High frequency of precursors of anomalous killer cells in human peripheral blood: evidence for T-cell regulation

The frequency of precursors (P) of the anomalous killer (AK) cells able to kill a melanoma target cell line without prior sensitization was determined by limiting dilution analysis. The frequencies obtained from the peripheral blood mononuclear cells of six healthy individuals ranged from 1/250 to 1/750, which was considerably higher than those of alloreactive cytolytic T-lymphocyte (CTL) precursors induced in the same cultures (range 1/900 to 1/7500). The presence of phytohemagglutinin (PHA) inhibited the appearance of both CTL and AK in bulk cocultures, and in limiting dilution analysis the presence of the lectin resulted in multiphasic cell dose-response curves rather than linear single hit responses for both types of precursor cells. The results suggest that AK-P are under the same type of regulation as are CTL-P.     

In vitro immunization to KLH. II. Limiting dilution analysis of antigen-reactive cells in primary and secondary culture

Limiting dilution analysis was used to estimate the frequency of human peripheral blood T lymphocytes that proliferate in response to in vitro immunization with keyhole limpet hemocyanin (KLH). Antigen-reactive cells (ARC) were estimated 9 days after primary immunization with KLH. The ARC frequency of lymphocytes from 12 subjects ranged from 1:23,800 to 1:52,631. Lymphocytes from five of these subjects were also primed for 12 days with KLH, rechallenged in secondary culture with fresh adherent cells and KLH, and assayed 4 days later. The ARC frequency increased to 1:1,123 to 1:7,247, indicating that T cell clones responsive to KLH had expanded during primary culture. In addition, we observed that the proliferative response of lymphocytes from 5 of the 12 subjects were inhibited at high cell concentrations. Depletion of OKT8+ T cells before culturing with KLH however did not alter the inhibitory effect of high concentrations of T cells.     

No evidence for genotoxic effects from 24 h exposure of human leukocytes to 1.9 GHz radiofrequency fields

The current study extends our previous investigations of 2-h radiofrequency (RF)-field exposures on genotoxicity in human blood cell cultures by examining the effect of 24-h continuous-wave (CW) and pulsed-wave (PW) 1.9 GHz RF-field exposures on both primary DNA damage and micronucleus induction in human leukocyte cultures. Mean specific absorption rates (SARs) ranged from 0 to 10 W/kg, and the temperature within the cultures was maintained at 37.0 +/- 1.0 degrees C for the duration of the 24-h exposure period. No significant differences in primary DNA damage were observed between the sham-treated controls and any of the CW or PW 1.9 GHz RF-field-exposed cultures when processed immediately after the exposure period by the alkaline comet assay. Similarly, no significant differences were observed in the incidence of micronuclei, incidence of micronucleated binucleated cells, frequency of binucleated cells, or proliferation index between the sham-treated controls and any of the CW or PW 1.9 GHz RF-field-exposed cultures. In conclusion, the current study found no evidence of 1.9 GHz RF-field-induced genotoxicity in human blood cell cultures after a 24-h exposure period.     

Low frequencies of apparently fragile X chromosomes in normal control cultures: a possible explanation

Low frequencies of apparently fragile X [fra(X)] chromosomes have been reported in normal control, short-term, whole blood cultures, and they have been noted in both amniocyte and fetal blood cultures. However, there is currently no universal agreement on the lowest frequency for fra(X)(q27) that is diagnostic for the fragile X syndrome. Here, we present our observations on low levels of apparently fra(X) chromosomes in normal samples. We observed frequencies of 0.5% in short-term whole blood cultures and 0.9% in amniotic fluid cell cultures. In 1982, Steinbach et al. described nonspecific telomeric structural changes (TSC) and suggested that such low frequencies of apparently fra(X) chromosomes in normal material may be occurring by the same mechanism that is responsible for TSC formation. To determine if TSC formation can explain the significant baseline frequencies of fra(X) in normal controls, 10,457 cells were screened from 178 individuals referred for fra(X) analysis. Our findings indicated that TSC are not randomly distributed across chromosomes but tend to occur at specific sites. Based on our observations, we offer the hypothesis that the low frequency of apparent fra(X) in normal individuals may be due to nonrandom TSC distribution.     

