Quantitative Cytofluorometry Research Articles

Peripheral blood T4 cell surface CCR5 density as a marker of activity in rheumatoid arthritis treated with anti‐CD20 monoclonal antibody

The chemokine (C-C motif) receptor CCR5 and its ligand CCL5 play key roles in the intra-articular recruitment of peripheral blood mononuclear cells (PBMC) in rheumatoid arthritis (RA). Therefore, using quantitative cytofluorometry, we followed T4 cell surface CCR5 density in 27 subjects with RA before and after treatment with the anti-CD20 monoclonal antibody rituximab. We observed low T4 cell surface CCR5 densities before treatment, which correlated positively with disease activity, as determined using a disease activity score evaluated on 28 joints (DAS 28), and negatively with CCL5 mRNA concentrations in PBMC, contrasting with a high proportion of intracellular CCR5 molecules, a pattern compatible with ligand-induced CCR5 internalization. At 3 months post-treatment, CCL5 mRNA expression in PBMC declined, whereas T4 cell surface CCR5 densities increased proportionally to the decrease in DAS 28. Thus, peripheral blood T4 cell surface CCR5 density is a good surrogate marker of RA activity and of the efficiency of anti-CD20 therapy.

Cytofluorometry as a Diagnosis of Protoporphyria

Protoporphyria is a disorder that usually has cutaneous symptoms. Only in a few cases liver disease develops, invariably progressing to cirrhosis and liver failure. A case of a patient who suffered from photosensitivity as a result of protoporphyria since the age of 2 is described. At age 33, she first presented with cholestatic hepatitis. At age 40, liver failure developed requiring liver transplantation. Quantitative cytofluorometry proved to be a quick and simple method in the follow-up of her erythrocyte protoporphyrin levels before, during, and after transplantation. Fluorescence correlated well with the protoporphyrin levels obtained by high-performance liquid chromatography (r = 0.98), a comparably cumbersome and complicated method for the determination of protoporphyrin. In addition, single cell analysis enabled us to follow the effects of transfusion therapy on protoporphyrin levels of the patient's own erythrocytes, which has not been possible by previous methods. Thus, cytofluorometry might prove to be elegant and useful for screening and future studies on therapy and pathophysiology of protoporphyria.

DAPI staining improved for quantitative cytofluorometry.

DNA-DAPI complexes emit strong bluish white fluorescence when excited by ultraviolet light so that even very small amounts of DNA such as those in mitochondria, chloroplasts, and virus particles can be visualized. Moreover, the staining procedure with DAPI is very simple and requires no hydrolysis. However, DAPI staining was considered unsuitable for quantitative purpose; nonspecific cytoplasmic fluorescence, scattering of strong emission light, and fading of the fluorescence under UV excitation were major problems of DAPI staining in quantitative cytofluorometry. We found that (1) nonspecific cytoplasmic fluorescence could be eliminated by reducing the DAPI concentration to 50 ng/ml, (2) fluorescence decay was markedly decreased by adding electron donors and molecules containing SH radicals in the mounting media, and (3) light scattering became negligible after reducing the intensity of the excitation light. Thus satisfactory precision could be obtained in DNA quantification by epifluorescent cytophotometry on DAPI stained specimens.

A Cytofluorometric Analysis of Polymer-Induced Mast Cell Secretion

Quantitative cytofluorometry was used to study the mechanism of mast cell secretion induced by polymyxin B <i>in vitro. </i>We measured 5-hydroxytryptamine (5-HT) and heparin in mast cells and individual mast cell granules, extruded from the cells or obtained from the interior of the cells by micromanipulation. Mast cell granules of untreated control cells contained both 5-HT and heparin. After polymyxin stimulation both intracellular and extruded mast cell granules contained heparin, but some intracellular and all extruded granules lacked 5-HT. These results indicate that the mast cell amines may be released from granules while they are still within the cellular domain. The mast cell granules did not release heparin whether extruded from the cell or not. The release of heparin from mast cells was thus determined by the number of extruded granules. Our results further suggest that the granules first released are those situated in the periphery of the cell and contain the largest quantities of amines and heparin. In agreement with the granule measurements, fluorescence measurements on stimulated mast cells showed a dose-dependent decrease of both heparin and 5-HT, but the 5-HT content decreased proportionally more than that of heparin.

