A new approach for fat removal in a discontinuous autotransfusion device-concept and evaluation.

BACKGROUND: Fat present during blood salvage in orthopaedic or cardiac surgery can pose a risk of fat embolism and should be eliminated before transfusion. Based on observations of central fat accumulation at the bottom of Latham bowls, a fat reduction program was developed using two volume displacements, where blood temporarily is removed and respun in the bowl to force the fat through the RBC sediment. MATERIALS AND METHODS: Pooled ABO-matched RBC and FFP were adjusted to a haematocrit of 10%, and human fat tissue added to a concentration of 1·25 vol%. In six experiments, blood was processed with the new-generation cell salvage device CS Elite in a newly developed fat reduction program in bowls of three sizes. Volumetric quantification of fat was performed after centrifugation of blood samples in Pasteur pipettes. From volumes, haematocrits and the concentrations of fat, RBC recovery and fat elimination rates were calculated. RESULTS: Fat removal rates of 93·2 ± 2·8, 97·0 ± 2·1 and 99·6 ± 0·3% were observed with a 70-ml, 125-ml and 225-ml bowl, respectively, and even higher rates when removal rates were calculated one cycle. At the same time, high RBC recovery and plasma elimination rates were maintained, not significantly different to the default program mode. CONCLUSION: Modifications in process parameters and sequence led to a fat reduction program that significantly improves fat removal with the Cell Saver Elite from 77·4 ± 5·1% in the default mode to an average of 98·6 ± 1·1%, yielding results equivalent to the continuous cell salvage system (C.A.T.S).

Synthesis and characterisation of an unsymmetrical, ether-linked, fluorinated bimesogen exhibiting a new polymorphism containing the N(TB) or 'twist-bend' phase.

In this article we report on the liquid crystal phases and properties of the bimesogen 4-((11-((4'-fluoro-[1,1'-biphenyl]-4-yl)oxy)undecyl)oxy)-2,3-difluoro-4'-(4-propylcyclohexyl)-1,1'-biphenyl. This material was shown to exhibit an Iso Liq-N-NTB-SmA phase sequence, thereby clearly indicating that the NTB phase possesses an ordering of the constituent molecules that is between that of a conventional nematic and the smectic A phase. This compound allows us to better understand the relationship between molecular structure and the NTB phase, and we conclude it is the gross topology that dictates the incidence of this fascinating phase and not molecular properties such as dipole moment and bend angle.

Recombinant human Monocyte/Neutrophil elastase inhibitor protects rat lungs against injury from cystic fibrosis airway secretions.

Human monocyte/neutrophil elastase inhibitor (M/NEI) is a fast-acting stoichiometric inhibitor of neutrophil elastase (NE), cathepsin-G, and proteinase-3. Recombinant M/NEI (rM/NEI) was evaluated with a rat model of NE-induced lung damage. rM/NEI was found to protect against pulmonary injury caused by instilled human NE or by a preparation from airway secretions (sputum) of cystic fibrosis patients (CF sol). Human NE instilled into rat lungs produced dose-dependent hemorrhage and increased epithelial permeability, whereas NE incubated in vitro with rM/NEI did neither. Similarly, hemorrhage was induced by CF sol, but not by CF sol incubated in vitro with rM/NEI. To examine its distribution and survival time in airways, rM/NEI was labeled with the fluorochrome Texas Red (rM/NEI-TR) and instilled into rat lungs. Confocal microscopy showed that rM/NEI-TR could be detected on large airways (300 microm) at 5 min, 1 h, 4 h, and 24 h after instillation. Pretreating rats with rM/NEI was found to provide extended protection upon subsequent NE challenge, reducing hemorrhage by 98, 96, and 73%, respectively, at 1, 4, and 24 h after rM/NEI pretreatment. Pretreating rats with rM/NEI similarly conferred protection against subsequent exposure to CF sol, reducing hemorrhage by 95, 86, and 87%, respectively, at 1, 4, and 24 h after pretreatment. The findings that rM/NEI (1) mitigates protease-induced lung injury and (2) remains present and active in the lungs for 24 h after instillation strongly support its potential for treating patients with neutrophil protease-induced inflammatory lung damage, such as occurs in CF and other diseases.

