CYP2D6 genotype determination in the Danish population

CYP2D6 genotyping was carried out by XbaI restriction fragment length polymorphism analysis and polymerase chain reaction in 168 healthy Danish volunteers, 77 extensive metabolizers (EM) and 91 poor metabolizers (PM) of sparteine. All EM were genotyped correctly as heterozygous or homozygous for the functional (wild type) gene, D6-wt. However, the D6-wt gene was apparently also present in 11 (12%) of the PM who accordingly were incorrectly genotyped as EM. The specificity of genotyping PM thus was 100% but the sensitivity was only 88%. The most common allele was the D6-wt with an apparent frequency of 0.741 (0.026) in the Danish population and the second most common allele was the D6-B with an apparent frequency of 0.194 (0.024). The median (range) of the sparteine metabolic ratio (MR) in 47 homozygous D6-wt EM was 0.28 (0.11–4.10) and the corresponding value in heterozygous EM was 0.36 (0.11–9.10). The median difference was 0.09 (95% confidence interval: 0.02–0.16). CYP2D6 phenotyping is a promising tool in tailoring the individual dose of tricyclic antidepressants, some neuroleplics and some antiarrhythmics. However if the genotype test could be improved with regard to both sensitivity in PM and the ability to predict CYP2D6 activity in EM then it would be of even greater clinical value in therapeutic drug monitoring.     

Amino acid sequences and structures of chicken and turkey beta 2-microglobulin

The complete amino acid sequences of chicken and turkey beta 2-microglobulins have been determined by analyses of tryptic, V8-proteolytic and cyanogen bromide fragments, and by N-terminal sequencing. Mass spectrometric analysis of chicken beta 2-microglobulin supports the sequence-derived Mr of 11,048. The higher apparent Mr obtained for the avian beta 2-microglobulins as compared to human beta 2-microglobulin by SDS-PAGE is not understood. Chicken and turkey beta 2-microglobulin consist of 98 residues and deviate at seven positions: 60, 66, 74-76, 78 and 82. The chicken and turkey sequences are identical to human beta 2-microglobulin at 46 and 47 positions, respectively, and to bovine beta 2-microglobulin at 47 positions, i.e. there is about 47% identity between avian and mammalian beta 2-microglobulins. The known X-ray crystallographic structures of bovine beta 2-microglobulin and human HLA-A2 complex suggest that the seven chicken to turkey differences are exposed to solvent in the avian MHC class I complex. The key residues of beta 2-microglobulin involved in alpha chain contacts within the MHC class I molecule are highly conserved between chicken and man. This explains that heterologous human beta 2-microglobulin can substitute the chicken beta 2-microglobulin in exchange studies with B-F (chicken MHC class I molecule), and suggests that the MHC class I structure is conserved over long evolutionary distances.     

The use of post-embedding immunoelectron microscopy in the diagnosis of glomerular diseases. Comparison of immunoelectron microscopic and immunofluorescence studies.

Fifty renal biopsies were studied by immunoelectron microscopy after embedding in a partly hydrophilic polyacrylic resin (LR White). Immunofluorescence studies were carried out on frozen sections of parallel tissue samples. Polyacrylic embedding gave good preservation of the renal ultrastructure and precise localization of immunoglobulin and C3c antibodies within glomerular electron-dense deposits. Non-specific staining of plasma proteins within vascular lumina could easily be detected. There was good correlation between immunoelectron and immunofluorescence microscopy. Immunoelectron microscopy is a very sensitive method, which can detect small amounts of antigen. More cases were, however, positive by immunofluorescence than by immunoelectron microscopy. This discrepancy may be explained by difference in sample size, and by difference in resolution of morphological details (electron microscopy versus fluorescence microscopy).     

One-year results: palatal implants for the treatment of obstructive sleep apnea.