Induction and Specificity of Antisera from Mice Immunized with Syngeneic UV-Induced Tumors

Abstract Sera from mice inoculated with syngeneic fibrosarcomas induced by ultraviolet light (UV) were tested to determine if antibodies specific for the immunizing tumor were present. The assay used for this determination involved indirect immunofluorescence binding of anti-sera to tissue culture cells derived from the appropriate UV-induced tumor. The degree of binding of a given antiserum was determined by either fluorescence microscopy or quantitative cytofluorometry. Antisera specific for the syngeneic UV-induced tumor used for immunization were produced. Binding by such antisera was tumor specific and was not due to the presence of contaminating antibodies directed against AKR murine leukemia viruses (MuLV). Only two instances of cross-reactivity were observed, and these involved allogeneic UV-induced tumors. These sera did not react with embryonic fibroblasts, tail skin epidermal cell lines, methylcholanthrene (MCA)-induced fibrosarcomas, or spontaneous fibrosarcomas. On the other hand, anti-UV-tumor sera, which also contained antibodies that react with MuLV, were cross-reactive with a number of tumors. Upon removal of anti-MuLV antibodies by appropriate adsorptions, the cross-reactive activity of these sera was lost, but the specific activity against the immunizing UV-induced tumor remained. We conclude that syngeneic mice challenged with UV-induced tumors can make a tumor-specific humoral immune response to individual tumors.

Phylogenetic origins of immune recognition: lymphocyte surface immunoglobulins and antigen binding in the genus Carassius (Teleostii)

AbstractVirtually all lymphocytes (from thymus, spleen and head nephros) of two closely related cyprinoid fish, C. auratus and C. carassius, were shown to bear surface immunoglobulin (Ig) detectable by immunofluorescence. Nonlymphoid cells (for example, those of the granulocytic and erythrocytic series) showed insignificant staining compared with lymphocytes. Rabbit antisera used in these studies were raised to purified serum IgM of C. auratus. The serum IgM of C. auratus and C. carassius expressed identical determinants as recognized by this antibody in radioimmunoassay, but with a lower level of expression on the IgM of C. carassius. Similarly, with the use of these reagents in quantitative cytofluorometry, the lymphoid cells of C. carassius stained less brightly than those of C. auratus. However, in both species, a hierarchy of fluorescence intensity could be observed, such that spleen ⩾ head nephros > thymus.A small percentage of lymphocytes in all organs of both species bound either myoglobin, keyhole limpet hemocyanin, or horse spleen ferritin antigens. All antigen‐binding lymphocytes were also positive for membrane Ig. Analysis of the distribution of antigen and Ig patches on these cells showed that antigen patches were always coincident with Ig patches.These results provide further evidence of lymphoid heterogeneity in the genus Carassius, and are compatible with the suggestion that lymphocyte surface Ig functions as a primary recognition molecule for all lymphocytes in Carassius species.

Shared idiotypic determinants on B and T lymphocytes reactive against the same antigenic determinants. II. Determination of frequency and characteristics of idiotypic T and B lymphocytes in normal rats using direct visualization.

Anti-idiotypic antibodies made against the antigen-binding receptors of T lymphocytes for a given antigen (Ag-B locus antigens in rats) can be shown to react with IgG antibodies of the same antigen-binding reactivity. Using such anti-idiotypic antibodies, normal Lewis T lymphocytes of B and T type can be visualized by the use of anti-(Lewis-anti-DA) antibodies. Visualization was made possible by the use of direct fluorescent antibody tests or by autoradiography. Using the first technique and naked eye observations 6.2% of normal Lewis T lymphocytes expressed idiotypic markers signifying anti-DA reactivity, whereas anti-DA-reactive B lymphocytes as measured by this approach was in the order of 1.1%. Autoradiography was purified normal Lewis T lymphocytes gave similar figures. When comparing the intensity of fluorescence at the single cell level using quantitative cytofluorometry anti-idiotypic antibodies reactive with T lymphocytes gave a similar degree of intensity as was obtained using anti-Ig antibodies against B lymphocytes.