Production of recombinant human monocyte/neutrophil elastase inhibitor (rM/NEI).

Recombinant human monocyte/neutrophil elastase inhibitor (rM/NEI) was expressed with a baculovirus expression system. The purified recombinant protein was shown to inhibit human neutrophil elastase by the formation of a stable equimolar complex, as had been shown for M/NEI isolated from monocyte-derived cell lines. rM/NEI was remarkably stable in aqueous buffers from pH 6 to pH 8, but not in buffers below pH 6. rM/NEI activity was stable when subjected to freeze-thaw cycles and low temperature storage in Tris or phosphate buffers. rM/NEI could also be lyophilized without significant loss of activity. A 1.6-g batch of greater than 95% purity in rM/NEI was obtained by anion exchange and size exclusion chromatography with yields of 7 to 8 mg per liter of cultured insect cells. Methods and protocols were chosen for compatibility with large-scale cGMP production and were suitable for biochemical characterization and preclinical evaluation of rM/NEI as a therapeutic agent for cystic fibrosis. The availability of large amounts of purified rM/NEI will facilitate clinical evaluation of rM/NEI for prevention of the elastase-mediated destruction of lung tissue associated with the morbidity and mortality of cystic fibrosis.

An HMG-I protein from human endothelial cells apparently is secreted and impairs activation of Hageman factor (factor XII).

A highly purified protein from lysates of human umbilical vein endothelial cells (HUVECs) inhibited the activation of factor XII [Hageman factor (HF)] and removed factor XIIa from an activating surface, thus impairing HF-dependent coagulation and kinin-releasing activities. Two tryptic peptides from this protein had 100% identity with amino acids 31-44 and 89-101 of a nonhistone DNA-binding protein known as high-mobility group protein (HMG-I). In specific antibody experiments, the clot-inhibiting property in purified lysate protein from HUVECs was associated with HMG-I. The molecular weight of the protein that inhibited clotting was consistent with that predicted for HMG-I. Protein that inhibited contact activation and had antigenic properties of HMG-I and HUVEC lysate protein also was found in conditioned media from unchallenged cultured HUVECs. After HUVECs were incubated with 14C lysine, conditioned media contained immunoprecipitable radiolabeled protein with the same molecular weight as that recovered from cell lysates, suggesting that this high-mobility group protein (HMG-I) may be secreted. Purified factor XII antigens were displaced from a glass surface by HMG-I from lysates in proportion to the amount of HMG-I protein that was added. This HMG-I probably inhibits factor XII functions because its high positive charge favors competitive binding to an activating substance.

Expression and purification of enzymatically active recombinant granzyme B in a baculovirus system.

Granzyme B (GranB), a serine protease stored in the granules of cytotoxic T lymphocytes and natural killer cells, can initiate target cell apoptosis. To produce large amounts of purified active enzyme, recombinant murine granzyme B (rGranB) was expressed from baculovirus in insect cells. The expressed rGranB is secreted into the culture medium and can be readily purified to homogeneity by one-step affinity chromatography to yield 1.5 mg enzyme per liter insect cell medium. RGranB is recognized by a GranB-specific anti-peptide antibody and is active against synthetic substrate Boc-Ala-Ala-Asp-SBzl with kinetic constant (kcat/Km 45,000 M-1s-1) comparable to purified human GranB, RGranB processes the caspase pro-CPP32 into its enzymatically active form and induces DNA fragmentation in isolated nuclei in the presence of cytosolic factors. The ability to express enzymatically active rGranB using the baculovirus system will help elucidate the role of this granzyme in the immune response.

Acquired C1 inhibitor deficiency as a result of an autoantibody to the reactive center region of C1 inhibitor.

