Severity of articular cartilage abnormality in patients with osteoarthritis: evaluation with fast spin-echo MR vs arthroscopy.

OBJECTIVE: The purpose of this study was to assess the accuracy of fast spin-echo MR imaging for depicting the severity of articular cartilage abnormalities in patients with osteoarthritis. SUBJECTS AND METHODS: Twenty-three subjects (10 volunteers less than 35 years old and 13 patients with proved, symptomatic, idiopathic osteoarthritis of the knee of 6 months' to 10 years' clinical duration) underwent fast spin-echo MR imaging of the knee. Two observers graded each articular surface using a five-category scale that took into account abnormalities in the signal intensity of cartilage as well as thickness and contour. The 13 patients also underwent arthroscopic evaluation (as part of a separate protocol) in which cartilage abnormalities were graded by using a similar five-category grading scale, without the graders knowing the results of MR imaging. Articular cartilage was assumed to be normal in the volunteers. RESULTS: One hundred thirty-seven joint surfaces were graded; one surface was obscured by artifact and was excluded. The Spearman rank linear correlation between arthroscopic and MR grading was highly significant (p < .002) for each of the six articular regions evaluated. The MR and arthroscopic grades were the same in 93 (68%) of 137 joint surfaces, they were the same or differed by one grade in 123 surfaces (90%), and they were the same or differed by one or two grades in 129 surfaces (94%). CONCLUSION: Our results suggest that fat-presaturated fast spin-echo MR imaging depicts the severity of articular cartilage abnormalities in osteoarthritis with reasonable accuracy, as compared with arthroscopic evaluation as the standard of reference.

Analysis of the epitopes of proliferating cell nuclear antigen recognized by monoclonal antibodies.

BACKGROUND: Monoclonal antibodies to proliferating cell nuclear antigen (PCNA) are increasingly used for evaluation of cell proliferation especially in diagnostic pathology. To understand the different staining characteristics of such antibodies, we found it important to map the reactive epitopes in detail. EXPERIMENTAL DESIGN: Overlapping, synthetic 15-mer peptides encoding the entire PCNA amino acid sequence were analyzed for reactivity with 6 anti-PCNA monoclonal antibodies using enzyme-linked immunosorbent assay and flow cytometric competition analysis. One immunodominant region was studied using a set of peptides with single amino acid substitutions. Further epitope localization was obtained by immunoblotting of fusion protein constructs. RESULTS: The epitopes recognized by the antibodies could be assigned to specific peptides. Five monoclonal antibodies (19A2, 19F4, TO17, TO30, PC10) reacted with the same protein region (aa 111-125) whereas one antibody (TOB7) recognized a separate region of the protein (aa 181-195). The reactivity pattern with 8 recombinant PCNA-fusion proteins agreed with the peptide data. The immunodominant aa 111-125 peptide reactive antibodies differed concerning immunofluorescence patterns. These differences could be attributed to epitope microheterogeneity within this peptide as determined by reactivity with Ala-substituted peptides. The 19A2, 19F4 and PC10 antibodies showed nuclear fluorescence and similar but not identical peptide recognition patterns. Compared with 19A2 and 19F4, the PC10 antibody was directed against a simpler epitope. TO17 and TO30 gave cytoplasmic filamentous staining and their epitopes were different from those defined by the other monoclonal antibodies. CONCLUSIONS: The results indicate that induced anti-PCNA antibodies recognize well defined linear epitopes, in contrast to PCNA autoepitopes which are strongly dependent on the protein conformation.

Elevated plasma levels of hyaluronate in patients with osteoarthritis and rheumatoid arthritis.

Plasma levels of hyaluronate (HA) in patients with rheumatoid arthritis (RA) and patients with osteoarthritis (OA), measured by enzyme-linked immunosorbent assay, were compared with levels in a healthy, age-matched non-arthritic control group, in a retrospective study. Compared with the controls, the mean level of plasma HA was sevenfold higher in the RA group and twofold higher in the OA group. There was no statistically significant correlation between HA levels and 7 other clinical and biochemical parameters in patients with RA. In the OA group, however, plasma HA levels were found to correlate with an objective functional capacity score and with an articular index based on the total amount of cartilage in involved joints. In a retrospective longitudinal study of 6 patients with RA, plasma levels of HA did not show a significant correlation with plasma levels of elastase or with the erythrocyte sedimentation rate. These data support in part the contention that plasma HA may be unique as a marker, in that it may be a reflection of synovial involvement and inflammation, rather than only of inflammation, in arthritis.

Optimization of routine transformation of Escherichia coli with plasmid DNA.

