Promotion of experimental thrombus formation by the procoagulant activity of breast cancer cells.

The routine observation of tumor emboli in the peripheral blood of patients with carcinomas raises questions about the clinical relevance of these circulating tumor cells. Thrombosis is a common clinical manifestation of cancer, and circulating tumor cells may play a pathogenetic role in this process. The presence of coagulation-associated molecules on cancer cells has been described, but the mechanisms by which circulating tumor cells augment or alter coagulation remains unclear. In this study we utilized suspensions of a metastatic adenocarcinoma cell line, MDA-MB-231, and a non-metastatic breast epithelial cell line, MCF-10A, as models of circulating tumor cells to determine the thrombogenic activity of these blood-foreign cells. In human plasma, both metastatic MDA-MB-231 cells and non-metastatic MCF-10A cells significantly enhanced clotting kinetics. The effect of MDA-MB-231 and MCF-10A cells on clotting times was cell number-dependent and inhibited by a neutralizing antibody to tissue factor (TF) as well as inhibitors of activated factor X and thrombin. Using fluorescence microscopy, we found that both MDA-MB-231 and MCF-10A cells supported the binding of fluorescently labeled thrombin. Furthermore, in a model of thrombus formation under pressure-driven flow, MDA-MB-231 and MCF-10A cells significantly decreased the time to occlusion. Our findings indicate that the presence of breast epithelial cells in blood can stimulate coagulation in a TF-dependent manner, suggesting that tumor cells that enter the circulation may promote the formation of occlusive thrombi under shear flow conditions.

Modification of the tensor fasciae latae flap for reconstruction of the abdominal wall: a case report.

Full-thickness periumbilical wounds after extensive abdominal surgery present a major challenge. In this case report the traditional tensor fasciae latae flap was modified, improving flap mobility while also facilitating donor defect repair.

Regulation of bradykinin sensitivity in peripheral sensory fibres of the neonatal rat by nitric oxide and cyclic GMP.

Bradykinin-induced activation of peripheral sensory fibres was studied using an in vitro preparation of the neonatal rat spinal cord with attached tail. Noxious heat stimulation, as well as the applications of bradykinin and capsaicin, to the tail evoked reproducible responses recorded as a depolarization of a lumbar ventral root. Prolonged administration of a supramaximal concentration of bradykinin invariably induced a complete but selective desensitization to a subsequent bradykinin challenge. Bradykinin-induced desensitization was significantly attenuated by concanavalin-A and the effect of concanavalin-A was prevented by alpha-methyl mannoside. Both cyclic GMP and sodium nitroprusside induced a long lasting reduction of bradykinin responsiveness in peripheral fibres. The effect of nitroprusside was prevented by concanavalin-A, and by methylene blue, an inhibitor of guanylyl cyclase. Methylene blue also reduced bradykinin-induced desensitization. L-arginine, but not D-arginine, induced a desensitization to bradykinin. On the other hand, 7-nitroindazole (7-NI, 200-500 nM), an inhibitor of NOS, reduced the desensitization of bradykinin responses but higher concentrations of 7-NI (IC50 = 6.7 +/- 0.9 microM) selectively attenuated responses to bradykinin. The effects of 7-NI were attenuated by L-arginine pretreatment. These data suggest that bradykinin-induced desensitization of peripheral sensory fibres is mediated in part via NO and cyclic GMP dependent mechanisms: possibly NO production is required for guanylate cyclase activation.

Bradykinin-induced activation of nociceptors: receptor and mechanistic studies on the neonatal rat spinal cord-tail preparation in vitro.

