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PubMed1998

The immunohistological diagnosis of E. coli O157:H7 colitis: possible association with colonic ischemia.

OBJECTIVE: E. coli O157:H7 may cause hemorrhagic colitis resembling ischemic colitis. Diagnosis is usually made by finding sorbitol-negative colonies on MacConkey agar that react with O157 and H7 antisera. Most ischemic colitis is idiopathic, but some may be caused by E. coli O157:H7, inasmuch as this organism can produce fibrin thrombi in colon vasculature. The objectives of this study were to determine whether E. coli O157:H7 infection can be diagnosed retrospectively from paraffin blocks of colon sections and whether an association exists between E. coli O157:H7 infection and colonic ischemia. METHODS: Paraffin-embedded sections of normal colon (n = 2) and various colitides [ischemic (n = 11), E. coli O157:H7 (n = 2), IBD (n = 8) and pseudomembranous (n = 3)] were used. Sections were deparaffinized, rehydrated, incubated with 3% peroxide in methanol, rinsed, and incubated with peroxidase-labeled antibody isolated from goats immunized with whole E. coli O157:H7. Sections were stained with peroxidase chromagen reagent and counterstained with hematoxylin. Coarse, granular, orange-brown staining was considered positive. To determine the localization of the chromagen deposits, three cases that stained positive, including one of the culture-proved E. coli O157:H7 colitis and two of colonic ischemia, were processed for electron microscopy. RESULTS: Both cases (100%) of E. coli O157:H7 colitis and three of 11 (27.3%) cases of ischemic colitis stained positive by light microscopy. In one culture-proved case, electron microscopy demonstrated staining of bacillary structures; in two cases of colonic ischemia, extensive deposits of chromagen material were present that were associated neither with inflammatory cells nor with bacterial forms. CONCLUSIONS: Immunoperoxidase staining of archival sections may be used to diagnose E. coli O157:H7 infection. An etiological role for this organism is possible in some cases of colonic ischemia.

Aged↗
PubMed1997

Streptococcus uberis acquires plasmin activity following growth in the presence of bovine plasminogen through the action of its specific plasminogen activator.

Three (0140J, C197C and EF20) out of four strains of Streptococcus uberis exhibited high levels of bound plasmin activity following growth in the presence of bovine plasminogen. The remaining strain (C197) bound considerably less plasmin following growth in the same medium. In contrast to the others, this strain was unable to activate bovine plasminogen. Following growth of strain C197 in the presence of bovine plasminogen and a source of plasminogen activator (urokinase or culture filtrate from strain 0140J) high levels of bacterially associated plasmin were detected. None of the strains was able to activate human plasminogen and only trace levels of plasmin activity were detected in association with the S. uberis following growth in the presence of human plasminogen. All strains were able to bind plasmin activity following incubation in the presence of either bovine or human plasmin. However, in each case the level of activity detected following incubation in human plasmin was approximately five-fold less than that observed following incubation with bovine plasmin. None of the strains bound detectable levels of either human or bovine plasminogen. It is concluded that activation of plasminogen is required prior to binding of plasmin by S. uberis.

Animals↗
PubMed1996

Oral fluid elastase as an indicator of periodontal health.

The study was designed to find out whether oral elastase activity could be used as a simple biochemical indicator of periodontal health. Both stimulated whole saliva and water rinse samples were collected from subjects with different degrees of adult periodontitis, gingivitis or healthy periodontium. In both sample types, elastase was mostly bound to insoluble fraction and preferred valine containing synthetic substrate, similar to neutrophil elastase. The elastase measurement required very little manipulation or time and its reproducibility was found to be good. The elastase levels were found to be negligible in edentulous subjects and usually very low in subjects with healthy periodontium. In about 85% of periodontitis cases having at least 1 deep periodontal pocket ( > or = 6 mm), clearly elevated elastases levels were detected in both the saliva and r rinse samples. In advanced periodontitis cases, the colour reaction took place in 0.5 to 2 h. In localized periodontitis cases, 2- to 18-h incubations were required for positive reaction. There was a good correlation between the elastase activity and the number of deep periodontal pockets and the average community periodontal index of the subjects. Elastase activity was not a good indicator of gingivitis. About 45% of gingivitis cases were positive with the elastase test, and the enzyme values were not significantly increased in experimental gingivitis. In a longitudinal study on advanced periodontitis cases, elastase levels dropped dramatically as a result of clinically successful therapy, close to the values of healthy subjects. The oral elastase test could serve as a valuable adjunct in periodontal screening and assessment of treatment efficacy.