Identification,paracrine generation,and possible function of human breast carcinoma myofibroblasts in culture

Summary Myofibroblasts from human breast carcinomas were identified and experimentally generated in culture, and a possible function was examined. The frequency ofα-smooth muscle actin immunoreactive cells was evaluated as a measure of myofibroblast differentiation in primary culture. Few or noα-smooth muscle actin-positive stromal cells (6.1 ± 8.4%) were identified in primary cultures from normal breast tissue (n=9). In contrast, high frequencies (68.8 ± 15.1%) were observed in primary cultures from carcinomas (n=19). The frequencies of myofibroblasts in primary cultures were almost identical to those obtained in the corresponding cryostat sections (69.1 vs. 68.8%). A possible precursor cell to the myofibroblast was looked for among typical fibroblasts and vascular smooth muscle cells. Purified blood vessels containing both fibroblasts and vascular smooth muscle cells were embedded in collagen gel and incubated with medium conditioned by breast epithelial cells. Fibroblasts rather than smooth muscle cells were recruited from the blood vessels. In medium conditioned by carcinoma cell lines or in co-cultures of carcinoma cell lines and purified fibroblasts,α-smooth muscle actin and the typical myofibroblast phenotype were induced in otherwiseα-smooth muscle actin-negative fibroblasts. The effect of myofibroblasts on cellular movement—essential to neoplastic cells—was analyzed. Spontaneous motility of tumor cells (MCF-7) was entirely suppressed in a collagen gel assay. Under these conditions tumor cell motility was selectively mediated by direct cell-to-cell interaction between tumor cells and myofibroblasts. Under chemically defined conditions, interaction was dependent on the presence of plasminogen. Anti-plasminogen, soybean trypsin inhibitor, and anti-fibronectin partly neutralized the effect of plasminogen. It is concluded that elements of myofibroblast differentiation and function may be studied in culture.     

Quantitative Measurement of the Ability of Different Mutagens to Induce an Inherited Change in Phenotype to Allow Maltose Utilization in Suspension Cultures of the Soybean, GLYCINE MAX (L.) Merr

Using a newly developed plating system, we have measured cell survival and the frequencies of variation in an inherited trait after treatment of soybean cell suspensions with different mutagens: ethyl methanesulfonate (EMS), methyl methanesulfonate (MMS), N-Methyl-N'-nitro-N-nitroso-guanidine (MNNG), hycanthone (1-{[2-(diethylamino) ethyl] amino}-4-(hydroxymethyl)-9H-thioxanthen-9-one and ultraviolet light (UV).—The heritable variation selected for displays a phenotype of rapid growth on maltose as carbon source. The marker is stable in the absence of maltose, and prolonged growth of variant cells on sucrose has not shown reversions to slow growth. Doubling time in suspension cultures is decreased from 100 hr to ca. 30 hr by the mutation. Both wild-type and variant cells grow on sucrose with a 24-hr doubling time. Thus, lethality after mutagen treatment can be estimated rapidly by growth on sucrose, whereas variants are scored on maltose medium. The spontaneous frequency of variants was 1.2 x 10^-7; induced frequencies ranged from a low of 3.6 x 10^-5 for EMS to a high of 10^-3 for hycanthone. The high frequency of variants induced by hycanthone, a frame-shift mutagen, and the observation that UV induces variants in haploid cells with much higher frequency than in diploid cells suggests a recessive mutation.     

Limiting dilution analysis of alloreactive cytotoxic precursor cells in aging mice

The quantitative minimal estimate of the frequency of alloantigen specific cytotoxic T lymphocyte precursor cells (CTL-p) was determined in young and old C57BL/6 mice by limiting dilution analysis. Supernatant from phorbol myristate acetate-induced EL-4 cells was used as a source of IL 2 in these assays, which therefore were independent of the presence of the Lyt-2-, IL 2-producing cells known to be deficient in aging mice. These studies showed that 24-mo-old mice had approximately 10-fold fewer CTL-p than their young counterparts. Comparison of the limiting dilution assay (LDA) with IL 2-supplemented primary MLC shows that estimates of the frequency of CTL-p do not consistently agree with cytotoxic activity detected in the higher cell density primary MLC, and that the LDA is most likely a better estimate of the effect of age on the development of CTL.     

