Thromboxane mediation of pulmonary hemodynamic responses after neutralization of heparin by protamine in pigs

Protamine neutralization of heparin is often associated with severe hemodynamic side-effects, including pulmonary hypertension and systemic hypotension. Because prostanoids may be involved, the authors studied the role of arachidonic acid metabolites, especially thromboxane A2, in this process. During anesthesia with enflurane and fentanyl, four groups of pigs were studied: Group 1 (n = 10) received heparin (250 IU/kg), followed by protamine (100 mg) after 15 minutes to neutralize the heparin. The same protocol was used in group 2 (n = 11), except that the thromboxane A2 receptor antagonist BM 13.177 (10 mg/kg) was infused 5 minutes before the protamine. The protocol for group 1 was also used for group 3 (n = 7) except that these animals were pretreated with indomethacin (10 mg/kg). Animals in group 4 (n = 10) were given protamine only (100 mg). Pulmonary artery pressure and pulmonary vascular resistance increased significantly in group 1 after protamine neutralization of heparin. This was accompanied by significant increases in plasma concentrations of the cyclooxygenase products thromboxane B2, 6-keto-prostaglandin F1 alpha, and prostaglandin F2 alpha. Cyclooxygenase products increased to comparable degrees in group 2, but without hemodynamic effects. Leukocyte counts decreased comparably in both groups. Hemodynamic reactions, as well as changes in plasma prostanoid levels were absent in group 3, and group 4, but leukocyte counts were less affected in animals that received protamine alone. The results indicate that the hemodynamic side-effects of protamine are mediated by prostanoids and that thromboxane A2 release is the pivotal step, because side effects were effectively prevented by pretreatment with a thromboxane receptor antagonist.(ABSTRACT TRUNCATED AT 250 WORDS)     

252. Probleme der xenogenen Hämoperfusion isolierter Organe in entfernt stammesverwandten Speciessystemen

Zusammenfassung In einem Modell, bestehend aus einer Rollerpumpe, Wärmeaustauscher, venösem Reservoir and Capillaroxygenator wurden Rattennieren mit Hundevollblut perfundiert. Perfundiert man die Nieren unter konstantem Druck mit 10 ml heparinisiertem Hundevollblut in vitro, so beobachtet man analog zur Ex-vivo-Perfusion einen raschen Anstieg des Strömungswiderstandes. Nach durchschnittlich 25 min kam es zu einem Absinken des Perfusionsvolumens auf unter 0,5 ml/g × min (Kontrolle: homologe Perfusion mit Rattenblut: Perfusionsvolumina um 3 ml/g × min konnten über 60 min aufrechterhalten werden).Befund der xenogenen Perfusion: Makroskopisch: Ödeme, Stauung, Hämorrhagie. Mikroskopisch: Endothelzellennekrosen, insbesondere im Glomerularbereich. Das Modell erlaubte das Hämoperfusat zu varheren: Perfusion mit durch Erhitzen dekomplementierten Hundeserum führte zu einer signifikant verzögerten Abstoßung. Entfernung der Leukocyten and Thrombocyten aus dem Perfusat verstärkte diesen Effekt. Mit einem Perfusat bestehend aus Hundeerythrocyten and hitzedekomplementierten Serum konnten Perfusionsvolumina um 3 ml/g × min erzielt werden, die erst nach 60–90 min Perfusionszeit absinken.Im Serum von Hunden können regelmäßig Antikörper gegen Rattengewebe nachgewiesen werden: Agglutinierende Antikörper gegen Rattenerythrocyten, Leukocyten, Bowie komplementbindende Antikörper gegen Rattenherz, -niere and -leber mit Titerstufen um 1:16–64.Immunfluorescenzuntersuchungen der abgestoßenen Nieren nach Perfusion mit Hundeblut zeigte deutliche Ablagerungen von Hunde-Immunglobulin G sowie Komplement ( 1 c).Nach diesen Ergebnissen muß angenommen werden, daß als ursächlicher Faktor der hyperakuten xenogenen Abstoßungsreaktion die präformierten Antikörper, die zum Zeitpunkt der Transplantation bereits im Empfängerkreislauf zirkulieren, verantwortlich zu machen rind.Vermutlich werden Leukocyten and Thrombocyten sekundär in die ablaufende Antigen-Antikörper-Reaktion mit einbezogen.

