No abstract

A murine monoclonal antibody (mAb) S-Endo 1 has been produced to detect circulating endothelial cells detached from blood vessels in pathological conditions. We have demonstrated that the associated-antigen (S-Endo 1 Ag) was highly expressed on human vascular structure irrespective of tissue origin or vessel caliber. Its expression was not restricted to endothelium, since it was also detected at low level on smooth muscle cells, stroma cells and follicular dendritic cells. But its absence on hematopoietic cells made S-Endo 1 a helpful reagent to specifically discriminate endothelium from hematopoietic tissues. Biochemical characterization showed that S-Endo 1 recognizes a monomeric structure of approximately 118 kDa on cultured endothelial cells. S-Endo 1 was submitted to the 5th International Workshop (Boston, 1993) and did not cluster in any of the old or new endothelial clusters discussed at the conference, indicating its unique reactivity. Together with the data presented in this paper, this suggested that S-Endo 1 defines a previously undescribed endothelial molecule.

SummaryThe presence in whole blood of circulating endothelial cells (EC) has been a subject of debate for many years. It could represent a good marker of vessel injury. We demonstrate here that human endothelial cells can be directly isolated and identified in circulating blood by means of an endothelial cell specific monoclonal antibody, S-Endol, coupled to micromagnetic beads. The specificity and efficacy of the assay were established using normal blood samples with cultured EC added. Specific rosettes formed between EC and beads could subsequently be isolated with a magnet. The rosetted cells were recovered with a yield >80%. Their endothelial origin was confirmed by the positive labelling of von Willebrand factor and thrombomodulin, as well as the presence of Weibel-Palade bodies. We applied this method to demonstrate significantly increased levels of EC in venous and arterial human blood samples in patients undergoing heart catheterization. This new whole blood immuno-separation method may be useful in determining endothelial cell injury in vascular disorders.

The endothelial cell (EC) is the primary target for Rickettsia conorii (RC) in Mediterranean spotted fever (MSF). Clinical manifestations such as thrombosis and vasculitis are mediated by pathologic changes localized in blood vessels. To study the in vivo endothelial injury induced by RC, markers of endothelial damage, including circulating EC (CEC), plasmatic thrombomodulin (TM), and von Willebrand factor (vWF), were investigated in 12 patients with MSF. CEC were counted in whole blood by a new immunomagnetic separation assay using a specific anti-EC antibody, S-Endo 1. Plasmatic TM and vWF antigens were measured by enzyme-linked immunosorbent assay. High levels of CEC and cell fragments were found in patients with a severe or malignant form of MSF. Sequential studies of CEC showed a decrease from 162 +/- 454 cells/mL before treatment to 6 +/- 7 cells/mL during treatment and recovery. Mean plasma TM and vWF levels that were also elevated before therapy (TM, 106 +/- 27 ng/mL; vWF, 420% +/- 164%) decreased progressively (TM, 55 +/- 43 ng/mL; vWF, 148% +/- 26%) during treatment. The measurement of cellular and molecular markers of vascular damage such as CEC, plasmatic TM, and vWF contributes to the definition of the Rickettsia-induced endothelial injury in vivo.

