Chemical shift differences between free and Fab-bound peptide correlate with a two-stage selection of peptide sequences from a random phage display library to delineate critical and non-critical residues for antibody recognition

Epitope libraries provide a method to identify peptide ligands for antibodies, receptors or other binding proteins. As such, they provide a powerful tool to rapidly identify lead ligands in the drug discovery process. In an attempt to correlate structural information with the results from peptide screening, we have used NMR spectroscopy of peptide/antibody complexes to demonstrate that core residues identified through a two-stage selection process undergo a larger structural change upon binding antibody than do positions in the peptide amenable to a variety of side chains. The model system used was the M2 monoclonal antibody/Flag? octapeptide epitope system. We have analyzed two peptides: Ac-Asp-Tyr-Lys-Leu-Gly-Asp-Asp-Leu-NH₂ (peptide l), which contains several non-core positions randomized, and Ac-Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Leu-NH₂ (peptide 2), which closely corresponds to the original Flag? sequence. Enrichment of the peptides with ¹⁵N facilitated the investigation by permitting spectral editing of the peptide resonances in the presence of antibody. For peptide 1 the absolute shifts for the free vs. Fab-bound peptide were found to be largest for the amide groups of Asp-1 and Asp-6, in agreement with classification of these residues as critical by the phage display library selection process. For peptide 2 the largest absolute shifts were observed for Asp-1 and Asp-4, with the other aspartic acid residues also showing significant but smaller changes. © Munksgaard 1995.     

Four unusual cases of Spinal Cord Compression

Four cases of spinal cord compression are presented, the causes being tuberculoma, haemangioma, gouty tophus and exophytic glioma; the first three being extradural, extraosseous in location. A short discussion follows. These cases are presented to remind the reader to keep less common causes in mind when dealing with a case of spinal cord compression.     

Effect of inhaled atrial natriuretic peptide on methacholine bronchoconstriction in asthma

We have previously demonstrated that intravenous and inhaled atrial natriuretic peptide (ANP) significantly inhibits histamine induced bronchoconstriction in asthmatic patients. The current study was designed to determine whether inhaled ANP was also able to inhibit the effects of methacholine. Eight atopic asthmatic patients (five women) were studied: mean (SD) age 38.2 (8.3) years flow expiratory volume per second (FEV₁) 2.97 (0.60) litres, equivalent to 92 (13) % of the predicted. Each had demonstrated at least mild bronchial hyperreactivity to inhaled methacholine at screening (geometric mean PC20 l.02mg/ml; range 0.1l–6.54mg/ml). Patients attended for 3 study days and after baseline spirometry received 3.5 ml saline (placebo), 0.1 mg ANP or 1 mg ANP (ANP dissolved in 3.5ml saline) in a randomized, double-blind manner via a Mizer aerosol conservation device. Aerosolization took approximately 9 min and FEV₁ was repeated at 0.5, 1.5 and 3 min after completion. Immediately thereafter each patient received a 2 min inhalation of methacholine at a dose individually calculated to give a 25% fall in FEV₁ (as extrapolated at their initial screening visit) and the FEY₁ was followed over the next 20 min. Mean (SEM)% FEV₁ did not change significantly after ANP being -4.3 (1.7), -3.2 (2.7) and -2.4 (1.2) after placebo, 0.1 mg ANP and 1 mg ANP respectively. The mean (SEM) maximum fall in FEV₁ after methacholine was as follows: placebo 26.9 (5.7)%, 0-1 mg ANP 18.2 (4.3)% and 1.0mg 11.2(2 7)%, (P < 0.05 placebo vs 1 mg ANP). These results demonstrate that ANP offers significant protection against methacholine induced bronchoconstriction in asthmatic patients.     

A Comparison Between M.R.I. and C.T. in Acute Spinal Trauma

Magnetic Resonance Imaging (MRI) at 0.3T and Computed Tomography (CT) were compared in the retrospective evaluation of 34 patients with acute spinal cord injury. MRI was highly accurate in the imaging of vertebral body fracture, and spondylitk changes, and is the method of choice for imaging ligament injury, traumatic disc protrusion and spinal cord compression. It was also useful for the identification of subtle subluxations in the sagittal plane. CT remains the method of choice for imaging neural arch fractures. MRI at 0.3T is a valid technique for assessing patients with acute spinal trauma.     