1. The effects of bradykinin on nociceptors have been characterized on a preparation of the neonatal rat spinal cord with functionally connected tail maintained in vitro. Administration of bradykinin to the tail activated capsaicin-sensitive peripheral fibres and evoked a concentration-dependent (EC50 = 130 nM) depolarization recorded from a spinal ventral root (L3-L5). 2. The response to bradykinin was unaffected by the peptidase inhibitors, bestatin (0.4 mM), thiorphan (1 microM), phosphoramidon (1 microM) and MERGETPA (10 microM) or by the presence of calcium blocking agents, cadmium (200 microM) and nifedipine (10 microM). 3. Inhibition of cyclo-oxygenase with indomethacin (1-5 microM), aspirin (1-10 microM) and paracetamol (10-50 microM) consistently attenuated responses to bradykinin. 4. The effect of bradykinin was mimicked by the phorbol ester PDBu, an activator of protein kinase C. The response to bradykinin was attenuated following desensitization to PDBu but desensitization to bradykinin did not induce a cross-desensitization to PDBu. The protein kinase C inhibitor staurosporine (10-500 nM) consistently attenuated the effects of PDBu and bradykinin. 5. Bradykinin responses were reversibly enhanced by dibutyryl cyclic AMP (100 microM). However dibutyryl cyclic GMP (0.5 mM) and nitroprusside (10 microM) produced prolonged block of responsiveness to bradykinin. Prolonged superfusion with pertussis toxin did not affect responses to bradykinin. 6. The B1-receptor agonist des Arg9-bradykinin (10-100 microM) was ineffective alone or after prolonged exposure of the tail to lipopolysaccharide (100 ng ml-1) or epidermal growth factor (100 ng ml-1) to induce B1 receptors. The BI-receptor antagonist, des Arg9 Leu8-bradykinin (10 JM) did not attenuate the response to bradykinin. A number of bradykinin B2 antagonists selectively and reversibly attenuated the response to bradykinin. The rank order potency was Hoe 140> LysLys [Hyp3,Thi5 8,D-Phe7]-bradykinin> D-Arg[Hyp3, Thi5'8, D-Phe7]-bradykinin = D-Arg[Hyp2,Thi5'8, D-Phe7]-bradykinin.7. These data show that bradykinin produces concentration-dependent activation of peripheral nociceptors in the neonatal rat tail. The responses were unaffected by calcium channel block and were partially dependent on the production of prostanoids. Bradykinin-evoked responses were consistent with the activation of protein kinase C-dependent mechanisms. Cyclic GMP-dependent mechanisms may be involved in bradykinin-receptor desensitization whereas cyclic-AMP dependent mechanisms increase fibre excitability and facilitate bradykinin-induced responses. The effects of bradykinin were mediated by a B2 receptor.

Tachykinin induced regulation of excitatory amino acid responses in the rat spinal cord in vitro.

The interaction between neurokinin and excitatory amino acid receptors in the spinal cord have been characterised using the neonatal rat spinal cord in vitro preparation. Ventral root (VR) depolarization evoked by N-methyl-D-aspartate (NMDA) and quisqualate was reversibly enhanced in the presence of subthreshold concentrations of neurokinin A (NKA; 1.0-10 nM), but not by substance P (1.0-5.0 nM). When substance P (SP) was replaced by the metabolically stable substance P methyl ester (SPOMe), both NMDA and quisqualate responses were significantly enhanced. VR depolarization evoked by kainate was not altered by any of the neurokinin (NK) receptor agonists. In the presence of the endopeptidase inhibitors, bestatin, captopril and thiorphan (each 1.0 microM), SP significantly enhanced NMDA-evoked responses. The selective NK1 receptor antagonist (+/-) CP96 345 (100 nM) reversibly blocked the enhancement of NMDA-evoked depolarization by SPOMe. Furthermore, MEN10 376 (50 nM), a selective NK2 receptor antagonist blocked the enhancement of NMDA- and quisqualate-evoked depolarization by NKA. The protein kinase C and protein kinase A inhibitor staurosporine (1.0 microM) blocked the enhancement of excitatory amino acid-induced responses by NK-receptor activation. However, whilst NKA-evoked ventral root depolarization was completely abolished in the presence of staurosporine, SPOMe- and SP-induced depolarizations were unaffected. These data show that activation of NK1 or NK2 receptors enhances NMDA- and quisqualate-evoked ventral root depolarization in the neonatal rat spinal cord. The interaction between neurokinin and excitatory amino acid receptors involves protein kinase C activation.

Receptors mediating tachykinin-evoked depolarisations of neurons in the neonatal rat spinal cord.

