Degenerative spondylolisthesis. Developmental or acquired?

Degenerative spondylolisthesis is four times more common in women than in men. Although this gender difference has long been recognised there has been no explanation for it. We have examined the radiographs and CT scans of 118 patients over the age of 55 years and of a control group under the age of 46 years. Our findings confirmed the presence of more sagittally-orientated facet joints in patients with degenerative spondylolisthesis but did not show that the gender difference can be explained by the morphology of the facet joint. Furthermore, we conclude that the increased angle of the facet joint is the result of arthritic remodelling and not the primary cause of degenerative spondylolisthesis. It is more likely to be due to loss of soft-tissue resilience with subsequent failure of the facet joints which are acting as the last restraints to subluxation.

An angiographic assessment of alteplase: double-bolus and front-loaded infusion regimens in myocardial infarction. DouBLE Study Investigators. Double Bolus Lysis Efficacy.

BACKGROUND: This study was designed to investigate the efficacy of alteplase double-bolus dosing compared with the front-loaded 90-minute infusion regimen in patients with acute myocardial infarction. Recent pilot studies have suggested that bolus dosing may provide improved efficacy in establishing early, complete, and sustained patency of the infarct-related artery in the thrombolytic treatment of acute myocardial infarction. METHODS AND RESULTS: In this multicenter, randomized, open-label trial, 461 patients with acute myocardial infarction received 100 mg alteplase as a front-loaded 90-minute infusion (15 mg bolus, then 50 mg over a 30-minute period, then 35 mg over a 60-minute period) or double bolus (two 50 mg bolus injections 30 minutes apart). All patients also received intravenous heparin and oral aspirin during and after alteplase treatment. The 90-minute angiographic patency rates were 74.5% in the double-bolus group and 81.4% in the infusion group (p = 0.08). Patency rates were also comparable for the two groups at 60 minutes (76.8% vs 77.5%) and 24 hours (95.5% vs 93.5%) after initiation of treatment. In-hospital mortality rates were 4.5% in the bolus group and 1.3% in the infusion group (p = 0.04); 30-day mortality rates were 4.5% and 1.7%, respectively (p = NS). The two-groups were comparable in frequency of all other adverse events. CONCLUSIONS: Double-bolus alteplase administration produced reperfusion rates comparable to front-loaded infusion, but in-hospital and 30-day mortality rates were higher in the double-bolus group. These findings are in agreement with those of the COBALT megatrial, which also reported a trend to higher mortality rates with double-bolus dosing.

Mycobacterium avium intracellulare: vertebral osteomyelitis.

A case of vertebral osteomyelitis secondary to Mycobacterium avium intracellulare mimicking Pott's paraplegia is reviewed. To our knowledge, it represents the first published case in a patient without gross immunocompromise. The importance of early differentiation from tuberculous osteomyelitis is stressed as treatment regimens differ and outcomes may be affected.

Pharmacokinetics of a slower clearing tissue plasminogen activator variant, TNK-tPA, in patients with acute myocardial infarction.

The rapid clearance of t-PA from plasma requires administration by intravenous (I.V.) infusion. A slower clearing, fibrin-specific rt-PA variant may allow single intravenous bolus administration, thereby simplifying dosing. This study was designed to characterize the pharmacokinetics of the slower clearing, fibrin-specific tissue-plasminogen activator variant, TNK-tPA, in patients with acute myocardial infarction (AMI) following a single I.V. bolus injection. Single I.V. bolus doses of 5 to 50 mg of TNK-tPA were studied in an open-label, multicenter, dose escalation study. A total of 113 AMI patients were enrolled. Blood sampling for pharmacokinetics was conducted in eighty-two patients (72 men, 10 women), with 5 to 27 patients per dose. TNK-tPA was administered as an I.V. bolus over 5-10 s. Following I.V. bolus administration, there was a biphasic elimination of TNK-tPA from plasma. The initial phase had a mean half-life that ranged from 11 +/- 5 to 20 +/- 6 min and was followed by a terminal phase with a mean half-life that ranged from 41 +/- 16 to 138 +/- 84 min. Mean TNK-tPA plasma clearance was 125 +/- 25 - 216 +/- 98 ml/min, and the initial volume of distribution was 4.3 +/- 2 - 8.4 +/- 6 1. A decrease in TNK-tPA plasma clearance with increasing TNK-tPA dose was noted. In addition, women and patients with lower body weight or older age had a slower plasma clearance. In conclusion, TNK-tPA has a slower plasma clearance in patients with AMI than that reported for rt-PA, allowing administration as a single I.V. bolus.

