Flow cytometric analysis of human dental pulp tissue.

Existing knowledge regarding the cellular components of the dental pulp has been derived primarily from classical methods of histology and biochemistry. Since observations made from prepared tissue sections are static, it is not clear whether this accurately reflects the cellular dynamics of living pulp tissue. Therefore, we developed a method to analyze vital human pulpal tissue by flow cytometry. To test this method, two analyses of the prepared pulpal tissue were performed. First, the prepared tissue was stained with monoclonal antibodies to detect lymphocyte subpopulations. Second, the tissue was processed for DNA analysis of individual cells. Results demonstrated that lymphocytes bearing CD4 and CD8 antigens were clearly detected in pulpal tissue by this method. No B cells were found in any sample. DNA analysis revealed two distinct cell populations. Approximately 88% were small and 12% were large. According to DNA content, 90% of all cells were noncycling and 10% were cycling. These results demonstrate the feasibility of using flow cytometric analysis to examine, at a quantitative level, the cellular heterogeneity of the human dental pulp.

Hypertrophy of renal mitochondria.

Compensatory renal hypertrophy leads to an increase in the size and metabolic capacity of renal tubular cells. Increased transport and metabolic activities must be sustained by an augmented rate of energy production, which is largely dependent on mitochondrial processes. Although previous studies have suggested that mitochondria proliferate in the hypertrophying cell, the data to support this have not been convincing. This study was designed to determine whether the mitochondria of the hypertrophied renal proximal tubular cell undergo hypertrophy or proliferation. Flow cytometric analysis of proximal tubular cells obtained from the kidneys of uninephrectomized rabbits revealed an increase in cell size and RNA content compared with control cells but showed no change in DNA content and nuclear size and no evidence of entry into the S/G2/M phases of the cell cycle. Histomorphometric analysis of cortical proximal tubules revealed that although cytoplasmic volume increased, mitochondrial density remained constant, indicating that mitochondrial volume increases in proportion to the increase in cell volume. By day 14, mitochondrial volume had increased 66% above control values. Electron microscopic examination of isolated S2 proximal tubules from 5/6 nephrectomized rabbits with maximal hypertrophy revealed mitochondrial cristae which appeared to be more densely packed than that in normal cells. The size of the functional mitochondrial pool per cell was determined by rhodamine-123 fluorescence. This increased within 24 h of uninephrectomy, peaked at approximately 80% above control levels at 5 days, and remained elevated throughout the 16 days of observation. The initial increase (days 1 and 2) occurred before a measurable increase in mitochondrial volume occurred and presumably reflects an increase in mitochondrial membrane potential.(ABSTRACT TRUNCATED AT 250 WORDS)

Suppressor cell activity in a randomized trial of patients receiving active specific immunotherapy with melanoma cell vaccine and low dosages of cyclophosphamide.

Previous studies have shown that melanoma patients develop an immune response to cell surface melanoma-associated antigens. The presence of this antibody response to cell surface antigens has been correlated with a better clinical outcome when melanoma patients are treated with an allogeneic melanoma cell vaccine (MCV) as an active immunotherapy protocol. It was hypothesized that the inability to consistently induce or enhance existing immune responses to melanoma-associated antigens was related to the downregulation by suppressor cells. Patients received treatments of MCV 3 times in a 4-week interval and then every fourth week. The biological response modifier cyclophosphamide (CYP) is an immunomodulator of suppressor T-cell function. In this study we set out to determine whether CYP given prior to MCV could reduce suppressor cell activity during vaccination. In a randomized trial stage II and III melanoma patients (n = 41) were given MCV alone or in conjunction with CYP at dosages of 300, 150, or 75 mg/m2. CYP was given 3 days prior to each MCV treatment. Suppressor cell activity in patients was monitored by a concanavalin A suppressor assay using peripheral blood lymphocytes from serial phlebotomies during a 12-week period of treatment. In each trial group there were patients who had major reduction in suppressor cell activity (greater than 50%). Overall, the greatest reduction in suppressor cell activity occurred in patients receiving 300 mg/m2 CYP compared to the other CYP dosages or MCV alone. For the first two treatments at all CYP dosages there was a greater number of patients showing reduced suppressor cell activity compared to later treatments. In a comparison of patients receiving MCV alone to MCV + CYP 300 mg/m2 phenotypic analysis of lymphocyte subsets showed significant (P = 0.03) reduction in the CD8+CD11B+ (suppressor) cells of the latter group. These studies suggest that CYP can be used at low dosages in conjunction with MCV to reduce suppressor cell activity.