Phosphoglycolate phosphatase in several population groups in Israel

The genetic polymorphism of phosphoglycolate phosphatase (PGP) found in red blood cells has been investigated in several population groups in Israel: Ashkenazi Jews, non-Ashkenazi Jews from Iraq, Yemen, Turkey, Iran, Balkan, North Africa and Arabs. The distribution of the PGP genes was not homogeneous (chi 2 = 40.545; d.f. = 20; p less than 0.005). The PGP2 gene frequency varied between 0.0185 in the Yemenite and 0.0688 in the Iranian Jews. PGP3 gene frequency ranged between 0.0062 in the Iranian and 0.0547 in the Moroccan Jews. Depsite this heterogeneity all the Israeli population groups showed some unifying characteristics which differentiated them from a random European population sample, namely higher frequencies of PGP1 gene (92-97% as opposed to 82% in th European sample) and lower frequencies of PGP2 gene (1.8-6.8% compared to 12.9% among Europeans).     

Increased frequency of chromatid breaks in lymphocytes of heterozygotes of ataxia telangiectasia after in vitro treatment with caffeine

The frequencies of caffeine-induced chromosomal aberrations (CA), mainly chromatid (CdB) and chromosome (CB) breaks, were studied in lymphocyte cultures derived from 6 obligatory heterozygotes and 1 homozygote of ataxia telangiectasia (AT), and from 4 control adult healthy persons. Caffeine (CF, 1 mM) was added at the beginning of the cultures exposed to CF the frequency of CB was 1.9% and of CdB 1.3%. In cells of the AT homozygote, the frequency of CdB was 6.8% in the absence and 8.7% in the presence of caffeine, the frequencies of CB being 3.4 and 10.9%, respectively. In AT heterozygous cells treated with CF, CdB increased 13-fold as compared to a less than 3-fold increase in control cells. Comparing the frequencies of CF-induced chromosomal lesions in control and AT heterozygous cells, potentiation factors (Pf) for the effect of 1 AT gene on cell sensitivity of CF (Pf [AT]) were 3.5 for CB, 6.6 for CdB and 5.5 for CA. These data demonstrate that lymphocytes of AT heterozygotes are significantly more sensitive to caffeine treatment in vitro in terms of increased frequency of CdB than normala cells, which may be useful for the diagnosis of carriers of this defective gene.     

The occurrence and structure of primary cilia in a subline of Potorous tridactylus

The paper describes a subline of rat kangaroo Potorous tridactylus kidney (PtK₁) cells capable of producing a single primary (9+0) cilium during interphase. Antitubulin immunofluorescence, scanning electron microscopy (SEM) and high voltage electron microscopy (HVEM) were used to demonstrate the occurrence and structural features of the cilia. During repeated subculturing the frequency of cilia ranged from approx. 60% of the cells in a growing population to almost 100% in confluent cultures. We believe that the subline may provide excellent material for high resolution correlative light- and electron microscopic studies on the development, function and subsequent fate of primary cilia during the cell cycle.     

Limiting dilution analysis of the specificity of influenza-immune cytotoxic T cells

