Coagulopathy associated with massive cell salvage transfusion following aortic surgery.

Cell saver blood is used within the peri-operative setting of cardiothoracic surgery to reduce the need for transfusion of allogenic blood products. Several meta-analyses have proven a significant decrease in allogenic transfusion with the use of cell salvage techniques. Washing of red cells by the cell saver and subsequent transfusion of suspended red cells can occasionally cause coagulopathy, particularly when using high concentration heparin saline to wash the spilled blood. We present the case of a 74-year-old female who underwent complicated aortic surgery and was transfused large volumes of cell-saved blood due to post-operative bleeding, which subsequently led to coagulopathy.

Relaxin inhibits effective collagen deposition by cultured hepatic stellate cells and decreases rat liver fibrosis in vivo.

BACKGROUND: Following liver injury, hepatic stellate cells (HSC) transform into myofibroblast-like cells (activation) and are the major source of type I collagen and the potent collagenase inhibitors tissue inhibitors of metalloproteinases 1 and 2 (TIMP-1 and TIMP-2) in the fibrotic liver. The reproductive hormone relaxin has been reported to reduce collagen and TIMP-1 expression by dermal and lung fibroblasts and thus has potential antifibrotic activity in liver fibrosis. AIMS: To determine the effects of relaxin on activated HSC. METHODS: Following isolation, HSC were activated by culture on plastic and exposed to relaxin (1-100 ng/ml). Collagen deposition was determined by Sirius red dye binding and radiolabelled proline incorporation. Matrix metalloproteinase (MMP) and TIMP expression were assessed by zymography and northern analysis. Transforming growth factor beta1 (TGF-beta1) mRNA and protein levels were quantified by northern analysis and ELISA, respectively. RESULTS: Exposure of activated HSC to relaxin resulted in a concentration dependent decrease in both collagen synthesis and deposition. There was a parallel decrease in TIMP-1 and TIMP-2 secretion into the HSC conditioned media but no change in gelatinase expression was observed. Northern analysis demonstrated that primary HSC, continuously exposed to relaxin, had decreased TIMP-1 mRNA expression but unaltered type I collagen, collagenase (MMP-13), alpha smooth muscle actin, and TGF-beta1 mRNA expression. CONCLUSION: These data demonstrate that relaxin modulates effective collagen deposition by HSC, at least in part, due to changes in the pattern of matrix degradation.

Gliotoxin stimulates the apoptosis of human and rat hepatic stellate cells and enhances the resolution of liver fibrosis in rats.

BACKGROUND & AIMS: Hepatic stellate cells (HSCs) play a pivotal role in liver fibrosis and stimulating their apoptosis could be an effective treatment for liver fibrosis. METHODS: Activated HSCs, hepatocytes, and rats with liver fibrosis were treated with gliotoxin. RESULTS: Addition of gliotoxin to activated (alpha-smooth muscle actin positive) rat and human HSCs resulted in morphologic alterations typical of apoptosis. Within 2-3 hours of incubation, caspase 3 activity was markedly induced and caspase inhibitor 1 (Z-VAD-FMK)-sensitive oligonucleosome-length DNA fragments were detectable by gel electrophoresis of low molecular weight DNA. Apoptosis was widespread as judged by fluorescence-activated cell sorter analysis and terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling staining in both rat and human HSCs at concentrations that had no effect on the viability of rat hepatocytes. Gliotoxin treatment significantly reduced the number of activated stellate cells and mean thickness of bridging fibrotic septae in livers from rats treated with carbon tetrachloride. CONCLUSIONS: These data demonstrate proof-of-concept that by up-regulating HSC apoptosis, the extent of fibrosis can be decreased in inflammatory liver injury.

Apoptosis of hepatic stellate cells: involvement in resolution of biliary fibrosis and regulation by soluble growth factors.

