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We have previously shown in patch-clamp experiments on excised outside-out cytoplasmic membrane patches from human macrophages that the activation of a high-conductance Ca2+ - and voltage-dependent potassium channel, the MaxiK channel, is an early step in LPS-induced transmembrane signal transduction in macrophages. MaxiK can be activated by agonistically active LPS, and activation can be completely inhibited by LPS antagonists (e.g. synthetic compound 406) and by anti-CD14 antibodies. Furthermore, by inhibiting MaxiK with the specific MaxiK blocker paxilline, we could show that activation of MaxiK is essential for LPS-induced cytokine production. As shown by RT-PCR, blockade of MaxiK by paxilline also inhibits induction of the mRNA of TNF-α and IL-6. This observation together with the fact that all patch-clamp experiments were done on excised outside-out patches reveal that MaxiK activation is an early step in cell activation by endotoxins. Thus, since cells lacking TLR4 on their surface can also not be activated to produce cytokines, these data allow the conclusion that TLR4 and MaxiK are both essential for activation by LPS and may form a co-operative signaling complex. We have also shown that LBP not only exists as a soluble acute-phase serum protein, but is also incorporated as a transmembrane protein (mLBP) in the cytoplasmic membrane of MNC; in this configuration, it is obviously involved in the binding of endotoxin and its transfer to the transmembrane signaling proteins finally triggering cell activation. Complexation of soluble LBP and LPS in the serum prior to binding of LPS to mLBP, in contrast, leads to neutralization of LPS. Here, we provide evidence from fluorescence resonance energy transfer spectroscopy that endotoxin aggregates are intercalated into reconstituted membranes by mLBP. In addition, cell culture assays and patch-clamp experiments demonstrate that endotoxin activates macrophages and the MaxiK channel in the aggregated, but not in the monomeric, state at similar concentrations. [ABSTRACT FROM PUBLISHER]

We previously showed a clear correlation between the molecular conformation of the lipid A moiety of endotoxin molecules and their cytokine-inducing capacity in mononuclear cells. While conically shaped lipid A moieties exhibit a high agonistic activity, a shift to a more cylindrically shaped lipid A leads to a decrease in agonistic and increase in antagonistic activity of the endotoxin molecules. Here, we show the involvement of a high-conductance Ca2+-activated potassium (MaxiK) channel in LPS signaling in macrophages. Corresponding to their biological activity, endotoxins activate a MaxiK channel as shown in outside-out patch-clamp experiments. LPS antagonists and anti-CD14 antibodies inhibit the LPS-induced activation of the channel. Blocking of the channel by specific channel blockers in macrophage cultures leads to inhibition of cytokine mRNA production. In particular, this result implies that there is no other independent transmembrane signaling pathway operative in macrophages. A shift of the molecular conformation of an a priori antagonistic lipid A from a cylindrical to a conical shape by adding the membrane-active compound chlorpromazine increases the activity of the MaxiK channel and the biological activity of the lipid A. We conclude that the activation of the MaxiK channel is a very early step in LPS-induced signaling in macrophages. [ABSTRACT FROM PUBLISHER]

First-void urine samples from 392 Norwegian military conscripts were investigated for the presence of Chlamydia trachomatis by enzyme immunoassay (EIA) on day 1 and day 5 after collection. Positive samples were subsequently investigated by direct immunofluorescence (IF) microscopy for the presence of chlamydial elementary bodies (EBs) in the urine pellet, and urethral swab material taken from the EIA-positive individuals was cultured. 4.8 % (19/392) of the urine samples were EIA-positive on day 1, and 5.4 % (21/392) were positive on day 5, with a combined total of 6.6 % (26/392). Twenty-four of the 26 urine samples were confirmed as positive on IF microscopy. Urethral swabs were taken from 21 EIA-positive individuals. Six of the swabs were positive on cell culture, whereas nine were positive on IF microscopy of swab material, suggesting that these techniques perform better in symptomatic cases than in male Chlamydia trachomatis carriers. In the urine samples a notable discrepancy in EIA results was seen when the same refrigerated samples were retested on day 5 compared to day 1. This discrepancy was probably due to storage-related factors. [ABSTRACT FROM AUTHOR]