The frequency of memory T cells in the spleens of mice primed with the A/Puerto Rico/8/34/1 (H1N1) (PR8) influenza A virus was determined using limiting dilution protocols. The mean frequency of memory cytotoxic T lymphocytes (CTL) in spleen populations from mice primed with PR8 and restimulated in vitro with the same virus ranged, in six experiments, from 1 in 1600 to 1 in 4800. In the same experiments, the frequencies of CTL capable of lysing targets infected with the heterologous A/Hong Kong/×31/68 (H3N2) (HK) virus ranged from 1 in 1700 to 1 in 4700 nucleated spleen cells. Thus, at least 80% of PR8 (H1N1) influenza-specific cytotoxic T cells are lytic for both HK (H3N2)- and PR8-infected target cells. Further analysis of the specificity of a series of monoclonal influenza-specific CTL was achieved by expanding limit dilution cultures and then testing lytic capacity for targets infected with a range of influenza A viruses. This approach confirmed that the great majority of PR8-primed influenza-specific CTL are cross-reactive for a variety of influenza A subtypes. These experiments demonstrate the feasibility of quantitating different influenza-immune CTL specificities at a stage very close to removal of cells from the animal.     

Frequency of allogeneic helper T cells responding to whole H-2 differences and to an H-2K difference alone.

Limiting dilution analysis was used to determine the frequency of splenic T cells that are stimulated by alloantigen to give help in a primary antibody response to SRBC. Several haplotype combinations were tested. A semilogarithmic plot of the fraction of nonresponding culture as a function of the number of T cells added to excess B cells gave a straight line intercepting with the origin. Thus a single cell-type was limiting, which was required to help B cells respond to SRBC. The frequency of syngeneic precursors of T helper cells specific for SRBC ranged from 1/10,000 to 1/55,000 with a mean of about 1/20,000. Allohelpers generated by whole H-2 differences gave precursor frequencies that ranged from 1/1000 to 1/7000 with a mean of about 1/2500. Thus allohelpers to whole H-2 differences were approximately 8-fold more frequent than SRBC-specific helpers. When the stimulation was limited to the H-2K difference between the mutant B6.C-H-2ba and wild-type B6, frequencies of from 1/2600 to 1/7900 allohelpers were found with a mean of about 1/5000, approximately half the frequency of allohelpers to whole H-2 differences. Thus some, but probably not all, of the magnitude of allogeneic halp can be attributed to the high frequency of helper T cells that respond to a given alloantigen.     

Fetal nucleated cells in maternal peripheral blood: frequency and relationship to gestational age

To determine the frequency of fetal nucleated cells in maternal peripheral blood during different stages of pregnancy, 50 primigravidas were investigated by determining the frequency of cells with the Y chromosome using fluorescence in situ hybridization (FISH) of Y-specific repetitive sequences of the DYZ1 family. Polymerase chain reaction (PCR) amplifying the same part of the DYZ1 used as the probe in FISH and a single-copy Y-specific fragment was also carried out for genomic DNA from the same samples. Cells with the hybridization signal were detected by FISH at and after 15 weeks of pregnancy in all pregnant women who gave birth to boys. The ratio of cells with the signal to those without the signal ranged from 1 in 144,000 to 1 in 4,000 with a tendency to increase as the pregnancy advanced. The frequency of fetal cells estimated by the PCR experiments was significantly and positively correlated with that found by FISH. The present study suggests that fetal nucleated cells increase in maternal peripheral blood with advancing gestation, from less than 1 in 100,000 nucleated cells in the first trimester to around 1 in 10,000 at term. These frequencies were much lower than those reported by cytological methods.     

Evaluation of radiation-induced chromosomal aberrations in human peripheral blood lymphocytes in vitro results of an IAEA-coordinated programme

The results of an IAEA coordinated programme on radiation induced chromosomal aberrations in human peripheral blood lymphocytes in vitro are presented. In a master experiment, a whole blood sample from one donor was irradiated with 200 R of X-rays. Different fixation times from 46 to 82 h were used. The progression of cells into mitosis was monitored by BrdUrd incorporation. 14 investigators took part in the scoring of chromosomal aberrations. The main conclusions of this study are: (1) The mean frequencies of aberrations changed with fixation time. (2) The number of cells scored as aberrant by different laboratories was very similar, but there was variability in the number of aberrations scored per aberrant cell. (3) The differences in the frequencies of aberrations between laboratories were minimal when the scoring was restricted to the first major peak of mitotic activity and sufficient cells were scored.