Induction of hypertrophy in cultured proximal tubule cells by extracellular NH4Cl.

Ammonia production increases in several models of renal hypertrophy in vivo. The present study was designed to determine whether ammonia can directly modulate the growth of renal cells in the absence of a change in extracellular acidity. In serum-free media NH4Cl (0-20 mM) caused JTC cells and a primary culture of rabbit proximal tubule cells to hypertrophy (increase in cell protein content) in a dose-dependent fashion without a change in DNA synthesis. Studies in JTC cells revealed that the cell protein content increased as a result of both an increase in protein synthesis and a decrease in protein degradation. Total cell RNA content and ribosome number increased after NH4Cl exposure and the cell content of the lysosomal enzymes cathepsin B and L decreased. Inhibition of the Na+/H+ antiporter with amiloride did not prevent the hypertrophic response induced by NH4Cl. The results indicate that ammonia is an important modulator of renal cell growth and that hypertrophy can occur in the absence of functioning Na+/H+ antiport activity.

Relation between DNA ploidy and the clinical behavior of phyllodes tumors.

We studied the DNA histograms obtained by flow cytometry from a series of six giant fibroadenomas and ten phyllodes tumors to determine if the analysis of DNA ploidy would help to predict clinical behavior. We were unable to document any relation between ploidy and histologic appearance, recurrence, metastasis, lesion size, or patient age. DNA aneuploid stem cell lines were seen in 75% of histologically benign phyllodes tumors, 50% of histologically malignant phyllodes tumors, and approximately 33% of giant fibroadenomas.

Prognostic significance of the DNA content of renal carcinoma.

DNA analysis was performed on fresh frozen samples of the primary tumor in 32 patients with renal carcinoma (13 with apparently localized disease and 19 with metastases at presentation). A comparison of ploidy with staging and standard histologic variables was performed. None of the patients who presented without metastases died of disease during the follow-up period. Eleven of 13 patients of this group had a diploid/near diploid pattern, and metastases developed in only one patient. Patients with metastatic disease and a diploid/near diploid DNA content had a significantly better survival rate than those with aneuploid primary tumors. Statistical analysis showed that grade and ploidy contributed significant but independent prognostic information. We concluded that DNA content is a useful prognostic factor in renal carcinoma.

Patterns of mRNA expression during early cell growth differ in kidney epithelial cells destined to undergo compensatory hypertrophy versus regenerative hyperplasia.

An increase in cell size and protein content is characteristic of cells undergoing hypertrophy and of replicating cells prior to DNA synthesis. Cell enlargement in the two situations could be regulated by similar early events with an interruption of the cell cycle occurring in hypertrophy, or the two processes could be uncoupled. In vivo models were used to compare hypertrophy induced by unilateral nephrectomy and hyperplasia induced by folic acid injection in rabbit renal cortical cells. Within 48 hr, cell volume increased in both groups but the number of cells in the cell cycle and DNA synthesis was increased only after folic acid. Patterns of mRNA expression of the following three groups of cell cycle-dependent genes were analyzed: (i) protooncogenes (c-fos, c-myc, and c-Ha-ras), (ii) structural protein genes (vimentin and beta-actin), and (iii) transport protein genes (Na+, K+-ATPase, ADP-ATP translocase, and calcyclin). mRNAs for all genes, except calcyclin and c-Ha-ras, were detected in controls. Folic acid generally induced rapid, transient increases in mRNA levels, but after unilateral nephrectomy, expression of most mRNAs showed a gradual, progressive increase. These data indicate that gene expression in the early stages of cell enlargement differs in cells destined to undergo proliferation vs. hypertrophy. The term "sustained message amplification" is proposed to describe the hypertrophied cell.