We have examined the contribution of NK1, NK2 and NK3 receptors to the depolarisation of the neonatal rat spinal cord in vitro evoked by exogenously applied tachykinine. Potential changes were recorded extracellularly from a lumbar ventral root. The NK1 receptor selective agonists substance P methly ester (SPOMs), septide and/Sar9/-substance P-sulphone (/Sar9/-P-sulphone), perfused onto the cord for 20 s, evoked ventral root potentials (VRPs) with similar EC50 values of 7.2 nM (95% confidence limits, 4.4-10.9 nM), 4.6 nM (1.8-9.3 nM) and 3.1 nM (1.6-5.1 nM), respectively. The NK3 receptor selective agonist senktide also evoked VRPs with an EC50 of 12.0 nM (5.1-23.4 nM), whilst the NK2 receptor selective agonist/beta-AlaB/-neurokinin A(4-10) (/beta-AlaB/-NKA(4-10)) was much less potent (EC50 = 228.3 nM, 95% confidence limits 138.0-350.0 nM). The non-peptide NK1 receptor selective antagonist RP67580 inhibited responses to SPOMe, septide and/Sar9/-SP-sulphone to varying degrees with IC50 values against each of 16.0 nM (10.7-23.4 nM), 19.8 nM (8.9-37 nM) and 58.0 nM (41-89 nM), respectively. The NK1 receptor antagonist CP-96,345 similarly inhibited responses to these agonists, although with higher IC50 estimates of 0.84 microM (0.51-1.40 microM) against SPOMe, 0.79 microM (0.50-1.17 microM) against/Sar9/-SP-sulphone and 0.37 microM (0.27-0.51 microM) against septide. Further analysis of the activity of RP67580 yielded a significantly higher pKB estimate for antagonism of responses to septide (7.67 +/- 0.04) than of responses to/Sar9/-SP-sulphone (7.18 +/- 0.05). In both cases Schild analysis indicated competitive antagonism. RP67580 also reversibly inhibited responses to /beta-AlaB/-NKA(4-10). However, in this case the Schild alope was significantly different from unity (0.44 +/- 0.55; P < 0.001), although the potency of the antagonist appeared similar to that seen with the NK1 receptor agonists (pA2 = 7.52). There was no effect of RP67580 against responses to senktide or of the inactive isomer RP67581 against septide-evoked VRPs. The NK2 receptor antagonist MEN 10,376 at concentrations up to 1 microM produced a partial but reversible inhibition of responses to a submaximal concentration of /beta-AlaB/-NKA(4-10) (0.3 microM) with a maximum reduction in VRP amplitude of 25.6 +/- 7.3%. A similar inhibitory effect was seen against septide-evoked VRPs (30.2 +/- 5.6% inhibition), although there was no effect against responses to submaximal concentrations of /Sar9/-SP-sulphone or senktide. In contrast, the non-peptide NK2 receptor antagonist SR 48,968 (1 microM) produced a maximal 48.0 +/- 7.7% inhibition of/beta-AlaB/-NKA(4-10)-evoked VRP's with no effect against responses to a submaximal concentration of septide. These data show that NK1 and NK3 receptor activation mediates depolarisation of the neonatal rat spinal cord, and suggest the presence of two NK1 receptor populations showing preference for septide and/Sar9/-SP-sulphone. Depolarisations mediated by /beta-AlaB/-NKA(4-10), previously described as a selective NK2 receptor ligand, are mediated predominantly via an action at NK1 receptors with a lesser involvement of NK2 receptors.

Laminin promotes coagulation and thrombus formation in a factor XII-dependent manner.

BACKGROUND: Laminin is the most abundant non-collagenous protein in the basement membrane. Recent studies have shown that laminin supports platelet adhesion, activation and aggregation under flow conditions, highlighting a possible role for laminin in hemostasis. OBJECTIVE: To investigate the ability of laminin to promote coagulation and support thrombus formation under shear. RESULTS AND METHODS: Soluble laminin accelerated factor (F) XII activation in a purified system, and shortened the clotting time of recalcified plasma in a FXI- and FXII-dependent manner. Laminin promoted phosphatidylserine exposure on platelets and supported platelet adhesion and fibrin formation in recalcified blood under shear flow conditions. Fibrin formation in laminin-coated capillaries was abrogated by an antibody that interferes with FXI activation by activated FXII, or an antibody that blocks activated FXI activation of FIX. CONCLUSION: This study identifies a role for laminin in the initiation of coagulation and the formation of platelet-rich thrombi under shear conditions in a FXII-dependent manner.

Free dermis-fat graft to correct the whistle deformity in patients with cleft lip.

Vermilion notching or 'whistle deformity' is a common secondary deformity of the vermilion in patients with cleft lip. The free border of the lip is a composite structure consisting of the orbicularis oris and the overlying tissues, namely subcutaneous fat, vermilion and mucosa. A deficiency of all or one of these structures is responsible for the vermilion notch. An absolute shortage of the muscle or subcutaneous tissue necessitates the use of tissue from another source to correct the deformity. Dermis fat grafts have been used to augment the free border of the lip and correct this deformity. The technique is simple and reliable. The tissue is available in plenty, easy to harvest and could be repeated if necessary, i.e. in case of resorption. From June 1996 until January 2000 the technique was used in 10 patients. Of these 10, one was a bilateral cleft and nine were unilateral cleft lip deformities. A degree of graft resorption was seen in one patient, one had a partial graft loss due to ulceration and exposure. The first patient had a further augmentation.

Desensitization of bradykinin-induced activation of peripheral nociceptors.

Bradykinin-induced activation of peripheral nociceptors has been studied in an isolated spinal cord/tail preparation from the neonatal rat. Prolonged administration of bradykinin consistently produced a selective desensitization which could be prevented by concanavalin A but not by succinyl concanavalin A or phenylarsine oxide. These data indicate that mannose-containing glycoproteins occur in or close to the bradykinin receptor site. In addition the desensitization observed under the present conditions, did not involve the internalization of bradykinin receptors.