Production and characterization of a new monoclonal antibody effective in recognizing the CD3 T-cell associated antigen in formalin-fixed embedded tissue

Phenotypic analysis of lymphoproliferative disorders is now considered mandatory for accurate classification which is the basis for optimum patient management. This is presently carried out in most cases using a range of antibodies recognizing B and T-cell antigens effective in paraffin sections, and an antibody to CD3 is currently a key member of such panels, indicating T-cell phenotype. Current antibodies to CD3 are polyclonal with the inherent disadvantages of this type of reagent compared to monoclonal antibodies. In this study, we have used a recombinant fusion protein representing part of the epsilon subunit of the CD3 molecule to generate a novel monoclonal antibody (NCL-CD3-PS1) effective in paraffin sections. The antibody has been characterized biochemically and by immunohistochemistry using a wide range of normal and pathological tissues. Lineage and phenotype specificity have been supported in our study and results from other laboratories are awaited with interest.     

Binding of cardiolipin to polystyrene beads: evidence for a lamellar phase orientation

Summary. The association of cardiolipin with polystyrene beads was studied using ³¹P-NMR and electron microscopy. In the presence and absence of fetal calf serum, cardiolipin appeared to bind to the polystyrene beads in lamellar phase as assessed by ³¹P-NMR imaging. Electron microscopic analysis revealed an even coating of phospholipid about the beads with extensive micelle binding. Cardiolipin-coated beads challenged with ACA-positive sera followed by immunogold indicated antibody bound to micelles associated with the bead. Studies conducted with ACA IgG purified from patient sera indicated that some ACA bound to CL beads in the absence of a source of ACA cofactor (i.e. gelatin-blocked beads), some ACA required β₂-GPI for binding (i.e. no binding in the presence of β₂-GPI-depleted plasma), whereas other ACA which showed negliglible binding with gelatin-blocked beads, showed enhanced binding in the presence of /?₂-GPI-depleted plasma. The data indicate that: (1) cardiolipin binds to polystyrene beads in lamellar phase, (2) ACA bind to phospholipid micelles bound directly to the polystyrene beads, and (3) ACA differ between individuals displaying varying phospholipid and phospholipid/cofactor substrate specificities.     

Subchronic and Chronic Inhalation Toxicity of Antimony Trioxide in the Rat

Fischer 344 rats were exposed by inhalation to Sb₂O₃ (antimonytrioxide) dust at exposure levels of 0, 0.25, 1.08, 4.92, and23.46 mg/m³ for 6 hr/day, 5 days/week for 13 weeks followedby a 27-week observation period. Subsequently, an inhalationon-cogenicity study was conducted at exposure levels of 0, 0.06,0.51, and 4.50 mg/m³ for 12 months followed by a 12-month observationperiod. The Sb₂O₃ in the subchronic study had a mass medianaerodynamic diameter (MMAD) of 3.05 ± 0.21 microns (mean± SD) with a geometric standard deviation (GSD) of 1.57± 0.06. In the chronic study, the MMAD was 3.76 ±0.84 and the GSD was 1.79 ± 0.32. Except for the eyes,no adverse clinical observations were attributed to Sb₂O₃ ineither study. In the subchronic study, corneal irregularitieswere seen after about 2 weeks of exposure and did not abateduring the observation period. In the chronic study, ophthalmoscopicevaluation at 24 months revealed a dose-related increase incataracts of 11, 24, 28, and 32% (both sexes combined) for eachgroup, respectively. Body weights were significantly lower (6%)than the control group's weights in the 23.46 mg/m³ males inthe subchronic study. These rats did not recover this weightduring the 27-week observation period. Body weights of the femalesin both studies and males in the chronic study were unaffected.There were no Sb₂O₃ effects on clinical chemistry or he-matologyin either study. Mean absolute and relative lung weights weresignificantly increased in the 4.92 and 23.46 mg/m³ groups inthe subchronic study. The 23.46 mg/m³ group's lung weights didnot recover to control levels during the 27-week observationperiod. Lung weights for rats in the chronic study were unaffected.Microscopic changes in the lungs in the subchronic and chronicstudy were limited to subacute-chronic interstitial inflammation,increased numbers of alveolar-in-traalveolar macrophages, foreignmaterial in the alveolar-in-traalveolar macrophages in the peribronchialand perivascular (chronic study only) lymphoid aggregates andin the peribronchial lymph nodes, granulomatous inflammation/granulomas,and fibrosis. In the chronic study, any observed neoplasms occurredwith comparable incidence among all groups and were within thehistorical range for controls. Clearance of Sb₂O₃ from the lungwas burden dependent and was reduced by 80/ in the 4.50 mg/m³group in the chronic study. The previously reported studies,which found Sb₂O₃ to be a carcinogen, were run at higher lungburdens. Under the exposure conditions of the current study,Sb₂O₃ was not a carcinogen.     

