Surface exposure of phosphatidylserine during apoptosis of rat thymocytes precedes nuclear changes

Cell surface exposure of phosphatidylserine (PS) during apoptosis serves recognition and removal of the dying cell by phagocytes. Loss of phospholipid asymmetry and PS exposure is investigated by immunocytochemistry and related to morphological changes. Loss of membrane asymmetry was determined on dexamethasone-treated rat thymocytes using the PS specific probe annexin V. Thymocytes incubated in the presence of dexamethasone were studied in time series during the execution of the apoptotic program. Thymocytes first start to expose PS at their cell surface. At this initial stage the barrier function of the plasma membrane remains intact. At a later stage the plasma membrane becomes leaky for compounds like propidium iodide and subsequently the cell disintegrates into apoptotic bodies. Microscopical evaluation of dexamethasone-treated thymocytes showed that the cells with an apoptotic morphology all bound annexin V. The cells with a normal viable morphology lacked annexin V binding except for those cells that started to shed small vesicles. These vesicles were positive for annexin V, indicating a local disturbance of the phospholipid asymmetry. The local exposure of PS is considered to be a very early event of apoptosis, preceding the full sequence of morphological changes at the ultrastructural level

Immunohistochemical, morphological and ultrastructural resemblance between dendritic cells and folliculo-stellate cells in normal human and rat anterior pituitaries

Immunolabeling of cryo-sections of human anterior pituitaries obtained at autopsy, and of cryo-sections of freshly prepared rat anterior pituitaries, with a panel of monoclonal antibodies against markers of the monocyte/dendritic cell/macrophage lineage, reveals in both species a characteristic pattern of immunopositive cells, among which many cells with dendritic phenotype are found. Cells characterized by marker expression of MHC-class II determinants and a dendritic morphology are present in both human and rat anterior pituitary. Markers characteristic of dendritic cells such as the L25 antigen and the OX62 antigen were present in anterior pituitaries from human and rat respectively. The population of MHC-class II expressing dendritic cells of the rat anterior pituitary is compared at the ultrastructural level with the folliculo-stellate cell population, which cell type has been previously characterized by its distinctive ultrastructure and immunopositivity for the S100 protein. Using immune-electron microscopy of rat anterior pituitaries fixed with periodate-lysine-paraformaldehyde, we were able to distinguish non-granulated cells expressing MHC-class II determinants, whereas no MHC-class II expression was found in the granulated endocrine cells. Using double immunolabeling of cryo-sections of these rat AP with 25 nm and 15 nm gold labels, we demonstrated an overlap between the populations of MHC-class II-expressing and S100 protein-expressing cells. Furthermore, MHC-class II-expressing and S100-positive cells showed ultrastructural characteristics that have been previously ascribed to folliculo-stellate cells. At the light microscopical level in the rat AP, a proportion of 10 to 20% of the S100-positive cells was found immunopositive for the MHC-class II marker OX6. In the hu

Evaluation of a new fluorescent-enzyme immuno-assay for diagnosis and follow-up of ANCA-associated vasculitis

In this study we have evaluated a new, fully automated fluorescent-enzyme immuno-assay (FEIA) for detection and quantification of anti-PR3 and anti-MPO ANCA in diagnosis and follow-up of ANCA-associated small vessel vasculitis (AAV). PR3- and MPO-ANCA were determined by FEIA technology in (1) sera of 87 consecutive patients with biopsy-proven, pauci-immune necrotizing crescentic glomerulonephritis (NCGN) and 72 controls; (2) 120 sera (60 patients with Wegener's granulomatosis and 60 controls) that were previously used in a multicentre comparison of direct and capture ELISAs for PR3-ANCA; (3) in samples preceding relapse in 23 PR3-AAV patients with and 23 matched PR3-AAV patients without relapse for prediction of relapses. PR3- and/or MPO-ANCA detection in pauci-immune NCGN by FEIA revealed an overall sensitivity of 82.8%. The FEIA specificity was 96% and 100% for PR3- and MPO-ANCA, respectively. The overall sensitivity of MPO- and PR3-ANCA could be increased to 88.5% by lowering the cut-off values without affecting the specificity (ROC-curve analysis), which is similar to a multistep ANCA procedure that combines indirect immunofluorescence with direct and capture ELISAs. The sensitivity for Wegener's granulomatosis (WG) of the PR3-ANCA FEIA (60%) was more comparable to direct ELISAs (64%) than to capture ELISAs (74%). A rise of 100% in ANCA level as measured by FEIA appeared optimal (ROC-curve) for prediction of relapses and such a rise was observed in 26 patients. In 18 of these 26 patients the rise was followed by a relapse (PPV 69%), whereas in 15 of the 20 patients without a rise no relapse was observed (NPV 75%). In conclusion, detection of PR3- and MPO-ANCA by FEIA has excellent performance in terms of diagnosis of AAV patients. Furthermore, detection of rises in PR3-ANCA by FEIA for prediction of relapses gives results comparable to other techniques

Image analysis:a novel approach for the quantification of antineutrophil cytoplasmic antibody levels in patients with Wegener's granulomatosis

Rises in antineutrophil cytoplasmic antibodies (ANCA) directed against proteinase 3 (PR3) have predictive potential for a relapse of Wegener's granulomatosis (WG). To assess the value of image analysis for monitoring ANCA levels, we measured PR3-ANCA in a cohort of PR3-ANCA positive patients with WG that were prospectively followed in our clinic and compared findings with other techniques. ANCA levels were measured serially by quantitative image analysis, titration in indirect immunolluorescence (IIF), two different directly coated antigen-specific enzyme-linked immunosorbent assays (ELISA), and a capture ELISA using a PR3-specific monoclonal antibody in 16 consecutive WG patients prior to a renal relapse, and in 16 age-and sex-matched patients with inactive WG. The positive predictive value (PPV) of an increase in ANCA titers by image analysis for relapse was 69% (11 of 16). The PPV of an increase in ANCA was 61% (11 of 18) by IIF, 71% (12 of 17) by a commercial direct ELISA, 63% (12 of 19) by in-house direct ELISA, and 75% (12 of 16) by capture ELISA. The negative predictive value (NPV) of the absence of an increase in ANCA titers by image analysis for relapse was 69% (11 of 16). The NPV of the absence of an increase in ANCA was 64% (9 of 14) by IIF, 73% (11 of 15) by a commercial direct ELISA, 63% (9 of 13) by in-house direct ELISA, and 75% (12 of 16) by capture ELISA. In conclusions, quantitative image analysis is a novel technique based on the principle of IIF to quantify ANCA levels in a single dilution in a patient sample. No major differences were observed between image analysis and the other techniques in their capacity to predict relapses of disease activity. (C) 2002 Published by Elsevier Science B.V

Natural Forces

Seeds dropped by wind, water, and passing wildlife have sprouted and grown in the crack of the grooved rock on the Kelleys Island shore