M. Sakon

A NOVEL ENDOTOXIN-SPECIFIC ASSAY BY TURBIDIMETRY WITH LIMULUS AMOEBOCYTE LYSATE CONTAINING BETA -GLUCAN

The gelation of standard Limulus amoebocyte lysate (LAL) is triggered by the addition of a small amount of beta-glucan (1-1000 ng/ml plasma), but in the presence of an excessive amount of beta-glucan (1 mg/ml plasma), the gelation becomes insensitive to beta-glucan. Utilizing this property, a method to determine quantitatively the amount of endotoxin circulating in humans was developed. When a modified LAL, or LAL-ES, which contains an excessive amount of CM-curdlan as beta-glucan, was used for the assay, a linear relation in the logarithmic scales was obtained between the gelation time measured by the turbidimetry (min) and the concentration of endotoxin. This relation was not affected by a considerable amount of beta-glucan (100 ng/ml). The sensitivity of the endotoxin assay was estimated to be as low as 3 pg/ml. The following aspects of the method were found by clinical application to normal and febrile subjects. (1) Using both LAL and LAL-ES, it was possible to distinguish the effect of endotoxin from that of beta-glucan in plasma, i.e., bacterial sepsis from fungal sepsis. (2) The amount of circulating endotoxin determined by the present method showed good correlation to those obtained by chromogenic assay using modified LAL devoid of Factor G which could be activated by beta-glucan.

Ultrastructural analysis of pseudo-intimal hyperplasia of polytetrafluoroethylene prostheses implanted into the venous and arterial systems

To evaluate and compare the pathogenesis of pseudo-intimal hyperplasia (PH) of venous and arterial prostheses, a segment of the inferior vena cava (n = 16) or abdominal aorta (n = 16) was substituted by a 3 mm internal diameter polytetrafluoroethylene tube graft (PTFE, 3 cm long, 30 microns in nodal distance) in albino rabbits. At designated time intervals (3-28 days) after the replacement, graft patency was examined and the dry weights of the intraluminal deposits measured as an indicator of the degree of PH. The harvested grafts were then subjected to an ultrastructural analysis by means of light microscopy (LM), and scanning electron and transmission electron microscopy (SEM, TEM). All the grafts remained patent during the entire observation period. The PH judged by the dry weight was significantly more extensive in the venous than in the arterial prostheses. The PH on day 28, observed by light microscopes was apparently most extensive in the mid-portion of venous prostheses but in the arteria prostheses it was mostly seen at the anastomotic sites. The lining of the intraluminal surface of the prostheses with endothelial-like cells observed by SEM was faster and more extensive in venous than in arterial prostheses. The process of PH in venous prostheses observed by TEM may be divided into the following steps: early thrombosis, phagocytosis of the thrombus, appearance of fibroblasts, growth of endothelial-like cells, appearance of smooth muscle cells, and pseudointimal thickening and proliferation of fibroblasts producing collagen fibrils. The process in arterial prostheses was essentially identical to that in venous prostheses but was much slower and less extensive. From these observations, it was concluded that the formation of PH occurs much faster in venous than in arterial prostheses, although the mechanism of PH is mostly identical in venous and arterial prostheses.

Chromogenic assay of endotoxin in platelet poor or rich plasma

In order to determine which sample preparation, platelet rich plasma (PRP) or platelet poor plasma (PPP), is more suitable for clinical endotoxin assay, we investigated the binding of endotoxins to platelets by comparing the amount of endotoxin in PRP with that in PPP, using a newly developed colorimetric assay with chromogenic substrate (Boc-Leu-Gly-Arg-pNA). When purified endotoxins were added to human whole blood, the amount of endotoxin recovered in PPP was significantly lower than that in PRP for all endotoxins tested except that from E. coli 0111:B4 and their ability to bind to platelets was varied depending on the species of bacteria from which they were purified. However, the amount of endotoxin in PRP obtained from surgical patients (n = 50) was almost same as that in PPP with a correlation coefficient r = 0.95, indicating that natural endotoxins circulating in human blood may not bind to platelets and that PPP can be used for endotoxin assay as well as PRP.