An autoantibody that we hypothesize to react with the reactive center of the plasma serine proteinase inhibitor, C1 inhibitor (C1INH), has been found in a patient with acquired C1INH deficiency. The Ab blocks the ability of C1INH to inhibit the hydrolysis of N-carbobenzyloxy-L-lysine thiobenzylester by purified C1s. A cryoprecipitate from the patient's plasma as well as the Ig fraction were able to block C1INH inhibition of C1s. The immunoaffinity purified Ab to C1INH from the patient's plasma Ig fraction neutralizes the inhibitory activity of C1INH in a dose-dependent manner and blocks the ability of normal C1INH to form a complex with C1s. The neutralizing activity of the purified Ab is reversed by a synthetic peptide that corresponds to the amino acid sequence in the P1 to P15 positions of the reactive center of C1INH but not by a 34-amino-acid trypsin peptide or 37-amino-acid elastase peptide derived from the C-terminus of C1INH. Western blot analysis indicated that the Ab is an oligoclonal Ig with kappa light chains.

Inhibition of germ tube development of Aspergillus fumigatus in cell-free transudate produced in subcutaneous chambers in rabbits.

The interaction of Aspergillus fumigatus conidia with host factors produced in rabbits was studied by means of subcutaneous, perforated plastic chambers. Transudate fluid from spore-free chambers, sampled 30 days after implantation, supported germ tube development and rapid hyphal growth of A. fumigatus in an assay in vitro. Inoculation of spores into chambers implanted 30 days previously produced a rapid infiltration of leukocytes, predominately neutrophils, into the chamber fluid. Cell-free supernatants, prepared from transudates collected 5-6 days after inoculation, inhibited germ tube development in vitro. This inhibition was also demonstrated using lysates derived from 3.6 X 10(6) leukocytes obtained from chambers 5 days after inoculation with 1 X 10(7) spores. However, lysates derived from greater than 10(7) leukocytes obtained from pre-inoculated chambers as well as peritoneal exudate cells did not inhibit germ tube development in vitro. The inhibitory activity of cell free supernatants was not altered by heating at 56 degrees C for 30 min but was destroyed by pronase treatment as well as boiling. These results provide evidence for a host defense mechanism against rapid hyphal extension mediated by the extracellular release of inhibitory factors by leukocytes.

Hageman-factor-dependent fibrinolysis: generation of fibrinolytic activity by the interaction of human activated factor XI and plasminogen.

Human coagulation factor XI has been purified, and upon activation with Hageman factor fragments, was found to convert the fibrinolytic proenzyme plasminogen to plasmin. This proactivator activity was shown to be functionally and antigenically distinct from prekallikrein. When the gamma-globulin fractions of plasma deficient in Hageman factor, prekallikrein and factor XI were isolated, factor-XI-deficient plasma possessed two-thirds of the plasminogen proactivator activity of the Hageman-factor-deficient plasma, while prekallikrein deficient plasma had only one-third of the plasminogen proactivator activity. Thus, the Hageman-factor-dependent plasminogen proactivator previously reported to be present in the gamma-globulin fraction of normal human plasma is a function of prekallikrein and factor XI, while the activity observed in prekallikrein-deficient plasma is attributable to factor XI. When compared utilizing digestion of iodinated fibrin, prekallikrein and factor XIa had similar potency per active site; they were, however, far less active than urokinase.

Studies of binding of prekallikrein and Factor XI to high molecular weight kininogen and its light chain.

Prekallikrein and Factor XI have been reported to circulate as complexes with the coagulation cofactor high molecular weight (HMW)-kininogen. In this study we have shown that native HMW-kininogen possesses a strong binding site for prekallikrein and Factor XI with association constants of 3.4 x 10(7) M-1 and 4.2 x 10(8) M-1, respectively. The diminished binding of prekallikrein relative to Factor XI may, in part, account for the ability of kallikrein to leave the surface and interact with other molecules of Hageman factor and HMW-kininogen. Prekallikrein and Factor XI appear to compete for binding to HMW-kininogen, suggesting a single (or closely overlapping) binding site(s). The purified light chain derived from kinin-free HMW-kininogen is shown to compete with native MHW-kininogen for binding to Hageman factor substrates and direct binding of the isolated light chain to prekalikrein and Factor XI is demonstrated. This binding of the light chain to prekallikrein and Factor XI appears to be essential to the function of HMW-kininogen as a coagulation cofactor and further digestion of the light chain with excess kallikrein destroys its coagulant activity.

