). At 30 min thereafter, INR declined to £1.3 in 93% of patients. At all postinfusion time points through 48 h, median INR remained between 1.2 and 1.3. Clinical hemostatic efficacy was classified as very good or satisfactory in 42 patients (98%). Prompt and sustained increases in circulating coagulation factors and anticoagulant proteins were observed. One fatal suspected pulmonary embolism in a patient with metastatic cancer was judged to be possibly PCCrelated. Conclusions: PCC treatment serves as an effective rapid hemorrhage control resource in the emergency anticoagulant reversal setting. More widespread availability of PCC is warranted to ensure its benefits in appropriate patients.

Summary. Background: Although fibrinogen concentrate has been available for the treatment of congenital fibrinogen deficiency for years, knowledge of its pharmacokinetics comes from only two small studies. Objectives: To assess the pharmacokinetic (PK) profile, clot integrity and safety of fibrinogen concentrate (human) (FCH) in patients with afibrinogenemia. Patients and methods: A multinational, prospective, open-label, uncontrolled study of patients with afibrinogenemia ‡ 6 years of age was conducted in the USA and Italy. Plasma was collected before and after infusion for PK analyses and evaluation by rotational thromboelastometry of maximum clot firmness (MCF) to assess clot integrity. Safety was assessed on the basis of adverse events and laboratory parameters. Results: After a single dose of 70 mg kg )1 body weight (b.w.)FCH in 14 patients, median incremental in vivo recovery was a 1.7 mg dL )1 increase per mg kg )1 b.w., and median levels were 1.3 g L )1 for fibrinogen activity and antigen 1 h after infusion.Median half-life (t 1/2 ) was 77.1 h for fibrinogen activity and 88.0 h for antigen. Plasma recovery in children < 16 years old was similar to that in adults aged 16 to < 65 years, but the t 1/2 and area under the curve were decreased, with an increased steady-state volume and clearance. MCF increased by a mean of 8.9 mm from baseline to 1 h after infusion of FCH (P < 0.0001). All four adverse events reported were mild, and none was serious or related to study drug. Conclusions: These PK findings confirm a rapid increase in plasma fibrinogen levels after infusion with FCH. Together with the clot integrity and safety data and published data on efficacy, the results support the idea that FCH substitution can restore hemostasis with a good safety profile.

IL-18 is a regulator of NK cell function which utilizes the serine-threonine IL-1R-associated kinase signal transduction pathway and may activate additional not yet characterized signaling pathways. Here we evaluated IL-18-mediated signal transduction using the human NK cell line NK92 as a model. NK92 cells were shown by RT-PCR to express all three IL-18 receptor chains (IL-18R, accessory protein-like chain, IL-18-binding protein). Stimulation by IL-18 strongly enhanced tyrosine phosphorylation of STAT3 and of the mitogen-activated protein kinases (MAPK) p44erk-1and p42erk-2. In contrast, STAT5 was not activated. The cytolytic activity of NK92 against K562 target cells, which was augmented in a dose-dependent manner by IL-18 in the presence of trace amounts of IL-2, was suppressed by the specific inhibitors of MAPK pathways (PD098059 and SB203580). Similarly, the stimulatory effect of IL-18 on IFN-γ protein production, given in conjunction with IL-2, was counteracted by inhibition of MAPK. IL-18 alone failed to stimulate IFN-γ protein production despite inducing expression of IFN-γ mRNA. IL-2 alone stimulated neither IFN-γ mRNA expression nor IFN-γ protein production. IL-18 did not stimulate proliferation of NK92 cells, either alone or in combination with IL-2 or IL-12. Inhibition of the MAPK pathway did not significantly alter the IL-2- and IL-12-induced proliferation of NK92 cells, whereas the Janus kinase/STAT pathway inhibitor AG490 strongly suppressed proliferation. MAPK activation appears to play a prominent role in IL-18 signaling, being involved in transcription and translation of IL-18-induced IFN-γ mRNA and IL-18-induced cytolytic effects. In contrast, proliferation of NK92 cells is not affected by MAPK p44erk-1 and p42erk-2.

Responsiveness of IEC lines to LPS is positively correlated with TLR 4 expression. Strategies targeting TLR 4 expression or TLR 4 mediated signaling may antagonize IEC activation by LPS.