Variation in DNA content of mononuclear cells of patients with dementia of the Alzheimer type.

Flow cytometry studies to measure mean DNA content and intercellular variability around the mean were performed on mononuclear blood cells obtained from 21 patients with a clinical diagnosis of dementia of the Alzheimer type (DAT) and 25 cognitively intact individuals. There was no difference in the mean DNA content between both groups. However, patients with DAT had significantly increased intercellular variability compared to controls (3.52 vs. 3.06, respectively). These results are consistent with the reports of increased aneuploidy associated with DAT found on cytogenetic examination.

The application of flow cytometry in the study of natural killer cell cytotoxicity.

A flow cytometric assay was developed to detect the cytotoxic function of natural killer (NK) cells. This procedure employed a single fluorochrome and forward angle light scatter to differentiate dead cells from live cells. When results obtained by flow cytometry were compared with 51Cr release assay, this assay showed high specificity and sensitivity. When propidium iodide was used to stain target cells killed by NK cells, we found that NK cell cytotoxicity was not cell cycle specific.

Identification and characterization of liver nonparenchymal cells by flow cytometry.

Our objective was to correlate biochemical or functional properties that could be measured by fluorescence to individual nonparenchymal cell types by the simultaneous detection of forward angle light scatter and fluorescence. Cells were released from liver following selective digestion of hepatocytes by Pronase perfusion. To fractionate the cells according to size, isolates were subjected to unit gravity sedimentation on a 1 to 5% sucrose gradient. Flow cytometry was used to analyze the resulting fractions as well as the original unfractionated cells. This reaffirmed that forward angle light scatter is linearly related to the sedimentation velocity (size) of the cells and that it can be used to assess cell sizes in an unfractionated population. Endocytosis of fluorescently tagged particulates was studied by injecting either fluorescein isothiocyanate-tagged colloidal albumin or Coumarin-tagged latex beads (0.57 micron diameter) prior to Pronase perfusion. Beads were found only in the largest cells (Kupffer cells). Since endothelial cells could endocytose only colloidal albumin, they were readily differentiated from nonendocytosing, nonsinusoidal cells ("lymphocytes"). Staining cell protein with fluorescein isothiocyanate made possible the determination of relative protein content per cell ("lymphocyte": endothelial:Kupffer cell was 0.57:1.0:2.0). To determine relative esterase activities, mixed cell isolates were incubated in fluorescein diacetate ("lymphocyte":endothelial:Kupffer cells was 0.13:1.0:2.4). Uptake of rhodamine 123 was used to assess mitochondrial function ("lymphocyte":endothelial:Kupffer cells was 0.45:1.0:14.6). Mitochondrial volume per cell is known to be 1:2 for endothelial:Kupffer cells. Therefore, the large difference in rhodamine uptake is not related to volume.

Pattern of expression of HLA-DR and HLA-DQ antigens and mRNA in myeloid differentiation.

We examined the expression of HLA-DR and HLA-DQ antigens and mRNA from myeloid and lymphoid cells obtained from normal volunteers and established cell lines. Cytofluorometric analysis and immunoprecipitation were performed using murine monoclonal antibodies specific for HLA-DR (L-243) and HLA-DQ (Leu 10). The expression of mRNA for HLA-DR and HLA-DQ chains was determined by Northern blot and RNA dot-blot analysis. Lymphoid cell lines expressed both HLA-DR and HLA-DQ antigens, with consistently higher levels of expression of DR. Myeloid cell lines of early myeloblast or bipotent (myeloid-erythroid) phenotype (KG-1, KG-1a, HEL) expressed HLA-DR at high levels, whereas cell lines manifesting a greater degree of myeloid maturation (ML-3, HL-60, U937) expressed DR at low or undetectable levels. The HLA-DQ antigen was expressed at low levels on the surface of KG-1 and KG-1a cells but was not detectable on other myeloid cell lines. The expression of mRNA for HLA-DR and HLA-DQ chains paralleled the pattern of expression of the respective antigens. The HL-60 and U-937 cells stimulated to differentiate in vitro to macrophages with 1,25 dihydroxyvitamin D3 [1,25(OH)2D3] were induced to express detectable levels of HLA-DR antigens. Exposure to gamma-interferon (gamma-IFN) increased the expression of HLA-DR antigens by all myeloid cell lines. Induction of differentiation in vitro with either 1,25(OH)2D3 or dimethyl sulfoxide potentiated this effect of gamma-IFN. Expression of the HLA-DQ antigens was increased on KG-1 myeloblasts after exposure to gamma-IFN. HLA-DQ expression could not be detected on other myeloid cell lines after exposure to gamma-IFN, nor was HLA-DQ expression stimulated by gamma-IFN after HL-60 and U-937 cells were induced to differentiate to macrophagelike cells in vitro. These results provide additional evidence that expression of the HLA-DR and HLA-DQ genes may be independently regulated in human myeloid cells.