Characterization of human high molecular weight kininogen. Procoagulant activity associated with the light chain of kinin-free high molecular weight kininogen.

Human high molecular weight (HMW) kininogen has been isolated and was found to be a single chain protein of approximately equal to 120,000 daltons. Upon digestion with plasma kallikrein bradykinin is generated, and SDS gel electrophoresis of the kinin-free protein reveals an apparent loss in size of 15,000 daltons. The kinin-free kininogen retains full activity as a coagulation factor and consists of two chains: a heavy chain of approximately equal to 66,000 daltons disulfide-linked to a light chain of 37,000 daltons. The heavy chain of HMW kininogen shares antigenic determinants with LMW kininogen and possesses no detectable coagulant activity. The isolated light chain is shown to be responsible for the coagulant activity of HMW kininogen and contains a unique antigenic determinant that distinguishes HMW kininogen from low molecular weight kininogen.

Further studies of a humoral chemotactic abnormality in glomerulonephritis.

Chemotactic activity in the sera of patients with glomerulonephritis was compared under three simultaneously performed conditions: (1) incubation with buffer at 37 degrees C (CF-UNACT); (2) incubation with immune complexes at 37 degrees C (CF-ACT); (3) immediate heating at 56 degrees C (CF-56 degrees C). In all cases the generation of chemotactic factors was terminated by standard 'heat-inactivation' at 56 degrees C. Patients' CF-UNACT was similar to that of controls; patients' CF-ACT was significantly less than controls', but patients' CF-ACT and CF-56 degrees C was significantly greater than controls'. Patients CF-ACT and CF-56 degrees C were largely C5-dependent and were quantitatively similar. These divergent abnormalities could not be explained by spontaneous in vivo or in vitro (i.e., blood clotting) generation of complement chemotactic factors, the absence of Hageman factor-dependent chemotactic activity, or the presence of humoral inhibitors in patients' sera. It appears that inital 56 degrees C heating liberates C5-dependent chemotactic activity, a procedure that is usually believed to block its formation. Terminal 56 degrees C heating after 37 degrees C incubation did not generate such activity in CF-UNACT. The duration or sequence of heating at 56 degrees C, or both, are critical determinants for final expression of chemotactic activity in patients' sera, when viewed in relation to 37 degrees C incubation with immune complexes or buffer.

Association of factor XI and high molecular weight kininogen in human plasma.

Factor XI and high molecular weight kininogen were found associated in normal human plasma at mol wt 380,000 as assessed by gel filtration on Sephadex G-200. The molecular weight of Factor XI in high molecular weight kininogen-deficient plasma was 175,000, the same value obtained for purified Factor XI. When high molecular weight kininogen-deficient plasma was reconstituted with purified high molecular weight kininogen, all of the Factor XI was then found at mol wt 380,000. Complex formation was also demonstrable upon incubation of Factor XI and highly purified high molecular weight kininogen. This complex was distinct from the prekallikrein-high molecular weight kininogen complex; thus high molecular weight kininogen forms bimolecular complexes with either Factor XI or prekallikrein but does not form a trimolecular complex that includes both Factor XI and prekallikrein. Neither Hageman factor nor plasminogen were found associated with high molecular weight kininogen; binding to high molecular weight kininogen appeared to be a specific property of the Hageman factor substrates.

Characterization of ten species of mycobacteria by reaction-gas-liquid chromatography.

The methanolic tetramethylammonium hydroxide whole-cell lysates of nine species of mycobacteria and the "rhodochrous complex" were examined by gas-liquid chromatography. The gas chromatographic patterns produced 10 characteristic chromatographic groups that corresponded to the 10 species studied. The gas chromatograms of Mycobacterium tuberculosis were very easily distinguished from the other mycobacterial strains by high levels of a component that eluted much later than the other components. The remaining nine species could be distinguished on the basis of characteristic components and by different amounts of components common to more than one species. This study demonstrated that direct gas-liquid chromatographic characterization of M. tuberculosis and other myobacterial species was not only feasible but practical in the clinical laboratory.