Potential etiologic role of PAF in two major septic complications; Disseminated intravascular coagulation and multiple organ failure

A possible role of platelet-activating factor (PAF) in the occurrence of the two septic complications, i.e., disseminated intravascular coagulation (DIC) and multiple organ failure (MOF) was investigated, employing a rabbit model and a novel PAF antagonist E5880. By an instillation of fecal suspension into the common bile duct of the rabbit, manifestations of DIC and MOF were observed with high reproducibility by 9 hours after the septic insult. E5880 was intravenously administered to 12 rabbits for 1 hour after the septic insult at dose of 1 mg/kg (n = 6) or 3mg/kg (n = 6). All the rabbits were subjected to observation of vital signs and serial determination of laboratory tests for 9 hours and then lung, liver and kidney were removed for histological examination. Blood endotoxin level increased significantly by 9 hours after the septic insult. Although administration of E5880 did not affect the endotoxemia, the antagonist attenuated in a dose related manner laboratory manifestation of DIC such as thrombocytopenia and prolonged prothrombin time as well as that of MOF such as increase in serum bilirubin and creatinine level. The beneficial effect of E5880 on MOF was also confirmed by the histological evaluation. These observations indicated that PAF is deeply involved in the occurrence of DIC and MOF due to sepsis and E5880 may be one of the modalities to treat or prevent these two major septic complications.

Studies on liposome-encapsulated heparin

In order to prolong the anticoagulant activity of heparin in vivo, attempts were made to encapsulate heparin into liposomes. Liposome-encapsulated heparin (lipo-heparin) prepared was large multilamellar vesicles (0.5-4.0 micron in diameter). The activity of lipo-heparin was 1.6-5.2 X 10(3) U/g lipid with recovery rate ranged between 0.4 to 1.3% and stable in saline at 4 degrees C for at least two weeks. When intravenously administered into rats, the anticoagulant activity of lipo-heparin was significantly prolonged (approximately three times), as compared with that of untreated heparin. Furthermore, the activity of lipo-heparin could be neutralized by protamine sulfate. From these observations, it was concluded that liposome-encapsulation of heparin results in the prolonged anticoagulant effect in vivo and lipo-heparin may be applicable for clinical use, after further studies on side effects of liposomes are completed.

Phosphorylation of phosphoinositides in human platelets.

32P-labelling of phosphatidylinositol (PI), PI-4-monophosphate (PIP), PI-4,5-bisphosphate (PIP2) and phosphatidic acid (PA) in 32P-labelled intact human platelets was investigated in the presence of various agents which alter intracellular level of cAMP or Ca2+. Addition of dibutyryl cAMP to intact platelets pre- or pulse labelled with 32P resulted in increased 32P-labelling of PIP and in concomitant decreased 32P-labelling of PI without affecting that of PIP2 or PA. Similar changes were observed in intact platelets treated by prostaglandin I2 (PGI2) or a new low Km phosphodiesterase inhibitor (DN-9693). When intracellular Ca2+ was chelated by loading quin 2-AM to intact platelets, 32P-labelling of PIP was significantly increased in a dose related manner. From these observations it was concluded that PI kinase is activated by elevation of cAMP or chelation of Ca2+ in intact platelets.

Stimulation of human platelet Ca2+-ATPase and Ca2+ restoration by calpain

To clarify the possible role of calpain (calcium activated neutral protease; EC 3.4.22.17) in Ca2+ homeostasis of human platelets, we investigated the effects of cell permeable calpain inhibitors, calpeptin and E-64d (EST), on the restoration of cytoplasmic Ca2+ ([Ca2+]i) in both Fura-2 and aspirin (ASA) loaded platelets. Although neither calpeptin (30 microM) nor EST (250 microM) altered the increase of [Ca2+]i in thrombin (1 U/ml) stimulated platelets, both calpain inhibitors delayed the decrease of [Ca2+]i back towards the basal level. These observations suggested that calpain might be involved in Ca2+ restoration. Then, the activity of Ca(2+)-ATPase was examined in thrombin (2 U/ml) stimulated platelets. Thrombin produced a rapid rise in Ca(2+)-ATPase activity by 2-fold at 8 s of incubation, which then returned to below the basal activity within 2 min. Calpeptin inhibited transient Ca(2+)-ATPase activation induced by thrombin in a dose related manner. Ca(2+)-ATPase of isolated platelet membranes was digested by purified human platelet calpain-I and Ca(2+)-ATPase activity was investigated. With a short incubation (8-15 s), Ca(2+)-ATPase activity was increased about 2-fold and then it decreased below the basal level at longer incubations or at a higher calpain/membrane ratio. The initial rate of Ca2+ uptake was also increased by about 2-fold with a short incubation (8-15 s). For molecular characterization of the Ca(2+)-ATPase, the formation of the enzyme-phosphate complex (EP) was investigated. The membrane bound intact 105 kD Ca(2+)-ATPase was converted by calpain to a fragment of approximately 50 kD.(ABSTRACT TRUNCATED AT 250 WORDS)