ANTIBODIES MADE AGAINST A FORMALDEHYDE-PROTEIN ADDUCT CROSS REACT WITH AN ACETALDEHYDE-PROTEIN ADDUCT. IMPLICATIONS FOR THE ORIGIN OF ANTIBODIES IN HUMAN SERUM WHICH RECOGNIZE ACETALDEHYDE-PROTEIN ADDUCTS

Acetaldehyde, the major metabolite of ethanol, reacts with lysineand other free amino groups on proteins to form acetaldehyde-proteinadducts. The presence of antibodies which recognize such acetaldehyde-proteinadducts in sera from alcoholics has been attributed to an immuneresponse to such adducts. Complicating this conclusion is thefinding that sera from non-alcoholic control subjects also containantibodies which recognize acetaldehyde-protein adducts. Inthe current research we sought to determine whether antibodieswhich recognize epitopes formed by the reaction of a proteinwith acetaldehyde can be formed in response to a protein modifiedwith a structurally related protein adduct. We modified lysineresidues on apolipoprotein (apo) B-100 with acetaldehyde andformaldehyde under reducing conditions, to form -N-methyl-and-N-ethyl-lysine residues, and with acetic anhydride to form-N-acetyl-lysine residues, and made antibodies against thesemodified proteins in guinea-pigs. In ELISA assays antibodiesmade against methylated apoB-100 (Me-apoB) cross-reacted effectivelywith ethylated apoB-100 (Et-apoB), while antibodies made againstacetic anhydride-modified apoB-100 did not cross-react. We concludethat methyl-lysine shares one or more immunoreactive epitopeswith ethyl-lysine, and that antibodies which recognize acetaldehyde-modifiedproteins can be formed in response to formaldehyde-modifiedproteins. We demonstrate that sera from both alcoholics andnon-drinkers contain antibodies which recognize Me-apoB andEt-apoB and that the titres of these antibodies are comparable.These data raise the possibility that some human serum antibodieswhich recognize acetaldehyde-modified protein epitopes may havebeen made against formaldehyde-modified protein epitopes. Thesedata also illustrate the difficulty in assigning a unique causalrelationship between the presence of an antibody, and the immunogenresponsible for the formation of such antibody.     

Data sources, bed supply and performance in hospital systems: An Anglo-Czechoslovak comparison

Background: There were a number of similarities, except fortheir effectiveness, in the health care systems of Czechoslovakiaand England and Wales between the Second World War and the late1980s. In a comparison of Czechoslovakia with England and Wales,the objectives of this study were to examine data sources andto report time trends and regional distributions in hospitalbed supply, hospital doctor supply and hospital utilisation.Methods: For the specialties of general medicine and generalsurgery in both countries from 1960 to 1986, data were collatedon bed supply, hospital doctor supply, discharge rates and lengthof stay. Issues concerning the comparability of the data wereaddressed, for example those of the definitions of specialty,length of stay and casemix. Results: In the period 1960 to 1986,in the specialties of general medicine and general surgery,there was a relative excess in the supply of hospital doctorsand beds in Czechoslovakia compared with England and Wales.Hospital performance in terms of discharge rates, dischargesper bed and length of stay remained relatively static in Czechoslovakiaduring this period compared to marked increases in dischargerates and reduced length of stay in England and Wales. Bothcountries recorded reductions in the regional variation of bedand doctor supply and hospital utilisation. Conclusions: Internationalstudies of hospital utilisation need to be interpreted carefullyin the light of definitions of hospital stay, casemix, the useof day cases and the availability of other services. Subjectto these caveats, discharge rates were high and duration ofstay long In Czechoslovakia compared with England and Wales;however, both countries achieved important improvements in regionalequity.