It is concluded that using controlled experimental conditions, human peripheral blood lymphocytes can effectively be used as a reliable biological dosimeter for absorbed radiation dose.     

The characterization and transmissibility of chromosome aberrations induced in peripheral blood lymphocytes by in vitro alpha-particle radiation

Peripheral blood lymphocytes were irradiated in vitro with (213)Bi alpha particles at doses of 0, 10, 20, 50, 100, 200 and 500 mGy. Chromosome analysis was performed on 47-h cultures using single-color fluorescence in situ hybridization (FISH) to paint chromosomes 1, 3 and 5. The whole genome was analyzed for unstable aberrations to derive aberration frequencies and determine cell stability. The dose response for dicentrics was 33.60 +/- 0.47 x 10(-2) per Gy. A more detailed analysis revealed that the majority of aberrations scored as dicentrics were part of complex/multiple aberrations, with the proportion of cells containing complexes increasing with dose. Cells containing aberrations involving painted chromosomes (FISH aberrations) were further classified according to cell stability and complexity. The majority of cells with FISH aberrations were unstable. The proportion of aberrant FISH cells with complex/multiple aberrations ranged from 56% at 10 mGy to 89% at 500 mGy. A linear dose response for genomic frequencies of translocations in stable cells fitted the data from 0 to 200 mGy with a dose response of 7.90 +/- 0.98 x 10(-2) per Gy, thus indicating that they are likely to be observed in peripheral blood lymphocytes from individuals with past or chronic exposure to high-LET radiation. Comparisons with the dose response for low-LET radiation suggest an RBE of 13.6 for dicentrics in all cells and 3.2 for translocations in stable cells. Since stochastic effects of radiation are attributable to genetic changes in viable cells, translocations in stable cells may be a better measure when considering the comparative risks of different qualities of radiation.     

Specific immunity after congenital or neonatal infection with cytomegalovirus or herpes simplex virus

Infants or children who had congenital or neonatal infection with cytomegalovirus (CMV) or herpes simplex virus (HSV) have fewer than 1:30,000 mononuclear cells in their blood lymphocytes preparations that proliferate in cultures stimulated with the corresponding viral antigens. CMV and HSV responder cell frequencies in children and adults whose immunity followed postnatal infection with these viruses are 1:10,000 to 1:20,000. The low precursor frequency after congenital or neonatal infection is not associated with defective antigen processing by monocytes or nonspecific immunosuppression. Phenotypic changes in T cell subsets and the presence of antibody in the subjects suggests that the virus(es) do indeed elicit an immune response, but that this response is quantitatively deficient.     

Bromodeoxyuridine labelling as an alternative method to identify 6-thioguanine-resistant mutant lymphocytes in humans

6-Thioguanine-resistant (TG^R) mutant lymphocytes in human blood are usually enumerated by the cloning assay which allows the molecular characterisation of the HPRT mutations to be detected. A “short-term” alternative approach is provided by the anti-bromodeoxyuridine (anti-BrdU) technique in which TG^R lymphocytes are identified immunocytochemically by their ability to synthesise DNA in the presence of 6-thioguanine (TG). We have evaluated the influence of various experimental factors that could affect the frequency of TG^R lymphocytes. A standard protocol is proposed, based on 24-h cold storage of isolated lymphocytes at 4°C and 40-h culture with and without TG, the last 16 h with BrdU. The harvested cells are treated with hypotonic (0.075 M) KCl, fixed with methanol:acetic acid (3:1) and put on microscopic slides. For the TG cultures, all cells are prepared on the slides, while slides from the control cultures are made by a 1/50 dilution. DNA is denatured by formamide, and the BrdU label is identified by anti-BrdU antibody detected by immunoperoxidase staining using a peroxidase-conjugated secondary antibody with diaminobenzidine as substrate. In 10 donors, the frequency of TG^R lymphocytes (variant frequency, V_f) detected by this protocol ranged from 69.65×10⁻⁶ to 83.45×10⁻⁶, and split measurements showed a relatively small intra-assay variation in V_f values of each donor. BrdU in DNA was also detected by immunofluorescence using a fluorescein-conjugated anti-BrdU monoclonal antibody. This method, facilitating easy identification of positive cells and rapid microscopic scoring, may serve as a basis for an automated analysis of TG^R lymphocytes. V_f values detected by the anti-BrdU assay are higher than mutant frequencies obtained by the cloning assay, which has been assigned to the presence of non-mutant phenocopies considered to represent spontaneously cycling lymphocytes. Although the anti-BrdU assay is rapid and easy and has been shown to respond to genotoxic exposures, its true value could be evaluated only when it can be ascertained that phenocopies do not significantly contribute to the V_f values obtained.     

