Two GnRH peptides have recently been identified in brain extracts of the African catfish, chicken-II GnRH ([His5,Trp7,Tyr8]GnRH, cGnRH-II) and catfish GnRH ([His5,Asn8]GnRH, cfGnRH). Using three experimental approaches, we investigated whether both peptides are involved in the regulation of pituitary gonadotropin secretion. First, the presence of cfGnRH and cGnRH-II in the pituitary was studied by biochemical and immunocytochemical techniques, as GnRH reaches the pituitary via axonal transport in teleost fish. Pituitary extracts contained cfGnRH- and cGnRH-II-immunoreactive material, showing the same HPLC retention times as the respective synthetic GnRH peptides; cfGnRH was present in 37-fold higher amounts than cGnRH-II. Using single and double labeling immunocytochemical techniques, both peptides were localized in the same peptidergic nerve fibers and often within the same secretory granules in the vicinity of the gonadotropes. Second, the two peptides were tested for their capacity to induce an increased secretion of the LH-like gonadotropin-II (GTH-II). In vivo studies showed that both GnRHs released GTH-II, but 100-fold higher cfGnRH than cGnRH-II doses were necessary to induce similar increases in circulating GTH-II levels. In vitro experiments using pituitary tissue fragments in a perifusion system also revealed a clearly higher GTH-II-releasing capacity of cGnRH-II compared to that of cfGnRH. Third, the peptides were tested for their ability to displace [125I]salmon GnRH analog ([D-Arg6,Trp7,Leu8,Pro9-NEt] GnRH, sGnRHa), a high affinity GnRH receptor ligand, from catfish pituitary membrane preparations. Chicken GnRH-II competed with [125I]sGnRHa for pituitary GnRH-binding sites, whereas cfGnRH did so only slightly. The present data show that cGnRH-II is the more potent GTH-II secretagogue, although a role for cfGnRH in the regulation of GTH-II secretion cannot be excluded. The high biological activity of cGnRH-II may be related to the regulation of GTH-II secretion surges, such as those associated with spawning, whereas cfGnRH may be involved in regulating moderate changes in GTH-II plasma levels. The peptides' potency differences appear to be related to their different binding affinities for the pituitary GnRH receptor.

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High-resolution flow cytometers (hFCM) are used for the detection of extracellular vesicles (EV) in various biological fluids. Due to the increased sensitivity of hFCM, new artifacts with the potential of interfering with data interpretation are introduced, such as detection of antibody aggregates. The aim of this study was to investigate the extent of aggregates in labels commonly used for the characterization of EVs by hFCM. Furthermore, we aimed to compare the efficacy of centrifugation and filtering treatments to remove aggregates, as well as to quantify the effect of the treatments in reducing aggregates. For this purpose, we labeled phosphate buffered saline (PBS) with fluorescently conjugated protein labels and antibodies after submitting them to 5, 10, or 30 min centrifugation, filtering or washed filtering. We investigated samples by hFCM and quantified the amount of aggregates found in PBS labeled with untreated and pre-treated labels. We found a varying amount of aggregates in all labels investigated, and further that filtering is most efficient in removing all but the smallest aggregates. Filtering protein labels can reduce the extent of aggregates; however, how much remains depends on the specific labels and their combination. Therefore, it is still necessary to include appropriate controls in a hFCM study of EVs.

Aims: Monocytes/macrophages play a crucial role in the development, progression, and complication of atherosclerosis. In particular, foam cell formation driven by CD36 mediated internalization of oxLDL leads to activation of monocytes and subsequent release of microvesicles (MVs) derived from monocytes (MMVs). Further, pro-inflammatory leukotriene B4 (LTB4) derived from arachidonic acid promotes atherosclerosis through the high-affinity receptor BLTR1. Thus, we aimed to investigate the correlation between different MMV phenotypes (CD14+ MVs) on the one hand, and arachidonic acid and eicosapentaenoic acid contents in different compartments including atherosclerotic plaques, plasma, and granulocytes on the other.Methods and Results: Samples from patients with femoral atherosclerosis and healthy controls were analyzed on an Apogee A60 Micro-PLUS flow cytometer. Platelet-poor plasma was labeled with lactadherin-FITC, anti-CD14-APC, anti-CD36-PE, and anti-BLTR1-AF700. Eicosapentaenoic acid and arachidonic acid content in different compartments in patients were analyzed using gas chromatography. Compared to controls, patients had lower levels of BLTR1+ MVs (p = 0.007), CD14+BLTR1+ MVs (p = 0.007), and CD14+BLTR1+CD36+ MVs (p = 0.001). Further, in patients CD14+ MVs and CD14+CD36+ MVs correlated inversely with arachidonic acid in granulocytes (r = −0.302, p = 0.039 and r = −0.322, p = 0.028, respectively). Moreover, CD14+CD36+ MVs correlated inversely with arachidonic acid in plasma phospholipids in patients (r = −0.315, p = 0.029), and positively with triglyceride in both patients (r = 0.33, p = 0.019) and controls (r = 0.46, p = 0.022).Conclusion: This is the first study of its kind and thus the results are explorative and only indicative. BLTR1+ MVs and CD14+CD36+ MVs has potential as markers of atherosclerosis pathophysiology, but this needs further investigation.

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