BACKGROUND: Activated hepatic stellate cells (HSC) are central to the pathogenesis of liver fibrosis, both as a source of fibrillar collagens that characterise fibrosis and matrix degrading metalloproteinases and their tissue inhibitors, the TIMPs. AIMS: To test the hypothesis that HSC apoptosis is critical to recovery from biliary fibrosis and that soluble growth factors may regulate HSC survival and apoptosis. METHODS: Rats (n=15) were subjected to bile duct ligation for 21 days, after which biliodigestive anastomosis was undertaken (n=13). Livers were harvested at fixed time points of recovery for periods of up to 42 days. Numbers of activated HSCs were quantified after alpha smooth muscle actin staining and HSC apoptosis was detected by terminal UDP-nick end labelling (TUNEL) staining and quantified at each time point. HSC apoptosis was quantified in vitro in the presence or absence of insulin-like growth factor (IGF)-1, IGF-2, platelet derived growth factor (PDGF), and transforming growth factor beta1 (TGF-beta1). RESULTS: Following biliodigestive anastomosis after 21 days of bile duct ligation, rat liver demonstrated a progressive resolution of biliary fibrosis over 42 days, associated with a fivefold decrease in activated HSC determined by alpha smooth muscle actin staining. TUNEL staining indicated that loss of activated HSC resulted from an increase in the rate of apoptosis during the first two days post biliodigestive anastomosis. Serum deprivation and culture in the presence of 50 microM cycloheximide was associated with an increase in HSC apoptosis which was significantly inhibited by addition of 10 ng/ml and 100 ng/ml IGF-1, respectively (0.05>p, n=5). In contrast, 1 and 10 ng/ml of TGF-beta1 caused a significant increase in HSC apoptosis compared with serum free controls (p<0.05, n=4). PDGF and IGF-2 were neutral with respect to their effect on HSC apoptosis. CONCLUSION: HSC apoptosis plays a critical role in the spontaneous recovery from biliary fibrosis. Both survival and apoptosis of HSC are regulated by growth factors expressed during fibrotic liver injury.

Hepatic stellate cells express the low affinity nerve growth factor receptor p75 and undergo apoptosis in response to nerve growth factor stimulation.

We have examined the expression of p75, a member of the TNF receptor superfamily in hepatic stellate cells (HSC) and pancreatic stellate cells (PSC). Activated HSC and PSC were demonstrated by Western blot analysis to express p75. p75 was immunolocalized to cells with a myofibroblast-like morphology in the fibrotic bands of six fibrotic and cirrhotic liver biopsies and three biopsies of fibrotic human pancreas. Immunostaining of parallel sections indicated that these cells were alpha-smooth muscle actin-positive, identifying them as activated HSC and PSC, respectively. HSC apoptosis in tissue culture in the presence of serum was quantified after addition of 0.1 to 100 ng/ml of nerve growth factor (NGF) a ligand for p75, by in situ counting of apoptotic bodies after addition of acridine orange. HSC demonstrated a significant increase in apoptosis in response to 100 ng/ml NGF (0.05 > P by Wilcoxon's rank; n = 7) after 24 hours. NGF 100 ng/ml had no effect on HSC proliferation, but reduced total HSC DNA by 19% relative to control after 24 hours (n = 3). These data demonstrate that activated HSC express p75 and respond to NGF stimulation by undergoing apoptosis. We therefore report p75 as a novel marker of activated HSC and suggest that signaling via ligand binding to p75 may provide a mechanism for selective apoptosis of HSC.

The inhibitory action of tamoxifen on the contraction of Ascaris suum somatic muscle in response to acetylcholine.

The somatic muscle of Ascaris suum is principally under the excitatory control of neuromuscular junction transmitter, acetylcholine (ACh). However, it has recently been shown that neuropeptides also play an important role in the motor-nervous system and one of these, AF3 (AVPGVLRFamide), also contracts muscle. The events which trigger contraction to ACh and AF3 would appear to be different, with ACh activating a nicotinic acetylcholine receptor whilst the response to AF3 is most likely to involve a G-protein coupled receptor negatively coupled to adenylate cyclase. In order to further elucidate differences in the cellular signalling pathways through which ACh and AF3 elicit muscle contraction, we investigated the actions of protein kinase C inhibitors, tamoxifen and chelerythrine, on the dorsal somatic muscle strip of A. suum. Contractions in response to 1 microM AF3 were potentiated by 17% in the presence of 10 microM tamoxifen (P < 0.05; n = 8); however, contractions in response to 10 microM ACh were markedly inhibited (tamoxifen IC50 44 +/- 18 microM; n = 6). Tamoxifen also blocked muscle cell depolarizations to 5 microM ACh (IC50 4 +/- 1 microM; n = 6) and 1 microM levamisole (IC50 14 +/- 6 microM; n = 4). This was unlikely to be a non-specific effect on the membrane as hyperpolarizations to 10 microM GABA were unaffected (93% of control with 10 microM tamoxifen; n = 6; P > 0.05). However, another inhibitor of mammalian protein kinase C, chelerythrine, did not affect the response either to ACh or AF3 (n = 6).