SummaryProthrombin complexconcentrates (PCCs) arewidelyadministeredf or emergencyo ral anticoagulation reversal andf or coagulation defects in liver disease.Pharmacokinetic datamay help to optimize treatment.Theobjectiveofthis study was to characterizethe pharmacokineticsofaPCC (BeriplexP/N) containing coagulation factors II (FII),VII (FVII), IX (FIX)and X(FX) and anticoagulant proteins Ca nd S. Fifteen healthyv olunteersr eceived as ingle rapid 50 IU/kgi nfusiono fP CC and underwent frequent blood sampling until 144 hours (h) after infusion. Coagulationfactors and anticoagulantprotein pharmacokinetic parameterswereestimated by non-linear regression.The mean infusionrate of PCC was 7.9ml/min, equivalent to 196.4 IU/min. By the earliestpost-infusion sampling point at 5minutes(min), KeywordsProthrombin complexc oncentrates, pharmacokinetics, safety, blood coagulationfactors, anticoagulation reversal plasma FIX concentration increasedbyamedian of 73%. Median increasesi nF II, FVII and FX at 5m in were 122%,62% and 158%,respectively. Proteins Cand Salsoincreasedrapidly.The median terminal half-lifeofFIX was16.7 h, FII 59.7 h, FVII 4.2 h and FX 30.7 h. Them edian in-vivor ecoveryo fF IX was1 .57 %/IU/kga nd thato ft he othert hree coagulation factors >2 %/IU/kg. Plasma concentration of thrombogenicity marker D-dimer didnot increase,and therewas no clinical evidence of thrombosis.Through up to 12 weeks follow-up there were no laboratoryfindingsindicating PCC-relatedviralexposure.Rapid PCC infusionp roducedp rompts ustained increasesi nc oagulation factors and anticoagulantp roteins with no clinical evidence of thrombosis or viral transmission.

This in vitro study showed considerable differences between PCCs in terms of coagulation inhibitory capacity and purity.

Expression of IL‐18 in intestinal epithelial cells (IEC) has been implicated in Th1 cell‐mediated chronic intestinal inflammation and anti‐tumor immunity. However, physiological regulatory factors have not been identified. Besides their effects on proliferation and restitution, immunomodulatory functions have been attributed to short chain fatty acids (SCFA). We investigated the effect of SCFA (butyrate, propionate, acetate) on expression of IL‐18 in IEC in vitro and in vivo. Expression of IL‐18 mRNA and protein in human carcinoma‐derived HT‐29 and Caco‐2 cells was analyzed by reverse transcription‐PCR and Western blot. Transcriptional regulation of IL‐18 gene expression was determined by transient transfection of wild‐type and mutated IL‐18 promoter. Further, in vivo expression of IL‐18 in the intestine from butyrate‐treated and untreated mice was assessed by immunohistochemistry. IL‐18 mRNA and the IL‐18 protein were expressed in IEC, while IL‐18 secretion was not observed. Butyrate and acetate increased intracellular IL‐18 content in a time‐ and dose‐dependent fashion. In contrast to proinflammatory stimuli butyrate potently activated the IL‐18 promoter, indicating that IL‐18 is regulated at the transcriptional level by SCFA. Furthermore, a 108‐bp sequence in the proximal region was identified to be essential for IL‐18 promoter activation by butyrate. As proof of principle butyrate effects were confirmed in vivo by demonstration of increased IL‐18 protein expression in IEC from butyrate‐treated mice. In conclusion, SCFAup‐regulate IL‐18 protein expression in IEC, suggesting a potential regulatory contribution of these luminal constituents to T cell mediated inflammatory and neoplastic intestinal conditions.

To characterize a functional assay for circulating fibrinogen based on rotational thrombelastography. Maximum clot firmness was determined by rotational thrombelastography in normal human plasma pool, fibrinogen-deficient plasma pool, normal whole blood, and individual plasma samples from 17 patients with fibrinogen deficiency. Plasma samples spiked with varying concentrations of exogenous fibrinogen were also measured. Results were compared with enzyme-linked immunosorbent assay and Clauss assay. The impact of sample freezing and filtration and use of cytochalasin D were also investigated. Over the tested range of 0-3 mg/ml added exogenous fibrinogen, the maximum clot firmness standard curve for determination of fibrinogen in plasma pools (n = 7) was linear (r2 = 0.97). Maximum clot firmness was highly linearly correlated both with Clauss assay (r2 = 0.93) and enzyme-linked immunosorbent assay (r2 = 0.95). In unspiked plasma samples from individual patients with fibrinogen deficiency, fibrinogen was undetectable by rotational thromboelastography. By all evaluated methods, the response to spiking with fibrinogen in such samples coincided closely in patients with afibrinogenemia and hypofibrinogenemia. In dysfibrinogenemia, smaller Clauss assay responses to spiking were observed, whereas the enzyme-linked immunosorbent assay response was variable. Maximum clot firmness was the only evaluated method of fibrinogen assessment to yield consistent results across all categories of fibrinogen deficiency. These in-vitro results suggest the potential clinical utility of rotational thromboelastography as a versatile method for monitoring the response to fibrinogen concentrate among patients with fibrinogen deficiency. Clinical investigations using rotational thromboelastography after in-vivo fibrinogen administration to patients with congenital fibrinogen deficiency are warranted.