Adult↗
PubMed1997

Multifocal eosinophilic granuloma with sequential periodontitis-like lesions.

Eosinophilic granuloma (EG) is the localized and mildest form of the triad commonly known as Langerhans cell histiocytosis. This report describes a case manifesting itself as a periodontal problem with the localized osseous lesions in jawbones which was first diagnosed as early-onset periodontitis. Later on, the diagnosis of EG was established, relying on histopathological and immunohistochemical evaluations. Immunohistochemical findings confirm that a minor component of cell aggregates is phenotypically related to Langerhans cells among the sheet-like accumulations of histiocytes and eosinophils. The aetiology of the disease remains largely unknown. Although surgical curettage of lesions is usually effective in treatment of EG of bone, corticosteroids might be used as an adjunctive. This multifocal case of EG stresses the importance of clinical follow-up examinations, since the sequential lesions appear with irregular intervals, and this may cause diagnostic problems as well as a delay in starting the treatment regimen.

Adrenal Cortex Hormones↗
PubMed1997

beta-Lactamase producing bacteria in adult periodontitis.

In 23 untreated adult periodontitis patients, the occurrence of beta-lactamase producing periodontal bacteria was determined. In addition to non-selective isolation media, selective isolation and growth of beta-lactamase positive subgingival bacterial species was carried out on blood agar plates supplemented with amoxicillin and plates with amoxicillin+clavulanic acid. Porphyromonas gingivalis, Prevotella intermedia, Actinobacillus actinomycetemcomitans, Peptostreptococcus micros, Fusobacterium nucleatum, Bacteroides forsythus and Campylobacter rectus isolates from the non-selective medium were tested for beta-lactamase activity by a nitrocefin disk method (DrySlide) and by a laboratory chromogenic nitrocefin-based test. Isolates from the amoxicillin plates that were absent on the amoxicillin/clavulanic acid plates were identified and tested for beta-lactamase production. Based on the non-selective plates, six of 23 P. intermedia isolates, 2 of 19 B. forsythus isolates and 3 of 23 F. nucleatum isolates were beta-lactamase positive. The beta-lactamase positive species Prevotella loescheii, Prevotella buccae, Prevotella buccalis and Actinomyces spp were recovered from the selective amoxicillin plates. beta-Lactamase positive subgingival species were recovered from 17 of 23 patients (74%) but usually comprised low proportions of the subgingival microbiota (range < 0.01-15%). Comparison of the DrySlide test and the nitrocefin-based laboratory test revealed full agreement of test results. beta-Lactamase activity in whole subgingival plaque was detected in 12 patient samples (52%). It was concluded that beta-lactamase activity in subgingival bacteria in adult periodontitis is a common feature. However, since the majority of the samples showed only low-level enzymatic activity, the clinical relevance of this observation with regard to therapy with unprotected enzyme-susceptible beta-lactams is uncertain, though failure on the other hand, is difficult to rule out when a mechanism of resistance is present. The majority of beta-lactamase positive strains was found among species of the Prevotella genus.

Actinomyces↗
PubMed1998

A relationship between proteinase activity and clinical parameters in the treatment of periodontal disease.

