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Background: HIV infection increases susceptibility for marginal periodontitis, with horizontal and rapid loss of periodontal soft tissues and alveolar bone.Objectives: To examine whether numbers, distribution and some properties of mast cells, neutrophils and macrophages are normal in chronically inflamed gingiva of HIV-positive patients.Methods: Gingival biopsies were stained for mast cell tryptase and chymase, neutrophil elastase, CD68, human transforming growth factor beta(1), HLA-DR, FcgammaRI, FcgammaRII and FcgammaRIII and calprotectin.Results: Patients at all stages of HIV infection showed radically increased numbers of mast cells and neutrophils throughout the connective tissue, and of macrophages below the oral gingival epithelium (P < 0.05).Conclusion: HIV infection is associated with increased numbers of mast cells, macrophages and neutrophils in the chronic periodontal lesion. This may predispose for tissue destruction through the release of inflammatory mediators and effector molecules. The unusually heavy cell infiltrate throughout the gingival connective tissue may contribute to the diverging pattern of periodontal tissue loss in HIV-positive patients. (C) 2002 Lippincott Williams Wilkins.

BACKGROUND:Fecal calprotectin is elevated in patients with colorectal cancer (CRC). An improved method has been developed. The aim was to evaluate sensitivity and specificity for CRC with the new fecal calprotectin method and to compare the results with those of the original method.METHODS:The study comprised 453 subjects including symptomatic CRC patients and CRC high risk subjects with and without CRC. Complete colonoscopy was performed. Calprotectin was measured with an enzyme linked immunosorbent assay (ELISA) using small (50-100 mg) feces samples.RESULTS:Fecal calprotectin levels were significantly elevated in symptomatic CRC and in asymptomatic CRC detected in high risk subjects. Calprotectin levels were significantly decreased 3 months after cancer removal. A cut-off limit of 50 microg/g resulted in a sensitivity of 89% in CRC patients and 79% in high risk subjects, compared to 89% and 75%, respectively, with the original method, using 10 mg/l as cut-off limit. Specificity was improved with the new method to 68% and 91% at cut-off of 50 and 150 microg/g, compared to 66% and 88%, respectively. Negative predictive value (NPV) was 99% for cut-off of 50 microg/g in the high risk population. One stool sample was sufficient, but measurement of two spots in two stools increased sensitivity to 98% for symptomatic and 82% for asymptomatic CRC.CONCLUSION:The new simple method, using small samples of feces, had a higher diagnostic accuracy, suggesting that it should be preferred to the original one, in screening high risk groups for CRC.

OBJECTIVE: Fecal calprotectin concentration in stool has recently been proposed as a marker of colonic neoplasm and inflammation, but the intraindividual day-to-day variability has so far received little attention. The present study was undertaken to determine the biological variability of fecal calprotectin in patients referred for colonoscopy.METHODS: A prospective design was applied. In each of 14 consecutive patients submitted for colonoscopy, eight stool samples were collected before the endoscopy. A detailed questionnaire was used. Calprotectin was measured by quantitative enzyme-linked immunoassay, and standard deviation for the within-patient variability was estimated from one-way analysis of variance.RESULTS: In absence of colonic neoplasm and inflammation, two populations of patients emerged: one (36%) with remarkably low and stable fecal calprotectin values all within the recommended cut-off of 50 mug/g, and one (64%) with labile values also beyond this limit. In this latter group, fecal calprotectin was 70 mug/g (mean) (single tests ranged from 9 to 461), and SID within patients was 52 pglg, showin- considerable day-to-day variation. History, concurrent diseases, or findings at colonoscopy could not explain labile values. A similar pattern was observed for spot variation in one stool sample from healthy volunteers, suggesting that factors other than disease contribute to the significant intraindividual biological variation of fecal calprotectin.CONCLUSIONS: Day-to-day variation of fecal calprotectin is considerable in patients without colonic inflammation or neoplasm, for whom the pattern of stabile low fecal calprotectin may seem to be a valid negative predictor. The origin and pattern of fecal calprotectin excretion deserve further attention. (Am J Gastroenterol 2001;96:2683-2687. (C) 2001 by Am. Coll. of Gastroenterology).