TNK-tissue plasminogen activator in acute myocardial infarction. Results of the Thrombolysis in Myocardial Infarction (TIMI) 10A dose-ranging trial.

BACKGROUND: TNK-tissue plasminogen activator (TNK-TPA) is a genetically engineered variant of TPA, which in experimental models has a slower plasma clearance and greater fibrin specificity and is 80-fold more resistant to plasminogen activator inhibitor-1 than alteplase TPA. METHODS AND RESULTS: The thrombolysis in Myocardial Infarction (TIMI) 10A trial was a Phase 1, dose-ranging pilot trial designed to evaluate the pharmacokinetics, safety, and efficacy of TNK-TPA in patients with acute myocardial infarction. One hundred thirteen patients with acute ST-segment elevation myocardial infarction presenting within 12 hours and without contraindications to thrombolysis were enrolled and treated with a single bolus of TNK-TPA over 5 to 10 seconds with doses ranging from 5 to 50 mg. TNK-TPA demonstrated a plasma clearance of 151 +/- 55 mL/min and a half-life of 17 +/- 7 minutes. Comparable values for wild-type TPA are 572 +/- 132 mL/min and 3.5 +/- 1.4 minutes, respectively. Systemic fibrinogen and plasminogen levels fell by only 3% and 13%, respectively, at 1 hour after TNK-TPA administration. TIMI grade 3 flow at 90 minutes was achieved in 57% to 64% of patients at the 30- to 50-mg doses. Seven patients (6.2%) experienced a major hemorrhage, which occurred at a vascular access site in six patients. CONCLUSIONS: TNK-TPA has a prolonged half-life so it can be administered as a single bolus. TNK-TPA appears to be very fibrin specific, and the initial patency and safety profiles are encouraging. Further study of this new thrombolytic agent is ongoing.

High-level expression of antibody-plasminogen activator fusion proteins in hybridoma cells.

We show that the mouse gamma 2b heavy chain or human beta-globin 3' untranslated region can greatly enhance protein expression in myeloma cells transfected by genes coding for antibody-plasminogen activator fusion proteins. Expression plasmids were constructed containing a cloned genomic heavy chain variable region from fibrin-specific monoclonal antibody 59D8, a cloned genomic constant region of the mouse gamma 2b heavy chain, and DNA sequence coding for either tissue-type plasminogen activator (tPA) or a segment of urokinase (UK) and their respective 3' untranslated sequences. Cell lines transfected with these constructs, pSVtPA (tPA) and pSVUKG(UK), produced extremely low levels of mRNA and protein (0.008-0.06 micrograms/ml) in comparison with the parental 59D8 myeloma cell line (7.6-10 micrograms/ml). In vitro nuclear run-off analysis indicated that the low steady-state levels of mRNA encoded by pSVUKG(UK) did not result from a lower rate of transcription of the transfected gene (relative to the rate of transcription of the endogenous heavy chain gene in the 59D8 parent cells). In an attempt to increase protein secretion, we assembled the expression plasmids pSVtPA(Ig), pSVUKG(Ig), and pSVUKG(beta), in which the 3' untranslated region of the mouse gamma 2b heavy chain or human beta-globin gene was substituted for the 3' untranslated region of the plasminogen activator gene. Analysis of supernatant media from cell lines transfected with these constructs showed an increase in recombinant protein secretion of 68 to 100 fold in comparison with that from cell lines transfected with pSVtPA(tPA) or pSVUKG(UK).

A recombinant chimeric plasminogen activator with high affinity for fibrin has increased thrombolytic potency in vitro and in vivo.