The objective of this research was to determine the effectiveness of a biochemical assay which measures proteolytic enzyme activity in gingival crevicular fluid (GCF) and to relate this enzyme activity to clinical parameters traditionally utilized for periodontitis detection. A clinical trial was conducted on 8 periodontitis subjects with > or =4 sites exhibiting a loss of attachment of > or =5 mm and probing depths of > or =5 mm with bleeding on probing. On each subject, a plaque index was performed, followed by GCF sampling at those sites which exhibited a loss of attachment and probing depths. GCF was analyzed for activity against benzoyl-L-arginine-p-nitroanilide in the presence (BAPNA w/gly-gly) and the absence (BAPNA w/o gly-gly) of glycyl-glycine and against MeOSuc-Ala-Ala-Pro-Val-pNA and Suc-Ala-Ala-Pro-Phe-pNA for neutrophil serine proteinases activity (elastase and cathepsin G, respectively). Subsequently, a gingival index was performed, attachment levels and probing depths were recorded using a constant force probe with bleeding on probing being noted. A split-mouth design was employed and half mouths were randomly assigned to the following treatment groups: group A, half of the mouth received scaling/root planing and polishing: group B, half of the mouth received no treatment (control). Subjects were treated, then instructed on toothbrushing and interdental cleaning. After 4 weeks, subjects returned to receive a plaque index; GCF sampling, gingival index, attachment levels, probing depths and bleeding on probing as described above. Using a paired Student t-test, the findings suggest that BAPNA w/gly-gly was significantly less in treatment sites than in non-treated control sites (p=0.05). No such correlation was found for other activities, including neutrophil serine proteinases which were shown to occur in GCF in free, proteolytically active forms. In addition, significant treatment effects were detected for probing depths (p= 0.03) which reduced by 1.3 mm and attachment levels (p=0.02) which gained 0.7 mm. The reduction of P. gingivalis from treated periodontitis sites as detected by a significant decrease in BAPNA w/ gly-gly may prove to be a valuable marker for periodontal disease activity.

Benzoylarginine Nitroanilide↗
PubMed2006

Chromogenic in situ hybridization analysis of melastatin mRNA expression in melanomas from American Joint Committee on Cancer stage I and II patients with recurrent melanoma.

OBJECTIVE: To determine whether loss of melastatin (MLSN) is a universal phenomenon in American Joint Committee on Cancer (AJCC) stage I and II melanoma patients who experienced recurrence. MATERIAL AND METHODS: Paraffin blocks of primary melanomas (PMs) were retrieved from 30 patients who had a negative sentinel lymph node biopsy and developed recurrent melanoma (AJCC stage I and II). Chromogenic in situ hybridization (CISH) methods were utilized to evaluate the expression of MLSN mRNA. These results were correlated with clinicopathologic data. RESULTS: Variable, heterogeneous expression of MLSN mRNA was identified in normal, in situ and invasive melanocytes within and between cases. For the invasive PM component, 24 (80%) had focal, regional or complete loss of MLSN mRNA. The remaining 20% had either regional or total partial downregulation of MLSN mRNA. Intact MLSN mRNA expression was present regionally in 14/30 (47%), with mean relative tumor area of 38%, range 5-85%. Increasing loss of MLSN mRNA significantly correlated with increasing tumor depth and microsatellites (r = 0.1/0.4, p = 0.04). However, thin, AJCC T stage 1a PM had higher relative mean loss than intermediate AJCC T stage 2a/2b/3a thickness PM (65% vs. 34%/48%/25%). Increasing loss of MLSN mRNA significantly impacted on disease free survival (DFS) by multivariate analysis (58 vs. 0% 2 years DFS, < or = 75 vs. > 75% mRNA loss, p = 0.02). Decreased overall survival significantly correlated with increasing age and vascular invasion on multivariate analysis. CONCLUSION: Extensive loss of MLSN in PM correlated with aggressive metastatic melanoma. Ancillary testing for MLSN mRNA expression by CISH could offer a means to more accurately identify AJCC stage I and II patients at risk for metastatic disease, who could benefit from adjuvant therapy.

Adult↗
PubMed1983

Simplified assay for antithrombin III activity using chromogenic peptide substrate. Manual and automated method.

