The impact of long-term potentiation (LTP) in nociceptive pathways on somatosensory perception was examined by means of quantitative sensory testing (QST) in the ventral forearm of 12 healthy human subjects. Electrical high-frequency stimulation of the forearm skin (HFS; 5 x 1 s at 100 Hz and 10 x detection threshold) led to an abrupt increase of pain to single electrical test stimuli, which were applied through the same electrode (perceptual LTP +72%, p<0.01). Perceptual LTP outlasted the 1-h observation period. The effects of HFS on somatosensory perception of natural test stimuli in the conditioned skin area were restricted to mechanical submodalities. Subjects exhibited a significant decrease of pain threshold and an increase of pain ratings to suprathreshold pinprick stimuli (p<0.01). In 5 out of 12 subjects (42%) light tactile stimuli led to painful sensations (dynamic mechanical allodynia). Furthermore, a small but significant decrease of threshold to blunt pressure stimuli (p<0.05) was found. In contrast, all thermal modalities comprising cold and warm detection thresholds, cold and heat pain thresholds as well as pain summation (perceptual wind up) remained unaltered. These data show that HFS of peptidergic cutaneous C-fiber afferents predominantly modulates Adelta- and Abeta-fiber mediated somatosensory functions, suggesting that LTP in nociceptive pathways enhances human pain sensitivity via interaction of two afferent pathways (extrinsic sensitization).
VB17(+) TCR dominate in Ni-driven T cell cultures from highly Ni-sensitized patients. Using transfection of TCR from three CD4(+), VB17(+), Ni-specific human T cell clones, we studied their Ni-MHC contacts by site-directed TCR mutation and combination of alpha and ss chains between different TCR. All three TCR exhibited N-nucleotide-determined Arg-Asp motifs in their CDR3-ss sequences. Two of them were specifically restricted to HLA-DR13, while the third one accepted a variety of HLA-DR alleles. The highly similar alpha or ss chains of the DR13-restricted TCR were interchangable without loss of specificity, but alpha or ss chains of other TCR were not tolerated. Mutations of their Arg-Asp motif revealed loss of reactivity upon exchanging Asp for Glu or Ala and of Arg for Ala but not of Arg for Lys or the Ni binding His. Reactivity was also destroyed by mutation of alpha chain position 51, proposed as a general contact site for MHC. Hence, in these two TCR the Arg-Asp motif is clearly involved in contacting Ni-MHC complexes, and close cooperation between alpha and ss chain is required. In contrast, the third TCR retained Ni reactivity upon mutation of alpha chain position 51 or of its ss chain Arg-Asp motif, which rather affected the pattern of DR cross-restriction. Moreover, its alpha chain paired with various ss chains from other, even mouse TCR, irrespective of their specificity, retaining Ni reactivity as well as promiscuous HLA-DR restriction. This preponderance of an alpha chain in defining specificity indicates fundamental differences in Ni interactions of individual TCR and implies that ss chain similarities may not necessarily result from antigen selection.
Calcification in the iliac arteries appears to be a good marker for the risk of leakage after rectal anastomosis. The calcification scoring system is easy to calculate after computed tomography and may aid in patient selection to create a protective ileostomy.
Two-metal binding HIV-1 integrase inhibitors (INIs) are potent inhibitors of HIV-1 in vitro and in patients. We report here for the first time the kinetics of inhibition of integrase-catalyzed strand transfer. First, the IC(50) values for each of six structurally distinct INIs decreased when a preincubation was included: S-1360 (1.3 microM vs 0.12 microM), L-731,988 (130 nM vs 9 nM), L-870,810 (130 nM vs 4 nM), raltegravir (300 nM vs 9 nM), elvitegravir (90 nM vs 6 nM), and GSK364735 (90 nM vs 6 nM). When reactions with these INIs were initiated with integrase, progress curve analyses indicated time-dependent inhibition, which could be fitted to a two-step mechanism of binding. Overall fitted K(i) values matched the IC(50) values measured with a preincubation: S-1360 (0.17 microM), L-731,988 (34 nM), L-870,810 (2.4 nM), raltegravir (10 nM), elvitegravir (4.0 nM), and GSK364735 (2.5 nM). To begin to understand the mechanism for this slow onset of inhibition and its possible impact on drug resistance, studies of resistance mutations were initiated. T66I/M154I exhibited little if any time-dependent inhibition by any of the six INIs, as measured by differences in potency upon preincubation or by progress curve analysis. These data demonstrate that slow binding is a signature of two-metal binding INIs, and that the second slow step is required for full potency. We discuss a possible structural explanation of the second slow step of inhibition and also the relationship between loss of time-dependent inhibition and drug resistance of this important new class of HIV-1 antiretroviral drugs.
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