Structural and functional characteristics of the B-domain-deleted recombinant factor VIII protein, r-VIII SQ.

Recombinant factor VIII SQ (r-VIII SQ), ReFacto, is a recombinant factor VIII product similar to the smallest active factor VIII protein found in plasma-derived factor VIII (p-VIII) concentrates. The protein comprises two polypeptide chains of 80 and 90 kDa and lacks the major part of the heavily glycosylated B-domain i.e. amino acids Gln744 to Ser1637. r-VIII SQ retains six potential glycosylation sites for N-linked oligosaccharides at asparagine residues 41, 239, 582, 1685, 1810 and 2118. We describe a thorough comparison of the characteristics of r-VIII SQ with those of p-VIII. The primary and secondary structures of r-VIII SQ were in good agreement with that of B-domain-deleted p-VIII (p-VIII-LMW) as shown by SDS-PAGE, Western blotting with antifactor VIII antibodies, tryptic mapping, amino acid sequence analysis and circular dichroism spectroscopy. A few divergences also existed. Thus r-VIII SQ was shown to contain a small amount of the single chain primary translation product of 170 kDa and also the product specific sequence of 14 amino acids, the SQ-link, in the C-terminal end of the 90 kDa chain. It was shown that r-VIII SQ had a high specific activity of about 14,000 IU VIII:C/mg as determined by use of a chromogenic substrate assay. The r-VIII SQ protein was comparable to p-VIII forms with a retained B-domain, in terms of potency measured by a chromogenic substrate or a two-stage clotting assay, in interactions with thrombin, and with activated protein C (APC) in combination with Protein S. The ability of r-VIII SQ to participate as a cofactor in factor Xa generation in a mixture of factors IXa and X, phospholipid and calcium was in conformity with that of p-VIII. Furthermore r-VIII SQ had a good binding capacity for phospholipid vesicles and von Willebrand factor (vWF) as shown in gel filtration studies. The same kinetics in binding to von Willebrand factor was found for r-VIII SQ and p-VIII as determined by real-time biospecific interaction analysis (BIA) with use of the BIAcore instrument. The apparent association rate constant was 4 x 10(6) M(-1)s(-1). Two dissociation rate constants were found, 1 X 10(-2)s(-1) and 4 x 10(-4)s(-1). The results extend the present knowledge that the factor VIII B-domain is dispensable for the factor VIII cofactor function in hemostasis.

Novel forms of B-domain-deleted recombinant factor VIII molecules. Construction and biochemical characterization.

Recombinant molecules similar to the smallest active plasma-derived factor VIII molecule, a complex of an 80-kDa and a 90-kDa polypeptide chain lacking the B domain, have been produced using various factor VIII cDNA constructs in order to obtain primary translation products which were efficiently processed into the 80 + 90-kDa complex. Three types of single-chain cDNAs encoding B-domain-deleted derivatives factor VIII were designed, taking account of sites at Arg740 and Glu1649, assumed to be important for processing factor VIII. In the type 1 constructs, either Arg747, Arg752, or Arg776 in the N-terminal region of factor VIII B domain was fused to the N-terminus (Glu1649) of the 80-kDa subunit. In the type 2 construct r-VIII SQ, Ser743 was fused to Gln1638, creating a link of 14 amino acids between the C-terminus (Arg740) of the 90-kDa chain and N-terminus of the 80-kDa chain, whereas in type 2 r-VIII RH, Arg747 was fused to His1646. In the type 3 constructs, the B-domain was completely removed or replaced with 1-4 Arg residues. After expression in Chinese hamster ovary cells, the type 1 derivatives and the type 3 derivatives with 0-2 Arg residues inserted were found to be only partially processed and contained a large amount of the 170-kDa primary translation product. In contrast, most of the type 2 derivatives r-VIII SQ and r-VIII RH and the type 3 derivatives r-VIII R4 and r-VIII R5 containing three or four extra Arg residues preceding the N-terminus of the 80-kDa chain were processed into the desired 80 + 90-kDa chain complexes. The feature common to the most efficiently processed factor VIII deletion derivatives was that they contained the recognition motif for proteolytic cleavage by the membrane-bound subtilisin-like protease furin, which is expressed in most types of cells; that is, basic amino acid residues at positions -1 and -4 relative to the cleavage site at Glu1649. Biochemical studies of r-VIII SQ and r-VIII R5, two of the most effectively processed factor VIII derivatives, showed that both proteins had a normal factor VIII cofactor function, and had N- and C-termini of the 80-kDa and 90-kDa chains corresponding to those found in plasma-derived factor VIII.

[Partial versus total meniscectomy. A prospective, randomized study]. / Partiel versus total meniskektomi. Et prospektivt, randomiseret studie.

