Linear 2' O-Methyl RNA probes for the visualization of RNA in living cells.

U1snRNA, U3snRNA, 28 S ribosomal RNA, poly(A) RNA and a specific messenger RNA were visualized in living cells with microinjected fluorochrome-labeled 2' O-Methyl oligoribonucleotides (2' OMe RNA). Antisense 2' OMe RNA probes showed fast hybridization kinetics, whereas conventional oligodeoxyribonucleotide (DNA) probes did not. The nuclear distributions of the signals in living cells were similar to those found in fixed cells, indicating specific hybridization. Cytoplasmic ribosomal RNA, poly(A) RNA and mRNA could hardly be visualized, mainly due to a rapid entrapment of the injected probes in the nucleus. The performance of linear probes was compared with that of molecular beacons, which due to their structure should theoretically fluoresce only upon hybridization. No improvements were achieved however with the molecular beacons used in this study, suggesting opening of the beacons by mechanisms other than hybridization. The results show that linear 2' OMe RNA probes are well suited for RNA detection in living cells, and that these probes can be applied for dynamic studies of highly abundant nuclear RNA. Furthermore, it proved feasible to combine RNA detection with that of green fluorescent protein-labeled proteins in living cells. This was applied to show co-localization of RNA with proteins and should enable RNA-protein interaction studies.

Analysis of antifading reagents for fluorescence microscopy.

The utility of p-phenylenediamine, 1,4-di-azobicyclo-(2.2.2.)-octane, and the commercial products Citifluor, Slowfade, and Vectashield, antifading agents frequently used as mounting media for fluorescence in situ hybridization, was investigated. Fading curves for bound fluorochromes were recorded with digital microscopy, and relative fluorescence intensities of fluorochromes in solution were measured with an aperture defined measurement system. The three commonly used fluorochromes, fluorescein, tetramethyl rhodamine, and coumarin, were studied. Vectashield offered the best antifading properties for all three fluorochromes, although their relative fluorescence intensity was slightly less in Vectashield in comparison with other antifading agents. In Vectashield, fluorescein, tetramethyl rhodamine, and coumarin showed half-life times of 96, 330, and 106 s, respectively, whereas in 90% glycerol in PBS (pH 8.5), these half-life time values were 9, 7, and 25 s, respectively. Vectashield is particularly recommended as a mounting medium for quantitative digital imaging microscopy and for multicolor applications, where it is easy to have errors due to differences in fading rates of the fluorochromes.

A new, automated and accurate in vitro method to quantify endothelial cells attached to vascular prostheses.

Over a decade ago the idea of endothelial cell seeding was introduced in an attempt to improve the function of small caliber vascular prostheses. Although endothelial cell seeding is currently being applied clinically, several questions regarding the functional properties of the seeded endothelial cells remain. Evaluation of functional properties of endothelial cells on various types of vascular prostheses can be performed partly in vitro, but it is hampered by the fact that commonly used methods to quantify endothelial cells do not adequately apply to these cells on prosthetic materials. An accurate quantification method is described that is rapidly and easily applicable to endothelial cells attached to vascular prostheses. The method can also be used to quantify endothelial cells attached to culture dishes or microcarriers. Colorless, non-fluorescing, fluorescein-diacetate was used, which was taken up by the attached endothelial cells, and which was then intracellularly converted to yellow fluorescein, emitting green fluorescence. Subsequently, triton-X-100 was applicated to release fluorescein and levels of fluorescence were measured with the automated aperture-defined microvolume (ADM) method, using an inverted fluorescence microscope to which a photometer was connected. The measured level of fluorescence is linearly related to endothelial cell numbers attached to prostheses. The accuracy and the reproducibility of cell countings are high.

Early replication signals in nuclei of Chinese hamster ovary cells.