Hageman factor substrates. Human plasma prekallikrein: mechanism of activation by Hageman factor and participation in hageman factor-dependent fibrinolysis.

Two molecular forms of prekallikrein can be isolated from pooled normal human plasma. Their approximate molecular weights by sodium dodecyl sulfate-gel electrophoresis are 88,000 and 85,000. The two bands observed are shown to represent prekallikrein by functional, immunochemical, and structural criteria. Both forms are cleaved by activated Hageman factor, they appear to share antigenic determinants, they are not interconvertible upon incubation with activated Hageman factor or kallikrein, and the ratio of kinin-generating, and plasminogen-activating activities of the preparations are independent of the relative proportion of each band. Activated Factor XII converts prekallikrein to kallikrein by limited proteolysis and two disulfide-linked chains designated kallikrein heavy chain (Mr = 52,000) and kallikrein light chains (Mr = 36,000 or 33,000) are formed. The active site is associated with the light chains as assessed by incorporation of [3H]diisopropyl fluorophosphate. No dissociable fragments were observed in the absence of reducing agents. However, kallikrein could digest prekallikrein to diminish its molecular weight by 10,000. In addition, two factors capable of activating plasminogen to plasmin have been isolated; one is identified as kallikrein. The second principle fractionates with Factor XI and is demonstrable in normal and prekallikrein-deficient plasma.

Inhibition of complement by mouse serum: selective inactivation of the fourth component of human complement.

Bound human C4b on EAC4 is rapidly inactivated in the presence of murine serum reagents. The functional characteristics of this inactivation suggest that it is probably caused by factor(s) homologous to human C3bI-C4bI: inactivation is temperature-dependent and occurs without concomitant consumption of the inactivator(s); loss of hemolytic function is associated with the cleavage of the bound C4b into C4c, which is released, and C4d, which is retained on the cell membrane. Murine serum reagents inhibit bound human C4b far more efficiently than C3b and may therefore be employed to selectively inhibit C4b.

Identification of prekallikrein and high-molecular-weight kininogen as a complex in human plasma.

Prekallikrein and high-molecular-weight kininogen were found associated in normal human plasma at a molecular weight of 285,000 as assessed by gel filtration on Sephadex G-200. The molecular weight of prekallikrein in plasma that is deficient in high-molecular-weight kininogen was 115,000. This prekallikrein could be isolated at a molecular weight of 285,000 after plasma deficient in high-molecular-weight kininogen was combined with plasma that is congenitally deficient in prekallikrein. Addition of purified 125I-labeled prekallikrein and high-molecular-weight kininogen to the respective deficient plasma yielded a shift in the molecular weight of prekallikrein, and complex formation could be demonstrated by incubating prekallikrein with high-molecular weight kininogen. This study demonstrates that prekallikrein and high-molecular-weight kininogen are physically associated in plasma as a noncovalently linked complex and may therefore be adsorbed together during surface activation of Hageman factor. The complex is disrupted when these proteins are isolated by ion exchange chromatography.

Human C3 and C5: subunit structure and modifications by trypsin and C42-C423.

The subunit composition of human C3 and C5 was analyzed. Acrylamide gel electrophoresis of the fully reduced and dissociated proteins disclosed a similar structure, consisting of one alpha and beta subunit, linked together by one or more disulfide bonds. The approximate molecular weights for the alpha and beta subunits of C3 as well as C5 were 140,000 and 80,000 respectively. C42 caused cleavage solely of C3alpha, whereas trypsin affected both C3alpha as well as C3beta. A characteristic subunit modification by both enzmes indicated that C3alpha constitutes the source of C3a. C423 as well as trypsin exclusively affect C5alpha. C5a therefore appears to originate from the C5alpha subunit. The mode of primary cleavage by C423 and trypsin differs, giving rise to different forms of C5b. The questions is raised if multiple forms of C5a also exist. It appeared from our studies that certain forms of C5b may retain portions of the alpha subunit, which could potentially release some biologically active split products following secondary cleavage by the appropriate enzyme.