Analysis of nuclei from exponentially growing and conjugated Tetrahymena thermophila using the flow microfluorimeter.

Isolated nuclei of Tetrahymena thermophila from both exponentially growing cultures and from cells following conjugation have been analysed using a flow microfluorimeter. The macronuclei from a culture in exponential growth display a single broad distribution of DNA contents, without bimodal character. The micronuclei are virtually all in G2 phase (4C). The mean of the macronuclear DNA distribution is about 12.4 times the micronuclear mean (50C). When cells are starved in preparation for conjugation, the macronuclei DNA content is decreased about 30%, but the distribution remains similar to that of nuclei from a culture in exponential growth. Following conjugation, the macronuclear anlagen develop through a set of relatively synchronous endoreplications. At 12 h after the initiation of conjugation the anlagen are at a 4C stage and at 18 h they are virtually all at a 8C stage. If the culture is refed, anlagen development progresses to a 16C and 32C, but the synchrony is poorly conserved. Cells that are not refed are arrested at the 8C stage and only a fraction of the population ever become mature macronuclei. In general we do not observe distinct peaks of anlagen with DNA contents in excess of 32C. The amitotic division of macronuclei may obscure any endoreplications producing anlagen stages with higher DNA content.

Alterations in the immune response of nonresponders to the hepatitis B vaccine.

The phenotypic characteristics of peripheral blood lymphocytes (PBLs) from responders and nonresponders to hepatitis B (HB) vaccine and their response to pokeweed mitogen (PWM) stimulation in vitro were compared. The nonresponders had significantly higher absolute numbers and percentages of T11+, HNK-1+, and T8+ lymphocytes. Also, after PWM stimulation, PBLs from HB-vaccine nonresponders had impaired IgG and IgM production and failed to produce antibodies to HB surface antigen in vitro. These findings suggest that healthy individuals who fail to produce such antibodies have a higher population of suppressor lymphocytes that alters their normal immune response to HB vaccination.

Growth and aging in the rat: changes in total protein, cellularity, and polyploidy in various organs.

The objectives of this study were to determine the influence of growth and aging on ploidy, cell number, and protein content of various organs. Tissue homogenates were prepared at 3, 8, 25, 50, and 100 weeks of age. Samples were analyzed for DNA per nucleus (by flow cytofluorometry), nuclei number, and protein content. Livers of 8- and 100-week-old animals were also perfused with collagenase and the released cells separated into parenchymal and nonparenchymal populations by unit gravity sedimentation. Nuclei of these cells were also analyzed for DNA. In all four zones of the kidney and in thyroid, 4n nuclei diminished in percentage between 3 and 50 weeks and increased at 100 weeks. In the growth phase these probably are cycling cells and after 50 weeks represent an increasing population of nuclei arrested after synthesis of DNA. Constant levels of ploidy were found in brain, heart, rectus abdominis, and adrenal throughout the 3-100 weeks. A dramatic increase in 4n nuclei occurred between 3 and 8 weeks in liver with little change occurring thereafter. Ploidy is a property of only parenchymal cells in liver and this probably is also true in other organs. The 4n nuclei that remain in constant proportion to the total population are established early in life and are not related to aging. They are probably tetraploid and replicate into 4n daughter cells during growth. Cerebrum shows no changes in nuclei number but exhibits a 70% increase in protein between 3 and 100 weeks. Although kidney, liver and adrenal show large increases in number of nuclei (approximately equal to fourfold) with growth, these are not as great as increases in body weight (approximately equal to 11-fold). With regard to organ protein, only liver shows increases approximating those in body weight. Increases in organ nuclei appear to occur in concert for adrenal, kidney, and liver whereas increases in organ protein bear no relationship to each other. Protein content remains at stable levels in organs of 100-week-old animals and little (adrenal, liver) or no (brain, kidney) diminution occurs in nuclei numbers.