A simplified method for the assay of antithrombin III (AT) with the highly reactive thrombin substrate 2AcOH X H-D-CHG-Ala-Arg-pNA (substrate Th-1) is described. The assay may be performed at either 30 degrees C or 37 degrees C, and alternatively with the substrate H-D-Phe-Pip-Arg-pNA (S-2238). The standard curve is linear in the 12.5-150% range. For routine assays, 3 standard dilutions of plasma are sufficient, and these may be stored at -20 degrees C for 3 weeks. As only the test plasma must be diluted prior to the assay procedure, the test is more rapidly performed than previous manual assays. In 80 patients plasma samples, with AT in the 19-108% range, there was a high correlation with the results of immunoquantification (r = 0.96). There was also a high correlation between the results obtained with the manual method and the automated version described using the Cobas-Bio Centrifugal Analyser and substrate Th-1 (r = 0.96). Low AT levels in hereditary deficiency (particularly during heparin treatment), in liver cirrhosis, in disseminated intravascular coagulation (DIC), and heparin-treated thrombosis were confirmed.

Antithrombin III↗
PubMed1986

Clinical application of the chromogenic assay of factor VIII in haemophilia A, and different variants of von Willebrand's disease.

A chromogenic substrate kit for determination of factor VIII activity (COATEST Factor VIII) was compared to a one-stage clotting assay and the correlation was evaluated in different genetic variants of mild and moderate haemophilia A, in severe haemophilia A and in all known variants of von Willebrand's disease. In all these cases a high correlation between the two methods was obtained. A good correlation was also obtained after intranasal administration of DDAVP (1-desamino-8-D-arginine vasopressin) to patients with von Willebrand's disease. The chromogenic substrate method was performed using a microtray technique.

Chromogenic Compounds↗
PubMed1986

Heparin cofactor II activity in plasma: application of an automated assay method to the study of a normal adult population.

A manual chromogenic substrate method for the determination of heparin cofactor II (HC II) activity in plasma has been adapted to the Cobas Bio centrifugal analyzer. By reducing the dermatan sulfate concentration used, interference by opacity was avoided, and the correlation between the manual and the new automated method was high (r = 0.96). Plasma samples from 182 females and 197 males were examined. The mean values of HC II activities in this normal population was relatively high, with a total range of 43-150% and a standard deviation of 17%. 4 apparently healthy individuals had levels below 60%. In males, the mean HC II activity was higher in the 50-59 yr age group than in either the 20-29 yr group or in the group above 60 yr (p less than 0.05). In females, the postmenopausal age group had higher mean HC II activities, but the difference was not statistically significant.

Adult↗
PubMed1997

Quantitation and localization of cycling tumor cells in pleomorphic adenomas and myoepitheliomas: an immunohistochemical analysis.

Previous investigations have suggested that in certain salivary gland tumors only nonluminal (myoepithelial-like) tumor cells proliferate and that this may have histogenetic implications; however, no quantitative assessment of the cycling component is available. We decided, therefore, to enumerate the cycling luminal and nonluminal cells in 15 pleomorphic adenomas and six myoepitheliomas by using an antibody to proliferating cell nuclear antigen. The mean percentages of cycling luminal and nonluminal cells in pleomorphic adenomas were 2.3 +/- 1.4 and 2.4 +/- 2.3, respectively, and 2.1 +/- 0.7 of the tumor cells in myoepitheliomas. Even though duct-like structures in pleomorphic adenomas are considerably separated by more numerous nonluminal cells and, therefore, cycling cells may seem fewer, both cell types proliferate at similar rates. The results indicate that nonluminal cells are not the sole proliferative component in salivary gland tumors.

3,3'-Diaminobenzidine↗
PubMed1982

Demonstration of extracellular proteolytic enzymes from Legionella species strains by using synthetic chromogenic peptide substrates.

Crude concentrates of extracellular proteases from strains belonging to species within the genus Legionella were examined for their effect upon synthetic chromogenic tri- and tetrapeptides. For the species L. pneumophila, similar and reproducible substrate degradation patterns were found. Strains from the other Legionella species produced proteases with dissimilar proteolytic patterns, which were all distinct from those of the L. pneumophila proteases.

Chromogenic Compounds↗
PubMed1983

Legionella extracellular protease activity on chromogenic peptides: a simplified procedure for biochemical enzyme identification.