DNA replication sites generally known as replicon domains were resolved as individual replication signals in interphase nuclei of permeabilized Chinese hamster ovary cells by immunofluorescent microscopy. Biotin-11-dUTP was utilized as a tool to label newly replicated DNA in permeable cells and to study the distribution of nascent DNA in pulselabel and in pulsechase experiments. Active sites of DNA replication were visualized in exponentially growing cells and in synchronized cultures throughout the S phase. Fluorescent images of replication sites were analyzed by standard fluorescence microscopy and in three dimensions by confocal laser scanning microscopy. The rapid increase in number of discrete foci of newly replicated DNA is an indication that DNA synthesis starts at limited number of sites in mammalian nuclei rather than at thousands of foci at the same time.

Enumeration of Leu2a+, Leu2a+-DR+, and Leu2a+-Leu15+ cells in peripheral blood of renal transplant patients.

Immunological monitoring of peripheral blood cells of renal transplant patients may be helpful for diagnostic and prognostic purposes. We therefore enumerated the percentage of Leu2a+ cells as well as the occurrence of HLA-DR activation markers within this population. Since Leu2a+Leu15+ lymphocytes display suppressor functions in-vitro, this population was included in the study. Blood samples of 50 renal transplant patients within 3 months of transplantation and 41 pretransplant samples were investigated. As controls, peripheral blood cells from 10 healthy volunteers and 20 renal transplant patients with stable graft function more than 6 months after transplantation were examined. Just prior to transplantation the percentages of Leu2a+ cells, as well as the percentages of Leu15+ and DR+ cells within this population, varied considerably. Shortly after transplantation, clinical rejection episodes and cytomegalovirus (CMV) infections correlated with decreased percentages of Leu2a+ cells (P = 0.024 and 0.016, respectively). In nine patients with a decreased percentage of Leu2a+ cells shortly after transplantation but no rejection, the percentage of Leu2a+ cells normalized within 14 days. No correlation was found between increased percentages of Leu15+ or DR+ cells and rejection episodes, although the percentages of Leu2a+Leu15+ and Leu2a+DR+ cells increased significantly shortly before rejection (P less than 0.005). A decreased percentage of Leu2a+ cells, together with increased percentages of Leu15+ and DR+ cells, was seen in association with CMV infections (P less than 0.005). From this study we conclude that low percentages of Leu2a+ cells correlate with rejection episodes or CMV infections, whereas low percentages together with increased percentages of Leu2a+DR+ and Leu2a+Leu15+ cells are associated with CMV infections, especially in patients treated with high doses of cyclosporine.

The influence of cytomegalovirus carrier status on lymphocyte subsets and natural immunity.

The influence of the cytomegalovirus (CMV) carrier status on peripheral lymphocyte subsets was studied in 70 healthy individuals. IgG-class antibodies against CMV late antigen were used as markers for the presence of CMV in those individuals. The 39 CMV-seropositive individuals had significantly higher numbers of CD3+ (P = 0.009), CD8+ (P = 0.005) and HNK1+ (P = 0.002) cells than the 31 CMV-seronegative individuals. Two-colour immunofluorescence studies revealed that the HNK1+ cells coexpressing CD4 or high density CD8 were particularly increased in the number under the influence of CMV, but not the HNK1+ cells coexpressing CD16. Since HNK1 and CD16 are markers associated with natural killer (NK) activity and antibody-dependent cellular cytotoxicity (ADCC), we investigated the influence of the CMV carrier status on those functions. The NK and ADCC functions of peripheral blood mononuclear cells (PBMC), HNK1+ and HNK1- cells were correlated with the percentages of CD16+ cells among those cells. Although CMV-seropositive individuals had significantly less CD16+ cells among their HNK1+ cells than CMV-seronegative individuals (mean and s.d.: 39 and 19%, versus 58 and 11%, P = 0.003), the NK and ADCC functions of the HNK1+ cells were similar in both groups. Also, the CMV carrier status did not influence significantly those functions of PBMC and HNK1- cells. We conclude that the CMV carrier status, i.e. CMV-seropositivity, is associated with a significant increase in the numbers of HNK1+ lymphocytes coexpressing T cell markers. That situation may reflect the continuing interaction between CMV and the immune system of its host.