OBJECTIVE: To evaluate long-term effectiveness of palatal implants for treatment of mild to moderate obstructive sleep apnea (OSA). STUDY DESIGN: A prospective study of 26 referred patients with a pretreatment apnea-hypopnea index (AHI) of 10 to 30 and a body mass index of < or =30, representing an extended follow-up of a subset of 41 patients enrolled in previous short-term trials. RESULTS: Twenty-one of 26 patients (80.8%) experienced a decrease in AHI. Fifteen of 26 patients (57.7%) had a follow-up AHI <10 at 1 year, whereas 13 patients (50%) had a 50% or greater reduction to an AHI <10 at 1 year. Mean AHI was reduced from 16.5 +/- 4.5 at baseline to 12.5 +/- 10.5 at 3 months (P < 0.014) and to 12.3 +/- 12.7 at 1 year (P < 0.019). CONCLUSIONS: Patients initially responding to palatal implants with improved AHI maintained improvement through long-term follow-up at 1 year.     

Assay for the specificity of monoclonal antibodies in crossed immunoelectrophoresis

A method is described based on crossed immunoelectrophoresis of a complex antigen mixture in agarose gel followed by incubation of the gel with the monoclonal antibody. The bound monoclonal antibody is detected by the use of a secondary enzyme-labelled antibody.Using this technique we have been able to identify the precipitate arc in crossed immunoelectrophoresis of major histocompatibility complex (MHC) class I molecules in a mixture of all detergent solubilized cell membrane molecules by means of a monoclonal antibody, the specificity of which was known independently to be against MHC class I molecules. In other experiments using the same technique we demonstrated the reaction of a monoclonal antibody specific for chicken Ig light chains.     

Involvement of CD14 and beta2-integrins in activating cells with soluble and particulate lipopolysaccharides and mannuronic acid polymers

Lipopolysaccharide (LPS) and related bacterial products can be recognized by host inflammatory cells in a particulate, bacterium-bound form, as well as in various soluble, released forms. In the present study we have compared the mechanisms used by LPS, detoxified LPS (DLPS), and mannuronic acid polymers (M-polymers), in solution or covalently linked to particles, in stimulating monocytes to tumor necrosis factor (TNF) production. The addition of recombinant LPS binding protein (LBP) and/or soluble CD14 (sCD14) enhanced the production of TNF from monocytes stimulated with soluble LPS, DLPS, or M-polymer, but did not affect the response to M-polymer or DLPS attached to particles. Treatment of monocytes with antibody to CD14, CD18, or CD11b showed that CD14, but not CR3 (CD11b/CD18), mediated monocyte TNF production in response to the soluble antigens. In contrast, anti-CD14, anti-CD11b and anti-CD18 monoclonal antibodies all inhibited the response to the particulate stimuli. On the other hand, B975, a synthetic analog of Rhodobacter capsulatus lipid A, completely abrogated the monocyte TNF response induced by LPS but did not affect the TNF induction by DLPS or M-polymer, either in soluble or particulate forms. These data demonstrate that the engagement of immune receptors by bacterial products such as LPS, DLPS, and M-polymer is dependent upon the presentation form of their constituent carbohydrates, and that factors such as aggregation state, acylation, carbohydrate chain length, and solid versus liquid phase of bacterial ligands influence the mechanisms used by cells in mediating proinflammatory responses.     

The influence of pH on heat denaturation of anti-haemophilic cryoprecipitate

The influence of pH on heat denaturation of anti-haemophilic cryoprecipitate (CP) was studied by using phosphate and citrate buffers at three different pH levels for processing lyophilized, heat treated CP. The solubility and the content of FVIII:C and fibrinogen were determined following heating at 68 degrees C for 24 hours and compared to "ordinary", non-heated CP. Both the solubility and the biological activity were best preserved in the most acidic samples (pH 5.7-6.7), these batches equalled non-heated CP. At higher pH, heat treatment resulted in reduced solubility and a more pronounced loss of FVIII:C and fibrinogen. Amino acids (Syntamin 17) have previously been shown to stabilize CP during heat treatment. Some of the stabilizing effect seems to be due to a large buffering capacity, maintaining a low pH during lyophilization and heating. Raising the pH level in Syntamin-CP from 6.6 to 7.8 resulted in decreased solubility and FVIII:C content. The quality of heat treated, acidic Syntamin-CP was comparable to that of heated, acidic phosphate- and citrate-CP. We conclude that buffers used for processing heat treated CP should be of low pH, and that acidic buffers may replace Syntamin without lowering the quality of the heated product.