Impact of chronic catheterization and automated blood sampling (Accusampler) on serum corticosterone and fecal immunoreactive corticosterone metabolites and immunoglobulin A in male rats.

Jugular catheters were inserted in nine male rats under general isofluorane anesthesia and the catheters were connected to a commercially available computerized blood sampling device (Accusampler). Blood samples (150 microl) were collected every 4 h during the first 24 h after surgery and every 12 h during the following 72 h until 94 h after surgery, when the animals were killed. All fecal pellets were collected at blood sampling. Serum corticosterone and fecal concentrations of immunoreactive corticosterone metabolites and immunoglobulin A (IgA) were quantified by ELISAs. In blood, high corticosterone concentrations (>200 ng/ml) were recorded in the first samples obtained after surgery, but the concentrations decreased steadily during the day and became cyclical, showing a diurnal variation with high levels during evenings and low levels in the mornings. The automatic blood sampling itself did not result in recordable increases in serum corticosterone concentrations. The time delay between the presence of elevated corticosterone levels in blood and in feces was approximately 12 h. Fecal immunoreactive corticosterone metabolite levels remained elevated during the 94 h study period after surgery. The fecal concentrations of IgA showed substantial between-animal variation and decreased non-significantly after the surgery. Like serum corticosterone, fecal IgA showed a diurnal variation in amounts excreted, in this case with high values in the morning and low values in the evening. The concentrations of fecal corticosterone and IgA were negatively correlated in samples obtained before surgery but no correlation existed after surgery. This indicates that fecal immunoreactive corticosterone metabolites, but not IgA, constitute a good marker of acute stress. For immunoreactive corticosterone metabolites as well as for IgA, the concentration in feces correlated well with total excretion, making single fecal samplings usable as a measure of total secretion.

Identification of multiple sources of charge heterogeneity in a recombinant antibody.

Seven forms of a therapeutic recombinant antibody that binds to the her2/neu gene product were resolved by cation-exchange chromatography. Structural differences were assigned by peptide mapping and HIC after papain digestion. Deamidation of light chain asparagine 30 to aspartate in one or both light chains is responsible for two acidic forms. A low potency form is due to isomerization of heavy chain aspartate 102; the Asp102 succinimide is also present in a basic peak fraction. Forms with both Asn30 deamidation and Asp102 isomerization modifications were isolated. Deamidation of heavy chain Asn55 to isoaspartate was also detected. Isoelectric focusing in a polyacrylamide gel was used to verify the assignments. All modifications were found in complementarity determining regions.

Validation of an HPLC method for the analysis of the charge heterogeneity of the recombinant monoclonal antibody IDEC-C2B8 after papain digestion.

An HPLC procedure was validated for determining the purity with respect to the charge variant distribution of the recombinant monoclonal antibody (MAb) IDEC-C2B8 by high-performance ion-exchange chromatography. Papain was used to fragment the molecule into Fab and Fc fragments prior to chromatographic analysis. Fragmentation allowed the resolution of the variants arising from the cyclization of glutamine to pyroglutamate at the amino-terminus of the light and heavy chains (Fab-pE/Q variants) from the variants resulting from the processing of the carboxy-terminal lysine residues of the heavy chains (Fc-Lys variants). The assay demonstrated good linearity, yielding correlation coefficients of > 0.99 for total protein, Fc-Lys variants and Fab-pE/Q variants. Recovery of total protein from the column was 95.7%. Sample recovery studies demonstrated a mean accuracy of 102% for a Fab fragment over the range 2-10% of the total protein. The limit of detection was 0.2 microg and 0.1 microg for Fc and Fab variants, respectively. The repeatability of the assay and intermediate precision had relative standard deviation (RSD) values of < 1%. Parameters of the papain digest (time, digest stability, reagent stability, pH and papain vendor) and of the chromatography (mobile phase pH, stability, buffer concentration, and column lot and aging) were evaluated for robustness and determined to be acceptable. Data are presented demonstrating the suitability of the assay for determining the product purity of a recombinant MAb.

SEM and TEM analyses of isolated human retinal microvessel basement membranes in diabetic retinopathy.

Human retinas from persons with diabetic retinopathy and age-matched controls were rendered acellular by sequential detergent treatment. The resulting network of microvascular extracellular matrix (ECM) materials, including basement membranes (BMs), was compared by TEM and, following cryofracture, by SEM. Our study demonstrates that in diabetics, retinal capillary BM complexes are generally thickened and that their ECM subcomponents, including BM leaflets and BM-like pericytic matrix (PCM), are differentially altered. Two diabetic microvessel types were identified. In type A vessels, ECM expansion is manifested by loosely arranged combinations of concentric PCM layers and collagen fibrils with thickened subendothelial (EBM) and pericyte (PBM) BM leaflets. Type B vessels show densely compact central PCM masses and poorly recognizable EBMs and PBMs. In both types, Müller cell BMs (MBMs) are relatively unaffected. High-resolution SEM shows tissue-specific features in normal EBM and MBM surfaces, but disease-related topographic changes are not evident. It is possible that the ECM arrangements identified in our study relate to different microvessel domains and that their specific morphological features may play important roles in the pathogenesis of diabetic retinopathy including capillary closure and neovascularization.

Implant-fixed, dental bridges from carbon/graphite fibre reinforced poly(methyl methacrylate).

Conventional bridge frameworks of gold alloys are expensive. A method for preparing bridges from carbon/graphite reinforced poly(methyl methacrylate) on titanium implants is described. After a separate in vitro investigation of both heat- and auto polymerized systems with different quantities of carbon/graphite fibres, the material system was selected. Clinical data on 11 patients with observation periods from 11 to 24 mnth indicated that the carbon/graphite fibre reinforced polymer bridges functioned satisfactorily.

Ostectomy in the mandible.

Fourteen patients, diagnosed as cases of apertognathism, were subjected to ostectomy in the premolar or molar region on both sides. The operations were made with an intraoral approach in the premolar region anterior to the molar foramen and extraorally in the molar region. The mandibular segments were immobilized by cap-splints after the operations. A follow up, clinically and roentgenologically, was made after the splints had been removed and, in addition, one and five years later. The aesthetic as well as the functional results of the operations were very good. Only minor changes in the occlusion occurred during the follow up period. In a few patients minor unilateral decreases of sensibility were observed. The late results of the operations must therefore be regarded as good.