Legionella strains produce extracellular proteases. One method to demonstrate these is through the activity of broth culture filtrates on para-nitroanilide (pNA)-derivatized peptides. Previously, this method has comprised a 100-times concentration of the filtrates, in order to bring the protease activity, as measured by the liberation of free pNA from hydrolysed peptides, up to easily recordable values. By introducing a diazotation step, the sensitivity of the test has been increased sufficiently to omit the time-consuming concentration procedure. Accordingly, the analysis of extracellular Legionella proteases by pNA-derivatized peptides has become rapid and straight-forward.

Chromogenic Compounds↗
PubMed1985

Exotoxin A from various Pseudomonas aeruginosa cultures: in vitro activity measured by enzyme-linked immunosorbent assay (ELISA).

An enzyme-linked immunosorbent assay (ELISA) for the detection of Pseudomonas aeruginosa exotoxin A (toxin A) was developed. The level of toxin A produced by fresh P. aeruginosa isolates was compared to the level of toxin A produced by twelve-months-old subcultures derived from the same strains. Results obtained with the ELISA showed that toxin A was produced in similar amounts both by the fresh isolates and the subcultures. The production of toxin A seems to represent a stable property of the assayed P. aeruginosa strains. This is in contrast to other exoproducts from the same strains, such as extracellular proteinases. Some proteinases were produced at very different levels when freshly isolated P. aeruginosa cultures were compared with their twelve-months-old counterparts, confirming earlier reports in this respect.

ADP Ribose Transferases↗
PubMed1986

Quantitative determination of beta-lactamase production in Staphylococcus aureus strains compared to qualitative testing by a microbiological clover leaf test, a chromogenic cephalosporin test and a iodometric test.

One hundred and twelve beta-lactamase producing strains of Staphylococcus aureus with known quantitative production were investigated together with 45 non-beta-lactamase producing derivatives by three qualitative beta-lactamase tests: the microbiological clover leaf test, the iodometric tube test and a chromogenic cephalosporin disc test. For the beta-lactamase producing strains strong positive reactions in the qualitative tests were not correlated with high beta-lactamase production, large degree of extracellularity or high induction ratio in any of the tests. Low beta-lactamase production was not correlated to weak reactions. Best results in identifying beta-lactamase production were obtained using the iodometric test in which 109 of 112 beta-lactamase producing strains were positive and no false positive reactions were observed. Some strains had only weak positive reactions but they were easily read. The clover leaf test and the chromogenic cephalosporin test detected 105 and 107 of 112 strains, respectively. The chromogenic cephalosporin disc test had more weakly reacting strains, which were difficult to read, especially among strains of phage type 95. Three non-beta-lactamase producing strains of phage type 95 were positive in the clover leaf test. It is concluded that the iodometric tube test is the most useful one for qualitative determination of beta-lactamase production in S. aureus. It is recommended as control when normal susceptibility testing for penicillin has shown susceptibility or doubtful susceptibility. Furthermore, it is useful when no susceptibility testing has been carried out and a rapid result is needed.

Biological Assay↗
PubMed1991

Rapid identification of Legionella pneumophila zinc metalloprotease using chromogenic detection.

Strains of Legionella spp. produce extracellular proteases than can be detected using synthetic chromogenic peptides. Chromogenic tri- and tetrapeptides show a high degree of sensitivity, specificity and reagent stability when linked to para-nitroaniline (pNA). For example, SucOMe-Arg-Pro-Tyr.pNa (S-2586) is specifically hydrolysed by proteases of Legionella pneumophila and some other Legionella species. A paper disc method to sample protease directly from agar plates has been used to evaluate chromogenic peptides as reagents for diagnostic purposes. Strains of Legionella spp., Pseudomonas spp. and Enterobacteriaceae were examined, together with a recombinant Escherichia coli strain containing the cloned 38 kDa zinc metalloprotease from L. pneumophila, S-2586 was hydrolysed by 282 out of 283 L. pneumophila strains, and by the recombinant E. coli. Two of the six strains representing other Legionella species, and 22 of the 50 strains from the Pseudomonas group were also positive. No reaction was seen with any of the Enterobacteriaceae strains. Although there was functional homology between proteases from several bacterial groups, the high prevalence of S 2586-hydrolysing proteases within L. pneumophila indicates a potential usefulness for phenotypic identification.

Bacterial Proteins↗