Limiting dilution analysis of TNF producing cells in C3H/HeJ mice

A limiting dilution assay (LDA) that measures the frequency of TNF producing cells is described. LDA determination is based on the inhibition of growth of a highly TNF sensitive subline from the WEHI-164 fibrosarcoma by using a micro assay sensitive to single picogram amounts of recombinant murine TNF. Using such LDA, it was determined that the reported deficiency in LPS-induced TNF production in C3H/HeJ mice is a function of reduced frequency of TNF producing cells rather than a complete lack of responsiveness. In bulk culture, LPS-triggered TNF was produced by Thy-1.2 negative spleen cells with activity recovered in both G10 Sephadex adherent and nonadherent subpopulations. LPS stimulation of spleen cells from C3H/HeJ mice resulted in TNF mRNA expression as shown in both Northern blots and in situ hybridization. The frequency of TNF mRNA bearing cells in control of C3H/HeSnJ mice by in situ hybridization correlated with that found for TNF producing cells in LDA. In C3H/HeJ spleen, significantly higher numbers of TNF mRNA positive cells were found than were shown to produce TNF in LDA.     

The genotoxic and cytotoxic effects of ribavirin in rat bone marrow

The genotoxicity of the 2-furylethylene derivative 1-(5-bromofur-2-yl)-2-nitroethene (2-betaNF) has been evaluated in cultured human peripheral blood lymphocytes at concentrations ranging from 0.5 to 15microg/ml. The frequencies of micronuclei (MN) and sister-chromatid exchanges (SCEs) were used and scored as indicators of genetic damage. To asses the role of the metabolism mediated by the enzymes present in the S9 mix, over the possible genotoxic potential of the test agent, the cultures for MN and SCE demonstrations were treated for 3h in presence and in absence of rat liver microsomal fraction. The results indicate that, under the experimental conditions used, the test agent does not induce significant increases in the frequency of micronucleated cells, irrespective of the presence/absence of metabolic fraction. Nevertheless, a slight increase in the SCE frequency was observed in those cultures treated without the S9 mix; although this slight increase disappeared in the experiments carried out with the microsomal fraction. In addition, cytotoxic/cytostatic effects of (2-betaNF) were observed mainly in the cultures treated without the S9 fraction.     

Gestation stage-specific frequency of adipogenic cells in Syrian hamster cell cultures

High frequencies (up to 50%) of spontaneous adipocyte differentiation are observed in cultures of 9 day gestation Syrian hamster embryos (E9 cells) within six to eight population doublings after primary culture. This is in contrast to the absence of adipogenic cells in primary cultures derived from later gestation age Syrian hamster tissue. In addition, E9 primary cultures contain a transient subpopulation of presumptive mesenchymal stem or progenitor cells that lack density dependent inhibition of growth [contact-insensitive (CS-) cells]. Analysis of the temporal pattern of expression of the CS- and adipocyte phenotypes during the proliferative life span of E9 cells demonstrates that maximal expression of the CS- phenotype precedes maximal expression of adipocyte differentiation. In addition, lipid accumulation appears to occur primarily, if not exclusively, in the contact-sensitive (CS+) cells that are derived from CS- cells. These observations suggest that primary E9 cultures contain either adipoblasts or primordial mesenchymal cells that become determined to the adipocyte lineage early during the in vitro life span of the cultures, and that the CS- phenotype may be a marker for these earlier developmental cell stages.