Gamma-interferon induces expression of the HLA-D antigens on normal and leukemic human myeloid cells.

Gamma-Interferon (IFN-gamma) is a lymphokine produced by T lymphocytes. We find that recombinant human IFN-gamma induces expression of HLA-D antigens on human promyelocytic leukemia cells (HL-60) and enhances expression of HLA-D antigens on normal human monocytes and macrophages. Induction of both HLA-D antigen expression and HLA-D mRNA accumulation occurs within 1 day of exposure of HL-60 to IFN-gamma and is maximal by day 5. Maximal antigen expression occurs in the presence of 100-500 units of IFN-gamma per ml. IFN-gamma induces expression of DR but not DC antigens on HL-60, as confirmed by using four different murine monoclonal antibodies or one rabbit heterologous antibody. RNA blot data show that IFN-gamma-exposed HL-60 cells contain mRNA for DR alpha and DR beta polypeptides but not for DC alpha and DC beta polypeptides, which also suggests that HLA-DR and HLA-DC gene regions can be independently regulated. IFN-gamma induces 20% of HL-60 cells to differentiate to macrophage-like cells. However, IFN-gamma dose-response studies using both HL-60 cells and a nondifferentiation variant of HL-60 cells (HL-60 blast) clearly show that induction of transcription and expression of HLA-D gene products by IFN-gamma can be uncoupled from expression of other monocyte-macrophage characteristics. Further studies show that IFN-gamma enhances expression of HLA-D antigens on normal human monocytes and macrophages. Expression of the HLA-D antigens is necessary for the interaction of macrophages and T lymphocytes; IFN-gamma may play a fundamental role in this interaction.

Conversion of linear histogram flow cytometry data to a logarithmic display.

A routine is described that readily allows the rescaling of linear histographic data to a corresponding logarithmic histogram. This procedure significantly improves data display, particularly where a wide range in the measured parameter is encountered. The logarithmic scale displays peaks with band widths more proportional to their respective coefficients of variation than is the case in a linear display. Rescaling several linear histograms to a common logarithmic scale allows the combination of these linear data even though the linear ranges are different. This routine is presented as a program written in BASIC for execution on a microcomputer.

Proliferation of mitochondria during the cell cycle of the human cell line (HL-60).

Using rhodamine 123 to stain mitochondria of the human cell line HL-60, we have followed their increase over the cell cycle by flow cytometry. A near-linear synthesis of mitochondrial mass was shown to occur over the cell cycle. A comparison with the cell's DNA synthesis pattern obtained by the same technique established a common time-base. The mitochondrial synthesis curve changes with culture age. As a control, thd dye was tested for its binding specificity and for its use to resolve mitochondria microscopically. Its stoichiometric range was established and, above 0.25 microgram/ml, it was shown to reduce growth rate and cell viability in culture.

An ecdysteroid-induced alteration in the cell cycle of cultured Drosophila cells.

The addition of physiological concentrations of the arthropod molting hormone 20-hydroxyecdysone results in the cessation of cell division in the Kc cell line of Drosophila melanogaster. Fluorometric mononitoring of the cell cycle reveals that treatment of the cells with hormone for 12 hr causes a G2 arrest. The dose-response curves are in agreement with those obtained for other hormonal effects in both the Kc line and the intact animal. In the continual presence of hormone, cells remain G2-arrested for approximately 100 hr, resuming division by 120 hr. Cells which have responded once to ecdysteroids and subsequently reentered the cell cycle are insensitive to hormonal restimulation. This lack of response has been correlated with, and is probably due to, the loss of ecdysteroid receptors in stimulated cells.