An infection-resistant PTFE vascular graft; spiral coiling of the graft with ofloxacin-bonded PTFE thread

OBJECTIVE To develop an infection-resistant polytetrafluoroethylene (PTFE) vascular graft for potential clinical use in grafting in sites of bacterial contamination and in replacement of the infected grafts. SETTING Experimental study in rabbits. MATERIALS AND METHODS An antibiotic ofloxacin (OFLX) was bonded to a sheet of PTFE by impregnation, which was cut and twisted into fine threads. The in-vitro antibacterial activity of OFLX-PTFE thread was determined by measuring the zone of growth inhibition against Escherichia coli. The thread was spirally coiled around a ridged outerwall PTFE to make the OFLX-PTFE graft. OFLX-PTFE graft or control graft was interposed in the inferior vena cava (IVC) of rabbits and the entire graft was covered with fibrin containing a fixed number of E. coli. Three or 7 days after the grafting, the grafts with perigraft tissue were harvested and subjected to bacteriological studies. RESULTS In spite of early phase rapid elution of OFLX, a significant antibacterial activity was retained for more than 2 weeks. The antibacterial activity of OFLX-PTFE threads implanted in the subcutaneous space of rabbits decreased to 48% after 24 h and to approximately 1% after a week. The swab culture of all the control grafts was positive, while only one of 13 PTFE-OFLX grafts was positive. The number of viable bacteria in the perigraft tissue of OFLX-PTFE grafts was remarkably low in comparison with that of control grafts. Thus, the OFLX-PTFE grafts exhibited a marked in-vivo antibacterial activity. CONCLUSION By a unique method, it was possible to furnish PTFE graft with an excellent infection-resistant property, without affecting the original biological behaviour.

Calpains and Calpastatin in Human Blood Platelets

Calpain, a Ca(2+) activated intracellular protease and its endogeneous protein inhibitor, calpastatin are abundant in platelets. The structure and enzymological properties of calpain, including its isozymes, and of calpastatin in platelets have been fully characterized. Also, platelet calpain has been shown to cleave various endogeneous polypeptides. However, the mode of activation and the physiological function of platelet calpains have not been clarified. Our recent investigations on platelet calpains with cell permeable calpain antagonists and specific antibodies reveal that calpains are not involved in the early phases of platelet activation such as shape change and aggregation, but in the later phases of platelet activation such as cytoskeletal reorganization and Ca(2+) uptake.

Subendothelial layer of pseudointima of polytetrafluoroethylene graft is formed by transformation of fibroblasts migrated from extravascular space

A well organised pseudointima is formed in polytetrafluoroethylene (PTFE) grafts within 4 weeks after implantation into inferior vena cava (IVC) of rabbits. To investigate the process of the subendothelial organisation of pseudointima, an animal experiment was conducted. The outer wall of PTFE graft (30 microns fibril length, 3 mm inner diameter, 3 cm long) was coated with 10 um silicon film in the following ways to prevent cellular ingrowth from the extravascular space: non-coating; full-length coating; half-length coating; and full-length coating excluding 5 mm midportion. These grafts were implanted into rabbit IVC and were harvested 4 weeks later. All the grafts were patent but the lumen of the non-coated area was narrowed by pseudointimal hyperplasia. The degree of the hyperplasia estimated by dried tissue deposit was inversely proportional to the length of the coating. The coverage of the luminal surface with endothelial-like cells was noted at anastomotic areas and also at the surface corresponding to the non-coated area. Light microscopy and immunostaining studies on the non-coated midportion revealed the presence of fibroblasts in the interstices of PTFE and smooth muscle cells and myofibroblasts in the pseudointima. Transmission electron microscopy confirmed the presence of myofibroblasts in the midportion of the non-coated area. No transmural capillary ingrowth was observed in the midportion by histological and immunohistochemical analysis. These observations suggest that the subendothelial layer of pseudointima in PTFE grafts is formed by proliferation and transformation of fibroblasts migrating from the extravascular space and that endothelial-like cells may also be derived from such transformation.