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Problems encountered in the perfusion of isolated organs with blood of distantly related species

Summary A model consisting of a roller pump, heat exchanger, venous reservoir and capillary oxygenator was used to perfuse rat kidneys with whole blood from dogs. In kidneys perfused in vitro with 10 ml heparinized whole blood of dogs at a constant pressure, one observed a rapid increase of the circulatory resistance analogous to that observed in ex vivo perfusion. After approximately 25 min the perfusion volume fell to under 0.5 ml/g per min. (A control reading after homologous perfusion with rat blood showed perfusion volumes of 3 ml/g per min even after 60 min.)Findings after xenogenous perfusion: Macroscopic-oedema, congestion, haemorrhage, microscopic-endothelial cell necrosis, particularly in the glomerular region.The model permitted variations in the perfusate:Perfusion with dog serum de-complemented by heating led to a significant delay in the rejection. Removal of leucocytes and thrombocytes from the perfusate increased this effect. With a perfusate consisting of heat-decomplemented serum with dog erythrocytes, perfusion volumes of 3 ml/g per min could be reached, which fell only after perfusion times of 60–90 min.Antibodies against rat tissue can be traced regularly in dog serum. These are agglutinating antibodies against rat erythrocytes, leucocytes, and complementbinding antibodies against rat heart, liver and kidney, with titres of 1:16–64.Immune-fluorescence examination of rejected kidneys after perfusion with dog blood showed definite deposits of dog-immune globulin G, as well as complement(ß 1 c).On the basis of these observations it can be assumed that the causal factor of the hyper-acute rejection reaction must be the presence of pre-formed antibodies already circulating in the bloodstream of the recipient at the time of the transplantation.Leucocytes and thrombocytes are probably involved later in the course of the antigen-antibody-reaction.

     

The role of current product release criteria for identification of human islet preparations suitable for clinical transplantation

BACKGROUND: Alloimmunity, autoimmunity, and nonspecific inflammation are known to be potential determinants for long-term islet survival and insulin independence. Sufficient islet mass is a key determinant. But islet engraftment and posttransplant survival may also depend on functional characteristics of the graft. This study investigated the significance of current product release criteria for the transplantation outcome. METHODS: Fourty five consecutive transplanted human islet preparations and their functional outcomes were analyzed. Islet mass was determined according to standard criteria: purity by light microscopy, viability by dye exclusion and Insulin secretory response to static glucose incubation. Islet graft function was monitored for > or = 1 year. Islet function was defined as full (FF), partial (PF), or nonfunction (NF) based on serum C-peptide levels and insulin independence. RESULTS: All islet grafts displayed primary function. Islet mass [IEQ/kg BW]: 7331.3 +/- 679.7 (FF), 5821.3 +/- 546.7 (PF), 6468.6 +/- 658.5 (NF), (FF vs PF p = .032) Purity [%] 86.9 +/- 3.1 (FF), 76.0 +/- 2.87 (PF), 88.2 +/- 2.3 (NF) (FF vs PF P =.045, PF vs NF, P = 0.01). (4) Viability [%]:89.2 +/- 2 (FF), 86.2 +/- 1.7 (PF), 87.3 +/- 1.8 (NF) (ns). Stimulation index (SI): 20 +/- 6.3 (FF), 80.2 +/- 28.2 (PF), 21.6 +/- 3.5 (NF) (ns) No correlation was observed between SI and any other parameter nor between SI and C-peptide levels. Islet mass significantly correlated with C-peptide levels at 6 and 12 months after transplantation for functioning grafts. CONCLUSIONS: Stringent product release criteria allow identification of islet preparations suitable for clinical transplantation. However, currently used parameters are not predictive of long-term graft function, indicating that further refined quality assessments including apoptosis and resistance to early inflammation, are required to assess the primary engrafted islet mass.     

Successful human islet isolation utilizing recombinant collagenase

The enzymatic dissociation of acinar tissue by collagenase is a substantial step in the isolation of pancreatic islets. Although essential collagenase components have been purified, the variability in the activity of different batches limits long-term reproducibility of isolation success. The utilization of purified recombinant proteases would solve this problem. In the present study, pancreases from multiorgan donors were dissociated by means of digestion-filtration using either Liberase HI (n = 51) or a recombinant collagenase blend (n = 25). No significant differences were found regarding islet yield before and after purification, the percent of exocrine-attached islets, and final purity. However, the ratio between islet equivalents and islet numbers indicated a lesser fragmentation in islets isolated with recombinant collagenase (P < 0.01). In contrast, viability was slightly higher in islets isolated with Liberase (92.3 +/- 0.8 vs. 85.6 +/- 2.9%; P < 0.05). Insulin release during static glucose incubation was not different between experimental groups. Islet transplantation into diabetic nude mice resulted in sustained normoglycemia in either group until the graft was removed. These results demonstrated that viable human islets can be isolated using recombinant collagenase. Final optimization of this enzyme blend would offer continuous reproducibility of isolation success.     