A recombinant plasminogen activator with high fibrin affinity and specificity was expressed by transfecting hybridoma cells with a plasmid that combines sequence coding for low molecular mass (32 kDa) single-chain urokinase-type plasminogen activator [scuPA(32kDa)] and anti-fibrin monoclonal antibody 59D8. The expression of the recombinant molecule [r-scuPA(32kDa)-59D8] was optimized by replacing the 3' untranslated region (initially that of high molecular mass scuPA) in the plasmid with the 3' untranslated region of either beta-globin or mouse immunoglobulin. This modification resulted in a greater than 100-fold improvement in the level of protein expression. The 103-kDa r-scuPA(32kDa)-59D8 protein displayed catalytic activity indistinguishable from that of high molecular mass scuPA and fibrin binding comparable to that of native antibody 59D8. r-scuPA(32kDa)-59D8 was 6 times more potent than high molecular mass scuPA in lysing a human plasma clot in vitro and was 20 times more potent than high molecular mass scuPA in the rabbit jugular vein model of thrombolysis. Molecules of this type may serve as prototypes for highly specific, antibody-targeted enzymes suitable for human use.

Recombinant antibodies possessing novel effector functions.

Our method for constructing an antifibrin antibody-t-PA chimeric protein can be adapted to form other bifunctional, antibody-targeted proteins. Once an appropriate targeting antibody is obtained, the investigator can derive the heavy chain loss variant cell lines and clone the functional heavy chain rearrangement transcribed by the hybridoma. Other useful reagents include antisera directed against mouse Fab and antisera against whatever effector component is to be combined with the antibody. These are helpful during the screening of transfectants and the characterization of the secreted fusion protein, and they allow for protein purification by affinity chromatography. An assay of the functional activity of the effector domain is also desirable. The apparent retention of enzymatic activity and substrate specificity in our antibody-targeted plasminogen activator hybrid demonstrates that even complex molecules with strict folding requirements and multiple intrachain disulfide bonds can be used to form hybrid recombinant proteins. We have documented by electrophoretic transfer blotting that the heavy chain-t-PA fusion protein is secreted in association with light chain in the form of a 180-kDa dimer. The heavy chains appear to be attached by disulfide bonds at the hinge region, as is the case with the heavy chains of natural immunoglobulins. Our method can be adapted to various uses. More or less of the antibody constant region could be employed, depending on the desired geometry and the immunologic interactions mediated by the Fc domain. We have made a recombinant fusion peptide containing an additional 100 constant region amino acids but found that its targeting and catalytic abilities did not differ from those of the smaller molecule. Recent reports indicate that it is possible to express an antibody Fv that has full antigen recognition and binding properties; such small immunoglobulins could minimize potential immunogenicity while affording full targeting capability. The use of a human constant region sequence may also provide a less immunogenic molecule, and, by transferring the complementarity-determining regions of the monoclonal antibody into human variable region sequence, it may be possible to completely "humanize" an antibody-directed chimeric protein. The application of these and other innovative approaches should soon make antibodies an attractive means of targeting a wide range of molecules, both in scientific investigation and in medical therapy.

Discrete automated chemistry system with tableted reagents.

An instrument/reagent system with tableted reagents has been developed for performing ultraviolet/visible photometric assays. Preformed disposable plastic cuvettes pass through a stationary track every 5 s, providing a maximum throughput of 698 tests/h. The photometric system consists of a single light source with a separate set of light guides for each of eight photometer stations; bichromatic readings are possible at any of seven wavelengths between 340 and 630 nm. Tablets for 30 different analytes have been developed, each of which contains all necessary components for a single determination. Tablets are dispensed from disposable dispensers into cuvettes under the direction of the system's microprocessor and are completely dissolved within 45 s by ultrasonic agitation. Sample volumes range from 2 to 20 microL. Test requests are selected on a cathode-ray tube display, and sample-container bar-code labels are automatically printed. Sample entry is totally random. The system is designed to provide reagent quality control and also serum and reagent blank corrections for each test. Examples of equilibrium, zero-order, and first-order kinetic assays are presented.