Background-Faecal concentrations of the protein calprotectin have been found to be elevated in patients with colorectal neoplasia, suggesting that it might be used as a screening tool for colorectal cancer as well as adenomas.Aims-To measure the sensitivity and specificity of faecal calprotectin for the detection of adenomas in high risk individuals undergoing colonoscopy. Also, to investigate between and within stool variability of calprotectin concentrations.Subjects-A total of 814 patients planned for colonoscopy were included for the following indications: positive faecal occult blood test, 25; neoplasia surveillance, 605; newly detected polyp, 130; and family risk, 54.Methods-Two faecal samples from each of two stools were analysed using the PhiCal ELISA test device (Nycomed Pharma AS).Results-Adenoma patients had significantly higher calprotectin levels than normal subjects (median 9.1 (95% confidence interval 7.5-10.1) v 6.6 (5.6-7.4)mg/l). There was no significant decrease in calprotectin levels after polypectomy. Levels in cancer patients were significantly higher than those in all other subgroups (median 17.6 mg/l (11.5-31.0)). With a cut off limit of 10 mg/l, the sensitivity for cancer was 74% and for adenoma 43%. Corresponding specificity values were 64% for no cancer and 67% for no neoplasia (cancer+adenoma). Specificity varied from 71% for one stool sample to 63% for four samples. Stool variability was small, suggesting that two spots from one stool were as discriminative as two spots from each of two stools.Conclusions-The sensitivity and specificity of faecal calprotectin levels as a marker for colorectal adenoma and carcinoma justifies its use in high risk groups, but specificity is too low for screening of average risk persons. Lack of a decrease in levels after polypectomy may be due to a more widespread leucocyte migration into the intestinal lumen than that at the polyp site, and needs further investigation.

Fecal calprotectin is a marker of inflammatory and neoplastic disease in the lower gastrointestinal tract. A new fecal sample preparation procedure for the measurement of calprotectin has been developed, with higher calprotectin yield and lower contamination risk. Changes in the new method compared to the original [Roseth AG, Fagerhol MK, Aadland E, Schonsby H. Assessment of the neutrophil dominating protein calprotectin in feces. A methodologic study. Scand J Gastroenterol 1992;27(9):793-798] are smaller sample size, higher dilution of the sample, presence of dissociating agents in the extraction solution and procedure performed in closed disposable tubes. The extraction yield was 78% (41-100%) of total calprotectin, giving an overall five-fold increase compared to the original method. Samples with high calprotectin values were increased to a slightly higher degree, than low calprotectin samples, thus improving the separation between high and low calprotectin levels. Median calprotectin level in healthy subjects was 26 microg/g. Pathological samples with pancolitis showed levels up to 30000 microg/g. The mean C.V. (coefficient of variation) in blended feces was lower than that of unblended, suggesting uneven distribution of calprotectin. However, no significant difference between spot measurements was found when five samples from each of 47 stools were measured. Thus measurements of calprotectin in fecal samples were accurate and reproducible. No interference with foods or relevant oral pharmaceuticals or nutraceuticals was found.

An epitope may be defined as a specific site on an antigen module characterized by the binding of one monoclonal antibody (MAb). Epitope mapping by surface plasmon resonance in the BIAcore biosensor may be performed to characterize an antigen or a group of specific MAbs or both. This article describes the BIAcore instrument and methods for such mapping. Examples include molecular interaction studies with simple and complex proteins, such as myoglobin and calprotectin, respectively.