Two hundred patients with a meniscus lesion were peroperatively allocated to partial or total meniscectomy. The results were compared at one year and at 6.3-9.8 years (median 7.8). After one year, more patients with partial meniscectomy (90%) than with total meniscectomy (80%) had no complaints (p = 0.029). At the late review these figures were 62% and 52% respectively (p = 0.18). However, patients with partial meniscectomy had the highest functional scores. In five percent knee joint function had improved and in 35% deteriorated, in the latter group without detectable difference between partial and total meniscectomy. The incidence of lateral laxity rose from eight to 47%, most frequently seen after total meniscectomy. During the observation period radiological signs of knee joint degeneration changed from solely joint line narrowing into additional ridge formation and flattening of the femoral condyle, but unrelated to whether partial or total meniscectomy had been performed. Following partial meniscectomy posterior horn lesions had the poorest functional outcome, but only if more than one third of the meniscal surface had been removed. The amount of meniscal tissue excised was inversely correlated to the level of knee joint function except in bucket handle lesions treated with partial meniscectomy. These lesions had the largest areas of meniscal tissue removed, but higher functional scores than posterior horn lesions. Preservation of the peripheral rim of the meniscus following partial meniscectomy was essential for the functional outcome after surgery.

The long term outcome of open total and partial meniscectomy related to the quantity and site of the meniscus removed.

Two hundred patients were randomly selected for either a partial or a total meniscectomy for a meniscal tear during open operation. They were followed for a median of 7.8 years after operation. After partial meniscectomy, posterior horn tears had the worst outcome, but this was only apparent when more than two-thirds of the meniscus had been removed. The amount of meniscal tissue excised was inversely related to the function of the knee, except with bucket-handle tears treated by partial meniscectomy. Patients with bucket-handle, anterior and posterior horn tears had similar functional results after total meniscectomy. Preservation of the peripheral rim of the meniscus following partial meniscectomy produces the best functional results.

Partial versus total meniscectomy. A prospective, randomised study with long-term follow-up.

Two hundred patients with a meniscal lesion were peroperatively allocated to partial or total meniscectomy in a random manner. The results were compared at one year and at 6.3 to 9.8 years (median 7.8). After one year more patients with partial meniscectomy (90%) than with total meniscectomy (80%) had no complaints. At the later review these figures were 62% and 52%, respectively (p = 0.18). However, patients with partial meniscectomy had higher functional scores. The deterioration in function between the first review and the second showed no significant difference in the two treatment groups. The incidence of mediolateral instability rose from 8% to 47% and was more frequent after total than after partial meniscectomy. Between the two reviews the radiological signs of knee degeneration increased with no difference between the two treatment groups.

[Screening for diabetic retinopathy]. / Screening for diabetisk retinopati.

Photographic screening for diabetic retinopathy has been carried out for 40 months in an Eye Department. The results are described, with particular emphasis on cost-benefit. It is shown that the procedure finances itself with a wide margin. It probably provides better control of retinopathy than traditional clinical procedures. There may thus be a potential to reduce human suffering and the cost to society of visual impairment. The screening also provides the Department with better photographic services in general.

Detection and quantitation by lysis-filtration of bacteremia after different oral surgical procedures.

Patients with bacteremia after dental extraction, third-molar surgery, dental scaling, endodontic treatment, and bilateral tonsillectomy were studied by means of lysis-filtration of blood samples with subsequent aerobic and anaerobic incubation. Samples were obtained before, during, and 10 min after treatment. Bacteremia was observed in 100% of patients after dental extraction, 55% of patients after third-molar surgery, 70% of patients after dental scaling, 20% of patients after endodontic treatment, and 55% of patients after bilateral tonsillectomy. Anaerobic microorganisms were isolated more frequently than aerobic microorganisms were, and viridans group streptococci were the most commonly isolated bacteria. Ten minutes after treatment, the frequency as well as the magnitude of bacteremia showed pronounced reduction.

Plasmacytoma of the uvea in a case of multiple myeloma.

A case of plasmacytoma in the uvea, both clinically and grossly anatomically mimicking a malignant melanoma, is described in a 63-year-old man who died from multiple myeloma. This is an extremely rare condition in the eye. Only one report of a similar tumour has been found in the literature.

Spectral studies of iron coordination in hemeprotein complexes: difference spectroscopy below 250 millimicrons.

In order to evaluate the feasibility of observing the spectral behavior of protein groups in the coordination sphere of the iron in hemeproteins, criteria are developed to determine whether or not the application of difference absorption spectroscopy to the study of complex formation will be successful. Absolute absorption spectra, 300-1100 mmu, from bacterial catalase complexes are displayed, and the infrared bands correlated with magnetic susceptibility values of similar complexes of other hemeproteins. Dissociation constants for the formation of cyanide and azide complexes of metmyoglobin, methemoglobin, bacterial catalase, and horseradish peroxidase are given. Difference spectra, 210-280 mmu, are displayed for cyanide and azide complexes of these hemeproteins. A band at 235-241 mmu is found in the difference spectra of all low-spin vs. high-spin complexes. The factors which favor the assignment of this band to a transition involving a histidine residue are presented.