Protein kinase C delta activation and translocation to the nucleus are required for fatty acid-induced apoptosis of insulin-secreting cells

Insulin resistance as well as pancreatic beta-cell failure can be induced by elevated free fatty acid (FFA) levels. We studied the mechanisms of FFA-induced apoptosis in rat and human beta-cells. Chronic treatment with high physiological levels of saturated fatty acids (palmitate and stearate), but not with monounsaturated (palmitoleate and oleate) or polyunsaturated fatty acids (linoleate), triggers apoptosis in approximately 20% of cultured RIN1046-38 cells. Apoptosis restricted to saturated FFAs was also observed in primary cultured human beta-cells, suggesting that this mechanism is potentially relevant in vivo in humans. To further analyze FFA-induced signaling pathways leading to apoptosis, we used RIN1046-38 cells. Apoptosis was accompanied by a rapid (within 15 min) nuclear translocation of protein kinase C (PKC)-delta and subsequent lamin B1 disassembly. This translocation was impaired by the phospholipase C inhibitor U-73122, which also substantially reduced apoptosis. Furthermore, lamin B1 disassembly and apoptosis were decreased by cell transfection with a dominant-negative mutant form of PKC-delta. These data suggest that nuclear translocation and kinase activity of PKC-delta are both necessary for saturated fatty acid-induced apoptosis.     

Molecular impact of MinK on the enantiospecific block of I(Ks) by chromanols

Slowly activating I:(Ks) (KCNQ1/MinK) channels were expressed in Xenopous: oocytes and their sensitivity to chromanols was compared to homomeric KCNQ1 channels. To elucidate the contribution of the ss-subunit MinK on chromanol block, a formerly described chromanol HMR 1556 and its enantiomer S5557 were tested for enantio-specificity in blocking I:(Ks) and KCNQ1 as shown for the single enantiomers of chromanol 293B. Both enantiomers blocked homomeric KCNQ1 channels to a lesser extent than heteromeric I:(Ks) channels. Furthermore, we expressed both WT and mutant MinK subunits to examine the involvement of particular MinK protein regions in channel block by chromanols. Through a broad variety of MinK deletion and point mutants, we could not identify amino acids or regions where sensitivity was abolished or strikingly diminished (>2.5 fold). This could indicate that MinK does not directly take part in chromanol binding but acts allosterically to facilitate drug binding to the principal subunit KCNQ1.     

Endocrine and metabolic response in the human newborn to first feed of breast milk

The hormonal and metabolic response to the first feed of breast milk was studied in 12 infants at 4-6 hours of age. After the feed there was an increase in blood glucose concentration but no changes in the concentrations of lactate, pyruvate, alanine, or ketone bodies. The feed was followed by an increase in the concentrations of plasma insulin, growth hormone, gastrin, and enteroglucagon, but no change in levels of plasma glucagon or gastric inhibitory peptide. Several hormone systems are functionally active at birth and are stimulated by the first feed of milk.     

Imatinib mesylate and nilotinib (AMN107) exhibit high-affinity interaction with ABCG2 on primitive hematopoietic stem cells.

The majority of chronic phase chronic myeloid leukemia (CML) patients treated with the tyrosine kinase inhibitor (TKI) imatinib mesylate maintain durable responses to the drug. However, most patients relapse after withdrawal of imatinib and advanced stage patients often develop drug resistance. As CML is considered a hematopoietic stem cell cancer, it has been postulated that inherent protective mechanisms lead to relapse in patients. The ATP binding-cassette transporters ABCB1 (MDR-1; P-glycoprotein) and ABCG2 are highly expressed on primitive hematopoietic stem cells (HSCs) and have been shown to interact with TKIs. Herein we demonstrate a dose-dependent, reversible inhibition of ABCG2-mediated Hoechst 33342 dye efflux in primary human and murine HSC by both imatinib and nilotinib (AMN107), a novel aminopyrimidine inhibitor of BCR-ABL. ABCG2-transduced K562 cells were protected from imatinib and nilotinib-mediated cell death and from downregulation of P-CRKL. Moreover, photoaffinity labeling revealed interaction of both TKIs with ABCG2 at the substrate binding sites as they compete with the binding of [(125)I] IAAP and also stimulate the transporter's ATPase activity. Therefore, our evidence suggests for the role of ABC transporters in resistance to TKI on primitive HSCs and CML stem cells and provides a rationale how TKI resistance can be overcome in vivo.