The benefit of therapeutic drug monitoring (TDM) of immunosuppressants like sirolimus, everolimus, tacrolimus, and cyclosporine for the clinical outcome of transplant recipients has been shown in ...numerous studies. For the monitoring of cyclosporine, tacrolimus and mycophenolic acid immunoassays are mostly used. However, these have the disadvantage that on one hand the antibodies used cross-react to a varying extent with metabolites and on the other hand different immunosuppressants cannot be measured simultaneously. No commercial immunoassays are available for the quantification of sirolimus and everolimus. In modern immunosuppressive regimens, two or even more different immunosuppressants are combined, which allows a dose reduction of each single component. Analytical laboratories will consequently be challenged with blood samples containing a variety of different immunosuppressants in very low concentrations. Therefore, the high sensitive and specific detection techniques like liquid chromatography/liquid chromatography–mass spectrometry (LC–LC/MS), liquid chromatography/mass spectrometry (LC/MS) and LC/MS–MS seem to be the methods of choice now and in near future. However, the user of these new applications have to be aware of some pitfalls, as well.
The visible excitation and emission wave-lengths of the recently developed fluorescent Ca2+ indicator fluo-3 permit analysis of the intracellular Ca2+ concentration, Ca2+i, in flow cytometry with a ...488-nm argon laser. The role of Ca2+i in human polymorphonuclear leukocyte heterogeneity was investigated in response to formyl-methionyl-leucyl-phenylalanine (fMLP), C5a, and interleukin 8/neutrophil attractant/activation protein 1 (IL-8/NAP-1) by flow cytometry. The Ca2+i changes in different subpopulations within a heterogeneous cell suspension were resolved upon stimulation with fMLP. Using an anti-CD16 phycoerythrin-conjugated antibody and fluo-3 simultaneously, neutrophils affected and nonaffected in Ca2+ mobilization were distinguished in two patients suffering from glycogen storage disease type 1b. In normal neutrophils, a different time course of Ca2+ mobilization of neutrophil subpopulations immediately after stimulation with fMLP was detected. In addition, after stimulation with a low concentration of IL-8/NAP-1 (10(-10) M) two subsets of neutrophils appeared; one of them showed an increase in Ca2+i, while the other did not. These results indicate heterogeneity in the neutrophil signal transduction process involved in Ca2+ mobilization. Therefore, flow cytometric analyses can resolve changes in single-cell Ca2+i distribution patterns, which is important for the understanding of Ca2+i in neutrophil heterogeneous activation processes.
Both IL-1 alpha and IL-1 beta and TNF-alpha induced a time- and dose-dependent release of authentic PGE2 from cultured human glomerular mesangial cells (HMC). This release became significant only ...after a 4- to 6-h lag phase, and was abolished by inhibition of protein synthesis, and was not related to cell proliferation. Combinations of IL-1 and TNF-alpha when added simultaneously to HMC resulted in a dose-dependent synergistic increase in PGE2 production. These stimulatory effects were specifically inhibited by anticytokine antibodies and the synergistic effect required the simultaneous presence of both IL-1 and TNF-alpha. Arachidonic acid (AA) release experiments and measurement of cyclooxygenase activity, revealed that while both were increased by IL-1 beta and TNF-alpha alone (IL-1 beta greater than TNF-alpha), combinations of IL-1 beta and TNF-alpha resulted in only additive increases in AA release and cyclooxygenase activity. Taken together, these data suggest that stimulation of PGE2 in HMC, by combinations of these cytokines, is not rate limited by AA release or cyclooxygenase activation, but may be related to the induction of the distal enzymes controlling specific PG synthesis.
1. Serosally added synthetic endothelin-1 (ET-1) increased short-circuit current (Isc) across isolated muscle-stripped human
colonic mucosa in vitro. Bumetanide inhibited Isc responses, indicating ...that ET-1 stimulates electrogenic Cl- secretion. 2.
In isolated human jejunal mucosa, ET-1 exhibited a concentration-dependent dual action. At low concentrations it induced rapid
increases in Isc and these were inhibited by bumetanide. At a higher concentration (0.1 microM), ET-1 provoked a drastic and
progressive decrease in Isc below the baseline value. 3. Pretreatment with phlorizin or omission of glucose from the Krebs-Ringer
solution at the apical (luminal) side of the jejunal mucosa prevented the decreases in Isc evoked by ET-1, suggesting that
the peptide inhibits the glucose-coupled electrogenic Na+ absorption. Indeed, flux experiments with D-14Cglucose demonstrated
that ET-1 decreases jejunal glucose absorption by approximately 80% within 30 min. 4. Electron microprobe analyses of cryosections
of human jejunum showed that ET-1 (0.1 microM) evokes a significant decrease in intracellular Na+ concentrations of villus
(not crypt) epithelial cells, suggesting that the peptide attenuates apical Na(+)-glucose entry by reducing the activity of
the Na(+)-glucose cotransporter, SGLT1. 5. In the presence of tetrodotoxin (TTX), ET-1-induced Cl- secretion was significantly
reduced, in both human jejunal and colonic mucosa. However, the inhibitory effect on jejunal Na(+)-glucose absorption was
not affected by TTX. 6. ET-1 increases electrogenic Cl- secretion across human intestinal mucosa in vitro. This effect is
mediated in part via the activation of enteric nerves. Responses of the human jejunal mucosa to high ET-1 concentrations exhibit
a second component, namely the rapid inhibition of electrogenic Na(+)-glucose absorption, which might be mediated by an inhibition
of the transport activity of SGLT1. This effect is independent from neuronal mediators. Our results suggest different cellular
action sites for ET-1 in human small and large intestine.