Calprotectin is a calcium binding heterocomplex protein consisting of two heavy and one light chain.' 2 It belongs to the S-100 protein family and is derived predominantly from neu- trophils and monocytes.'This protein is distributed in myelomonocytic cells, epithelial cells, and keratinocytes and in various tissues and fluids in the body,' and is a putative protective protein.3Calprotectin and its subunits appear to have regulatory functions in the inflammatory process,5 6 and various bio- on August 18, 2023 by guest.

The interaction between human heart myoglobin and ten specific monoclonal antibodies was investigated with a new biosensor technology, real time biospecific interaction analysis (RT BIA), using surface plasmon resonance. Analysis of association and dissociation kinetics was monitored in real time, with unlabelled reactants. Antibody isotyping was rapid and simple. Epitope mapping with RT BIA confirmed, with substantial time saving, the sum of results obtained in conventional labelled systems. Monoclonal antibodies with four different epitope specificities and optimal binding function were selected for a myoglobin sandwich assay with enhanced sensitivity. BIAcore can be used directly as a diagnostic tool, or as an analytical tool in immunoassay development.

Staphylococcus aureus strains were separated from mastitis milk samples without cultivation by using monodisperse magnetic polymer particles coated with polyclonal antiserum against an encapsulated S. aureus strain. Exopolysaccharide was verified by transmission electron microscopy and the serum soft-agar culture technique. Capsular polysaccharide was found on virtually all clinical isolates. Surface protein A and S. aureus-specific cell wall components were masked when the strains were cultured on an exopolysaccharide-promoting medium. Masking of surface determinants was dependent on their concentration on the bacterial surface as well as on exopolysaccharide abundance. The polysaccharide layer on in vivo bacteria was reduced markedly after just one transfer from milk to blood agar plates but was reexpressed after culturing was done on a capsule-generating medium.

Lipopolysaccharide (LPS, endotoxin) was isolated from Bacteroides intermedius, Bacteroides gingivalis and Bacteroides loescheii by phenol extraction, purified by ultracentrifugation and investigated chemically by gas chromatography and mass spectrometry. Twenty different fatty acids were found. The fatty acid composition differed between the bacterial species, and intraspecies variation was also recorded. All endotoxins contained 3‐hydroxy‐15‐methylhexadecanoic acid and 3‐hydroxy‐hexadecanoic acid as major hydroxy fatty acids. Additional fatty acids were straight chain or iso‐ or anteiso‐branched fatty acids, and straight chain or iso‐branced hydroxylated fatty acids. The sugar composition showed great variability between different strains of the same species. Heptose and 2‐keto‐3‐deoxyoctonate (KDO) were detected in LPS from strains of B. gingivalis and B. intermedius. A tentatively identified glucosamine disaccharide was detected in all three Bacteroides species. This is indicative of lack of phosphorylation in the lipid A backbone, which is in concordance with the low general toxicity of Bacteroides LPS.

Monosized magnetic polystyrene beads, Dynabeads M‐450, coated with sheep IgG were used in an agglutination test for the detection of cell‐surface protein‐A, based on Fc‐protein‐A binding. Ninety‐three per cent of the bovine Staphylococcus aureus strains isolated from clinical mastitis expressed surface protein‐A. The agglutination test was rapid compared to FITC‐labelled IgG binding or dog‐serum agar precipitation test for protein‐A, and was significantly correlated with these methods. Scanning electron micrographs demonstrated binding of protein‐A positive bacteria to the particle surface, thus bridging the beads and forming the agglutination lattice. The microsphere agglutination assay described is a quick, simple and reproducible method for detecting surface protein‐A in staphylococci.

Mononuclear phagocytes(monocytes, macrophages) are multi-potent cells with immunomodulatory properties, as well as important effector activities in inflammatory reactions (1). Much interest is at present focused on endotoxins (lipopolysaccha-ride,LPS) as modulators of the Immune response, and particularly on the relations between structure and biological activity of LPS (2). The macrophage is suggested as the central effector cell in endotoxic reactions (3), and stimulatory as well as suppressive effects on mononuclear phagocytes have been reported (4,5).