In this paper the normal ranges of the expression of various differentiation antigens, referred to by their cluster of differentiation (CD) numbers, are described on the lymphoid, monocytic, and polymorphonuclear (PMN) blood populations in normal healthy individuals. The values expressed as antibody binding capacity per cell (ABC/cell) are related to the density of antigenic molecules expressed by these cells. These values have been quantitated by the quantitative indirect immunofluorescence (QlFI) test which renders the ABC/ cell values for the different antigens directly comparable and defines a "league table," i.e., an enumeration of antigen expression on the three main cell types studied. The values for occasional antigen that are expressed differently in adults and the elderly or men and women (CD5, CD8, and CD18) are also shown.Furthermore, the QlFl test is used in two-color immunofluorescence for defining the subset heterogeneity within the T lineage for the CD2 and CD7 antigens within the separately analyzed CD45RA and CD45RO subsets. These quantitative immune phenotype analyses, also referred to as quantimetry, show variations in ABC values if different monoclonal antibodies (MAbs) are used, although these differences are frequently minor. Therefore, using whole blood and well-characterized MAbs, we established values of antigen density in normal adults which can be regarded as control values for the various pathological conditions where CD antigen expression may be altered. o 1996 Witey-Liss, Inc.Key terms: leukocyte membrane antigen, antigen density, antibody binding capacity, flow cytometry, QlFl test, quantimetryRecently the stability and near linearity of the currently used flow cytometers have been well documented along the full range of fluorescence intensities (1). Collaborations have also shown that virtually identical flow cytometer settings can be obtained when multicenter studies adopt the same protocols and calibration procedures (2-4). Nevertheless, problems still arise from the fact that the preparation of antibodies and their conjugation with fluorochromes have not been fully standardized: antibodies differ depending on the fluorochromes used and are influenced by the conjugation methods, e.g., due to overconjugation or the use of spacer molecules during coupling, etc. These alterations can interfere with the binding of antibodies when compared to the features of the native antibody molecule.Despite these difficulties of standardization of immunofluorescence (IF), it is now accepted that the quantitation of molecules by monoclonal antibody (MAb) technology provides additional clinical information and is also useful for the precise characterization of lymphoid and hemopoietic cell populations. In certain malignant conditions precursor cell-associated antigens can be 0 1996 Wiley-Liss, Inc.overexpressed on malignant cells and can therefore be regarded as leukemia-associated features (5-7). In various pathological conditions, particularly in infectious diseases, the regulation of antigen ex...

In this paper the normal ranges of the expression of various differentiation antigens, referred to by their cluster of differentiation (CD) numbers, are described on the lymphoid, monocytic, and polymorphonuclear (PMN) blood populations in normal healthy individuals. The values expressed as antibody binding capacity per cell (ABC/cell) are related to the density of antigenic molecules expressed by these cells. These values have been quantitated by the quantitative indirect immunofluorescence (QIFI) test which renders the ABC/cell values for the different antigens directly comparable and defines a “league table,” i.e., an enumeration of antigen expression on the three main cell types studied. The values for occasional antigen that are expressed differently in adults and the elderly or men and women (CD5, CD8, and CD18) are also shown. Furthermore, the QIFI test is used in two‐color immunofluorescence for defining the subset heterogeneity within the T lineage for the CD2 and CD7 antigens within the separately analyzed CD45RA and CD45RO subsets. These quantitative immune phenotype analyses, also referred to as quantimetry, show variations in ABC values if different monoclonal antibodies (MAbs) are used, although these differences are frequently minor. Therefore, using whole blood and well‐characterized MAbs, we established values of antigen density in normal adults which can be regarded as control values for the various pathological conditions where CD antigen expression may be altered. © 1996 Wiley‐Liss, Inc.

No abstract

Rickettsia conorii, an obligate intracellular bacterium that infects vascular endothelial cells, is the etiologic agent of Mediterranean spotted fever (MSF). A new procedure using indirect immunofluorescence was used to directly detect R. conorii in circulating endothelial cells (CEC). CEC were separated from other blood components by using anti-endothelial cell monoclonal antibody-coated magnetic beads. An anti-R. conorii polyclonal rabbit antiserum was used to stain rickettsiae. The entire procedure took 3 h. R. conorii was detected in CEC from 9 of 12 patients ultimately confirmed as having MSF. Among the patients, 5 with R. conorii isolated by centrifugation-shell vial assay were also positive by the new technique. None of 3 patients whose diagnosis was other than MSF had R. conorii detected in CEC. The procedure may be helpful for rapid diagnosis of MSF and may lead to new technical approaches for the diagnosis of infectious diseases caused by intracellular pathogenic microorganisms.