Methods to optimize resources and transformation efficiency of routine daily transformations of DH1 Escherichia coli prepared by three calcium chloride methods were investigated and compared with polyethylene glycol and Hanahan methods. The benefit of a heat-shock step, a preplating incubation step to allow expression of antibiotic resistance, use of log phase bacteria and prolonged storage of bacteria were investigated using pBR322 and pUC18 plasmid DNAs. Bacteria prepared by CaCl2 methods consistently gave efficiencies of 4 x 10(6) transformants/microgram of plasmid DNA or better and were overall the most labor- and resource-efficient methods. Use of log phase bacteria, a heat shock and an incubation step were found to be beneficial for freshly prepared bacteria for all methods. Prolonged storage of up to 30 days of bacteria prepared by the CaCl2 methods was beneficial, resulting in a sustained increase in transformation efficiency when selection was by ampicillin but not when by tetracycline resistance. Also found when using bacteria stored three days or longer was an increased transformation efficiency of stationary vs. log phase bacteria and an unchanged or even increased efficiency when the preplating incubation step was omitted. The Hanahan methods were the most labor and resource intensive and routinely gave efficiencies of 2 x 10(7). Higher efficiencies of 10(8) were obtained only with repeated trial and error and were not consistently reproducible. The polyethylene glycol method consistently gave efficiencies of 2 x 10(7), and bacteria could easily be prepared daily or frozen with a minimal decrease in efficiency.

Insights into native epitopes of proliferating cell nuclear antigen using recombinant DNA protein products.

A cDNA clone encoding full-length human proliferating cell nuclear antigen (PCNA) was used to generate a panel of in vitro translated labeled protein products with COOH-terminal deletions and to construct a set of fusion proteins with COOH- and NH2-terminal deletions. A rabbit antiserum raised against an NH2-terminal peptide, a well-characterized murine monoclonal antibody (mAb), and 14 human lupus sera with autoantibody to PCNA were analyzed for their reactivity with the constructs using both immunoprecipitation and immunoblotting techniques. The rabbit antiserum reacted in immunoprecipitation and immunoblotting with constructs containing the appropriate NH2-terminal sequence and mAb reacted with a sequence from the midregion of PCNA. These experimentally induced antibodies also reacted with 15-mer synthetic peptides in enzyme-linked immunosorbent assay (ELISA). In contrast, none of the lupus sera reacted with synthetic peptides in ELISA. 9 of the 14 lupus sera also failed to react in Western immunoblotting with any recombinant fusion protein, although they all immunoprecipitated in vitro translated full-length protein. Four of the nine had variable patterns of immunoprecipitation with shorter constructs. The remaining five lupus sera were able to immunoprecipitate translation products as well as Western blot recombinant fusion proteins. From analysis of the patterns of reactivity of human lupus sera, it was deduced that the apparent heterogeneity of human autoantibodies to PCNA could be explained by immune response to highly conformational epitopes. These observations demonstrate that there might be special features in "native" epitopes of intranuclear antigens that are recognized by autoantibodies, and that these special features of native epitopes might not be present in prepared antigen used for experimental immunization. These features may be related to protein folding or to association of the antigen with other intranuclear proteins or nucleic acids, as might occur with antigens that are components of subcellular particles.

Complications of gastrostomy.

Despite its simplicity, gastrostomy has been associated with a relatively high incidence of complications. We have described two unusual complications: inadvertent duodenostomy, resulting in gastric outlet obstruction, and the migration of a gastrostomy feeding tube into the midportion of the jejunum, resulting in obstruction of the small bowel.

Synthesis and conformational properties of a synthetic cyclic peptide for the active site of alpha-chymotrypsin.

A nonapeptide Ac-His-Phe-Gly-Cys-D-Phe-Ser-Gly-Glu-Cys-NH2 (XI) cyclized through the cysteines at positions 4 and 9 is synthesized as a model active site for the enzyme alpha-chymotrypsin. A CPK model of XI indicates that the peptide will have a high probability of folding into a conformation in which the two beta-phenyls interact to form a hydrophobic site to one side of the cyclohexyl structure, and the Ser-His-Glu side chains form a hydrogen bonded triad over the plane of cyclopeptidyl structure. Substrates can then bind at the hydrophobic pocket formed by the beta-phenyls and be acted upon by the Ser-His-Glu catalytic triad, as in the enzyme. 1H. n.m.r. shows: (i) multiplet peaks for the phenyl protons in D2O that condense to a singlet in DMSO-d6, (ii) a perturbation of the phenyl protons chemical shift on proflavin association to XI, and (iii) perturbation of the His pKa to a higher value on association of proflavin to XI. These data support the existence of a hydrophobic site and a Glu-His interaction in the peptide. Furthermore, the greater than 10(2) better affinity of proflavin to XI than to AcTrp supports the existence of a hydrophobic site. However, no acceleration of p-nitrophenyl acetate or trans-cinnamoyl imidazole hydrolysis over that of imidazole is observed. The possible reasons for a lack of esterase activity in XI and other peptidyl models of serine protease active sites are discussed.