

FACS analysis of CHEM1-GPR54 cells before and after addition of human KP10.

<p>Representative results of FACS analysis of the calcium flux after addition of different concentrations (10⁻¹³ to 10⁻⁵ M) of human KP10 to CHEM1-GPR54. P4 (0-10S) and P5 (30-60S) were the time frames in which the fluorescence intensity of cells was registered and the means of baseline and stimulated levels were calculated. Human KP10 was added during the gap between P4 and P5, 10–30 seconds after the start of the FACS analysis. The concentrations of human KP10 are indicated in the upper right corner of each plot.</p

Dose response curves of p234, p271, p354 and p356 on CHEM1-GPR54 cells.

<p>Dose response curve of Ca²⁺ responses (median fluorescence; n = 3) of CHEM1-GPR54 cells concentrations ranges of kisspeptin antagonist p234, p271, p354, and p356. The error bars represents the range (min and max). * indicates a significant difference (P<0.05) from control (zero).</p