Resident mouse peritoneal macrophages synthesized and released prostaglandins (PGs) when challenged with 12-O-tetradecanoylphorbol 13-acetate (TPA) or 1,2-dioctanoyl-sn-glycerol (DiC8). Both stimuli ...were found to activate Ca2+/phospholipid-dependent protein kinase C (PKC). 1-(5-Isoquinolinesulphonyl)-2-methylpiperazine ('H-7') and D-sphingosine, known to inhibit PKC by different mechanisms, were able to decrease the PKC activity of macrophages in a dose-dependent manner. Addition of either PKC inhibitor decreased PG synthesis and also the release of arachidonic acid (AA) from phospholipids induced by TPA or DiC8. Simultaneously TPA or DiC8 also decreased incorporation of free AA into membrane phospholipids of macrophages. AA incorporation could be restored, however, by pretreatment with the PKC inhibitors. Our results demonstrate an involvement of PKC in the regulation of PG synthesis in mouse peritoneal macrophages and provide further evidence that reacylation of released fatty acids may be an important regulatory step.
This study was designed to analyze the role of postoperative donor cell chimerism for the induction and maintenance of transplantation tolerance in a porcine lung transplantation model.
Left-sided ...single lung transplantation from major histocompatibility mismatched male donors was performed in 27 female minipigs. All received a 28-day course of pharmacologic immunosuppression using various agents, some in combination with preoperative irradiation. Groups for eventual analysis were strictly defined by outcome, that is, pigs with acute rejection before postoperative day 178 (n=16) were allocated into one group, long-term surviving animals (n=11) into the other. Peripheral blood chimerism was monitored by flow cytometry and real-time polymerase chain reaction. Intragraft chimerism was detected from bronchoalveolar lavage fluid (BALF) by fluorescent in situ hybridization.
Blood chimerism peaked 1 hour after transplantation and was significantly higher in the group of long-term survivors at that time. Thereafter chimerism rapidly decreased, but tended to remain higher in long-term survivors. In case of acute rejection donor cells were lost, but remained detectable for up to 36 postoperative months in tolerant animals. In BALF, the percentage of male nuclei was equally high under immunosuppression in both groups. Rejecting animals showed a rapid decrease of Y-bearing cells in BALF after drug withdrawal and an almost complete loss when acute rejection occurred. In tolerant pigs, intragraft chimerism remained detectable throughout the follow-up.
This study demonstrates a clear correlation of donor leukocyte chimerism with long-term allograft survival in a porcine allogeneic lung transplantation model.
Availability of the common precursor arachidonic acid represents the fundamental prerequisite of the cellular eicosanoid synthesis. The amount of free arachidonic acid is regulated not only by ...phospholipases, which liberate this polyunsaturated fatty acid from lipid pools, but also by the reacylating enzyme acylCoA:lysophosphatide acyltransferase. We have previously shown (Goppelt-Strübe, G., C.-F. Körner, G. Hausmann, D. Gemsa, and K. Resch. Control of Prostanoid Synthesis: Role of Reincorporation of Released Precursor Fatty Acids. Prostaglandins 32:373. 1986.) that the organic mercury compound thimerosal in murine peritoneal macrophages inhibits arachidonic acid reincorporation into cellular lipids, thereby leading to an enhanced prostanoid synthesis. In this report we show that the production of leukotriene C4 was also increased after the addition of thimerosal to mouse peritoneal macrophages in a time and dose dependent manner. Concomitantly, thimerosal led to a significant rise of the intracellular calcium concentration as measured by fura-2 fluorescence. Simultaneous addition of thimerosal and indomethacin or exogeneous arachidonic acid to the cells resulted in a synergistic enhancement of leukotriene C4 synthesis. On the other hand, another sulfhydryl group blocking agent, ethacrynic acid, was found to be ineffective in increasing leukotriene C4 levels even in combination with exogeneous arachidonic acid. Thimerosal therefore provides a helpful tool in studying the basic regulatory mechanisms of the cellular leukotriene synthesis.
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