The role of cAMP in the actions of the peptide AF3 in the parasitic nematodes Ascaris suum and Ascaridia galli.

AF3 (AVPGVLRFamide) is an endogenous RFamide-like peptide isolated from the parasitic nematode Ascaris suum. It has a potent and long lasting excitatory effect in A. suum and Ascaridia galli. This is mediated by a mechanism independent of the nicotinic-like acetylcholine (ACh) receptor, which mediates excitatory transmission at the neuromuscular junction of both nematodes. In addition, AF3 has been found to sensitise A. suum muscle to the contractile effect of ACh. In this study, the involvement of the second messenger cAMP in mediating the action of AF3 on the somatic musculature of A. suum and A. galli has been investigated. Two approaches have been used; the effects of drugs which raise intracellular cAMP levels on the contractile responses to AF3 have been examined and biochemical assays have been used to measure the effects of AF3 on cAMP levels. AF3 contractions were inhibited in A. suum by 10 microM forskolin (by 22% of control; P < 0.05; n = 9) and by 500 microM isobutylmethylxanthine (IBMX, by 27% of control; P < 0.001; n = 6). AF3 decreased cAMP concentrations in A. suum somatic muscle (basal, 1721 +/- 134 pmol mg-1 protein; with 1 microM AF3, 1148 +/- 133 pmol mg-1 protein; P < 0.05, n = 5). AF3 (1 microM) also reduced the 10 microM forskolin induced potentiation of cAMP concentrations in A. suum (forskolin 3242 +/- 471 pmol mg-1 protein; forskolin and AF3, 1524 +/- 143 pmol mg-1 protein; P < 0.001, n = 6) and A. galli (forskolin 291 +/- 32 pmol mg-1 protein, forskolin +AF3, 185 +/- 12 pmol mg-1 protein; P < 0.005, n = 5). These data suggest that in both nematodes the contractile effect of AF3 is, at least in part, regulated by cAMP.

The effects of the peptides AF3 (AVPGVLRFamide) and AF4 (GDVPGVLRFamide) on the somatic muscle of the parasitic nematodes Ascaris suum and Ascaridia galli.

AF3 (AVPGVLRFamide) and AF4 (GDVPGVLRFamide) are endogenous RFamide-like peptides isolated from the parasitic nematode Ascaris suum. Here the actions of these peptides on the somatic musculature of Ascaris have been investigated and compared to the action of acetylcholine (ACh), the excitatory transmitter at the neuromuscular junction. ACh, AF3 and AF4 contracted muscle with EC50S of 13 +/- 1 microM, 24 +/- 6 nM and 37 +/- 2 nM, respectively (n = 6). The muscle cells were depolarized by ACh (3 microM; 5.2 +/- 0.4 mV, n = 42), AF3 (1 microM; 2.6 +/- 0.3 mV, n = 19) and AF4 (1 microM; 3.3 +/- 0.4 mV, n = 19). EC50S were 681 +/- 329 nM (AF3) and 901 +/- 229 nM (AF4), but an estimate could not be made for ACh due to muscle contraction at concentrations greater than 10 microM. The depolarization to 3 microM ACh was abolished by the nicotinic receptor antagonist mecamylamine (10 microM; n = 5) but the responses to the peptides were not (111 +/- 7% and 108 +/- 17% with respect to control; n = 5). The depolarization elicited by ACh was reduced to a greater extent by a 50% reduction in extracellular Na+ concentration than the response to AF3 and AF4 (P < 0.02). Cobalt was more effective at blocking the AF3 and AF4 depolarizations than those to ACh. These observations suggest that AF3 and AF4 contract Ascaris muscle without an action at the Ascaris nicotinic receptor. Furthermore, the ionic mechanism through which AF3 and AF4 depolarize Ascaris muscle is different from that for ACh. ACh, AF3 and AF4 were also found to contract Ascaridia galli somatic muscle with EC50S of 13 +/- 3 microM, 721 +/- 236 nM and 371 +/- 177 nM, respectively (n = 7). The muscle cells were depolarized by ACh (EC50 = 14 +/- 5 microM, n = 5), AF3 (EC50 = 5 +/- 3 microM, n = 4) and AF4 (EC50 = 10 +/- 5 microM, n = 4). Therefore the response to these peptides is not unique to Ascaris and they may subserve a functional role in the motor nervous system of parasitic nematodes.