Cytomegalovirus immunity and T lymphocytes in bone marrow donors and acute graft-versus-host disease.

T lymphocyte subset numbers in bone marrow grafts were correlated with the cytomegalovirus (CMV) antibody status of the donors and with the occurrence of acute graft-versus-host disease (GVHD) in their recipients. We studied whether or not the (previously reported) association between donor CMV antibodies and GVHD could be explained by CMV-related changes in T cell subsets in the marrow grafts. There were no significant correlations between any of the T cell subsets in the marrow grafts and the occurrence of grades II-IV GVHD. A particular subset of CD8+ T cells carrying the HNK1 marker was significantly increased in the marrow grafts of CMV-seropositive donors. Although recipients of marrow from CMV-seropositive donors received an average of five times more CD8+ HNK1+ T cells than those with CMV-seronegative donors, that situation was not associated significantly with the development of GVHD.

Reduction and repopulation of T-lymphocytes after cytoreductive therapy with or without autologous bone marrow rescue.

The reconstitution of peripheral-blood T-lymphocytes following cytoreductive therapy in standard (11 patients) or in high dosages (ten patients) was compared with that after supralethal cytoreductive therapy followed by autologous bone marrow rescue (ABMR, 20 patients). Along with the increasing cytotoxic potential of the three therapy protocols, T-cell counts fell to lower levels. Following all three forms of cytoreductive therapy, T8+ T-cell counts decreased to lower levels than T4+ T-cell counts. The greater relative reduction of T8+ T cells may indicate that T8+ T cells are more sensitive to cytoreductive therapy than T4+ T cells, and/or that T8+ T cells have shorter survival times. The contribution of residual (mainly T4+) T cells to the T-cell repopulation was significant in the patients on standard-dosage chemotherapy, less important in those on high-dosage chemotherapy, and minor in those receiving supralethal cytoreductive therapy and ABMR. The repopulation rates of T8+ T cells following ABMR exceeded those observed after chemotherapy without ABMR. The T3- (T3 negative) T-cell subset, which comprises only 5%-10% of peripheral T cells in normal individuals, decreased rapidly to low levels and remained so for the entire six-week observation period in both chemotherapy groups. Following ABMR, however, those T3- T cells rapidly increased again to normal levels. Since the T cells in bone marrow biopsies have a large T3- fraction, that rapid recovery of T3- T cells may reflect the contribution of marrow precursors in the marrow grafts to the improved T-cell regeneration following ABMR.

Flow cytometric analysis of tobacco and cowpea protoplasts infected in vivo and in vitro with alfalfa mosaic virus.

Immunofluorescence flow cytometry was used to study the distribution of viral antigen in protoplast populations. Protoplasts were isolated from healthy and alfalfa mosaic virus (AMV) infected tobacco leaves (designated in vivo infected). Furthermore isolated tobacco and cowpea protoplasts were infected in vitro with AMV. The FITC-conjugated antibodies could penetrate formaldehyde fixed protoplasts. The flow cytometric measurements were rapid and reproducible. Comparable immunofluorescence patterns were found for all infected samples (per sample 10(4) protoplasts were measured). Infectious virus could only be detected in in vivo infected tobacco protoplasts and in in vitro infected cowpea protoplasts.

Flow cytometry of human reticulocytes based on RNA fluorescence.

A fluorescent staining procedure based on pyronin Y is described. The technique has been used to stain RNA in human reticulocytes for subsequent flow analysis and sorting. Histograms of fluorescence values of mature red blood cells and reticulocytes obtained by flow cytometry of stained blood samples, contain more information than offered by conventional methods for counting reticulocytes. Analysis of the fluorescence histograms provides new descriptive parameters of the reticulocyte populations. The potential importance of this method in experimental and routine hematology is illustrated for a number of clinical cases.