Non-phagocytic adherent cells (NPAC) were found as contaminants in peritoneal macrophage cultures from mice kept in conventional animal houses. The cells were morphologically intermediates between macrophages and small lymphocytes. They showed a vigorous ameboid movement in vitro, and most of the cells detached during the first 24 h in culture. The cells were identified as B-lymphocytes by demonstrating surface immunoglobulin with fluorescence labelled goat-F(ab)2-anti-mouse IgG + IgM antibody, as non-phagocytes by latex beads phagocytosis test, and as non-T cells with an anti-Thy 1 monoclonal antibody. Macrophages were identified with M1/70 monoclonal antibody against the Mac-1 antigen. An increased number of NPAC was found in cultures harvested from the peritoneum of mice kept in conventional houses for one week or more, together with a rise in total peritoneal cell count. The keeping also resulted in increased functional activity of the macrophages, tested by morphology, lososomal enzyme activity and phagocytic functions. The rise in NPAC parallelled the activation of macrophages and may therefore serve as an indication of the functional state of the macrophage cultures.

Peritoneal macrophages from normal mice strains (C3H/Tif and C57BL/6J) and from the endotoxin (lipopolysaccharide, LPS) low responder strain (C3H/Hej were exposed to two structurally different endotoxins from Bacteroides intermedius and Escherichia coli in vitro. Intracellular activity of a lysosomal enzyme (acid phosphatase) and macrophage mediated cytotoxic activity against a tumor cell line (L929) were tested. Both endotoxins caused increased levels of acid phosphatase activity in normal mice macrophages. No change was obtained in the C3H/Hej macrophages exposed to E. coli LPS; however, the B. intermedius LPS was able to strongly elevate intracellular enzyme level in the C3H/Hej low responder macrophages. Cytotoxic activities were investigated in macrophage supernatants and in co-cultures of stimulated macrophages and target cells. Cytotoxic activity evaluated by measuring release of radioactivity from 14C-thymidine labelled tumor cells was increased with both endotoxins in normal mouse macrophages, but not in non-responder macrophages. When macrophage-mediated effects on tumor cells were tested by counting target cells left per culture, a reduction in target cell number was observed in endotoxin-treated low responder macrophage as well as in normal strain macrophage cultures more pronounced, however, in the normal strain. Cell contact between cytotoxic macrophages and target cells was verified by scanning electron microscopy. The results suggest that LPS effects on macrophages are dependent upon the functional parameters studied, and that the chemical composition of a particular LPS is important for its selective effects on macrophage functions.

Endotoxins (lipopolysaccharides, LPSs) from the strictly anaerobic bacterium Bacteroides intermedius and from Escherichia coli were compared with respect to effects on human monocytes. Exposure of monocyte monolayer cultures to the structurally different endotoxins caused morphological changes, demonstrated by large rounded cells containing cytoplasmic granules with the B. intermedius LPS, and smaller cells with several cytoplasmic protrusions with the E. coli LPS after 10 days of culture. The B. intermedius LPS was, at some doses, able to induce an increased level of the lysosomal enzyme acid phosphatase and increased C3b-receptor-mediated phagocytosis, while other doses gave no effect or weak suppression compared to control cultures. The stimulatory effects were donor-dependent. The E. coli LPS had predominantly suppressive effects on acid phosphatase level and C3b phagocytosis. Both endotoxins resulted in enhanced expression of the activation related cell surface antigen, characterized by the mononuclear phagocyte specific monoclonal antibody 1D5. Biphasic dose-response relationships were observed, particularly pronounced with B. intermedius LPS, giving stimulation of enzyme activity with low (1 ng/ml) and high (0.5 microgram/ml) doses, and suppressed enzyme activity with intermediate doses (0.01-0.10 microgram/ml). The results imply that the effects of endotoxins on different aspects of human monocytes vary considerably; they also suggest that the balance between stimulatory and suppressive properties of a particular endotoxin may influence its net effect on mononuclear phagocyte functions.