Human Blood Cultures Research Articles

Repair of sister-chromatid exchange-inducing lesions in mutagen-treated cultures of human whole blood and purified fresh or frozen lymphocytes

Repair of mutagen-induced lesions that result in sister-chromatid exchanges was evaluated in 10 normal individuals. The mutagens used were mitomycin C (MMC), 4-nitroquinoline 1-oxide (4NQO), and N-methyl- N′ nitro- N-nitrosoguanidine (MNNG). Cultures of whole blood, freshly purified lymphocytes, or purified lymphocytes cryopreserved for 6 months were analyzed after the mutagen treatments. All 3 mutagens induced reparable damage as evaluated by comparison of sister-chromatid exchanges between cultures that were given time to repair induced damage before 5-bromo-2′-deoxyuridine (BrdUrd)was added to the culture medium with those where BrdUrd was added immediately after the administration of the mutagens (MMC or 4NQO) or at culture initiation (MNNG). Repair of mutagen-induced DNA damage was detected in all 3 culture types; thus cryopreservation did not appear to alter the capacity of lymphocytes to repair mutagen-induced lesions. Quantitative differences in apparent repair capabilities were observed among individuals. Variability also existed among the different culture types within an individual, suggesting that caution should be exercised in interpreting these apparent differences.

Generation of High‐Rate Ovalbumin‐Specific Antibody‐Secreting Cells in Cultures of Human Peripheral Blood B Cells Obtained from Non‐Immunized Blood Donors

When human peripheral blood B cells are cultured for 6 days with the T cell-dependent peptide antigen ovalbumin (OA) in the presence of antigen-presenting cells and helper T cells, plaque-forming cells (PFC) are generated. These OA-induced PFC differ from the conventional high-rate antibody-secreting PFC formed after stimulation of B cells with recall antigens (e.g. tetanus toxoid) in that they secrete antibody at a very low level. Previous studies have shown that OA-induced PFC are B lymphocytes in an early activation state rather than cells that have differentiated into plasmablasts. The apparent arrest in the maturation of OA-induced PFC in an early activation phase can be overcome by simultaneous stimulation with interleukin 2 (IL-2) and gamma interferon (IFN-gamma). The isotype of the OA-specific antibodies secreted, however, are only of the IgM class, demonstrating that an isotype switch does not occur.

Baseline and mutagen-induced sister-chromatid exchanges in cultures of human whole blood and purified fresh or frozen lymphocytes

Baseline and mutagen-induced levels of sister-chromatid exchanges were evaluated in 10 normal individuals. Cultures with whole blood or purified lymphocytes, either freshly isolated or after 1 or 6 months of cryopreservation, were analyzed to determine whether frozen lymphocytes are suitable for SCE studies. Whole blood and freshly isolated lymphocytes were cultured from samples taken at the beginning of the study (Time 0) and 6 months later (Time 6). Cryopreserved lymphocytes were recovered after 1 month (time 1) and 6 months (time 6) of cryopreservation and t hen challenged with mutagens in culture. The mutagens used were mitomycin C, 4-nitroquinoline-1-oxide, and N-methyl- N′-nitro- N-nitrosoguanidine. Purified lymphocytes had consistently and significantly higher baseline SCE frequencies than cells from whole blood cultures and were more sensitive to N-emthyl N-′-nitro- N-nitrosoguanidine and 4-nitroquinoline-1-oxide. The response to mitomycin C was similar in all culture types. There was, overall, no consistent effect of freezing on baseline or induced sister-chromatid exchange frequencies in the purified lymphocytes. This suggests that purification and cryopreseration of human lymphocytes does not alter the baseline or mutagen-induced sister-chromatid exchange response and in certain epidemiological, occupational and monitoring situations may have logistical and technical advantages over the use of fresh whole blood.

Cytogenetic effects of penicillamine

Penicillamine (PA), a drug used for the treatment of rheumatoid arthritis induces sister-chromatid exchanges (SCEs) and chromosome aberrations in cultivated mammalian cells. PA in concentrations from 400 μg/ml upward induced SCEs and proliferative delay in human blood cultures when added for the last 24 h of the culture period. In V79 Chinese hamster cells SCE induction was found after acute exposure to PA before the addition of BrdUrd and after chronic exposure during one cell cycle in the presence of BrdUrd. The effect of PA on SCE frequencies occurred both after treatment in complete medium and in serum-free medium and was not influenced by the application of an S9 mix. The simultaneous addition of peroxidase reduced the PA-induced SCEs whereas catalase did not show any effect. Chromosome analysis in the first mitosis after PA treatment revealed a significant increase in the incidence of chromosome aberrations and endoreduplication. The results are discussed with respect to the cause and the significance of the observed effects in connection with mutagenicity testing.

The Role of Mast Cells in Inflammatory Processes: Evidence for Nerve/Mast Cell Interactions

In the rat, there is considerable evidence of mast cell/nerve interaction both in the normal and infected intestine. Between 67 and 87% of all mast cells in the intestinal lamina propria of rats infected 22-35 days earlier with Nippostrongylus brasiliensis were touching nerves. These membrane contacts were between subepithelial mast cells and nonmyelinated nerves containing substance P, calcitonin gene-related peptide and neurone specific enolase. 2.5S nerve growth factor (NGF) has a significant enhancement effect on antigen-induced histamine release without addition of phosphatidylserine, and the in vivo administration of NGF to rats causes both connective tissue and mucosal mast cells to dramatically increase in number. All of these effects are both dose dependent and NGF specific, as evidenced by inhibition with anti-NGF. 2.5S NGF also causes in vitro increase of colonies in methylcellulose cultures of human peripheral blood. The effects of NGF in this system are synergistic with other T cell-derived growth factors and relatively specific for metachromatic cell growth. These observations support the conclusions that nerves and mast cells may constantly communicate and provide a structural and conceptual framework whereby the central nervous system may communicate with inflammatory events.

Regulation of human T lymphocyte mitogenesis by antibodies to CD3.

The inhibitory and mitogenic effects of anti-CD3 antibodies (anti-CD3) were examined in cultures of human peripheral blood T cells. Resting T cells required the presence of accessory cells (AC) or phorbol myristate acetate (PMA) to be stimulated by soluble anti-CD3 (OKT3 and 64.1). Anti-CD3 was unable to induce activation of AC-depleted T cells as determined by IL 2 receptor expression, IL 2 production, cell cycle analysis, or detectable DNA synthesis. Although T cell responses to PHA also required AC, far fewer were necessary to generate responses. Anti-CD3 inhibited PHA-stimulated T cell IL 2 production, IL 2 receptor expression and proliferation in partially AC-depleted cultures. Moreover, anti-CD3 was able to inhibit PHA responses when added to culture as late as 24 to 42 hr after the initiation of a 96-hr incubation. Increasing concentrations of PHA reduced the inhibitory effect of anti-CD3 on PHA-stimulated T cell proliferation, whereas IL 2 production remained suppressed. Anti-CD3 linked to Sepharose beads effectively inhibited PHA-stimulated T cell DNA synthesis, indicating that internalization of the CD3 molecule was not required for inhibition of PHA responses. Although inhibition of IL 2 production was a major effect of anti-CD3 in PHA-stimulated cultures, it was not the only apparent inhibitory effect because the addition of exogenous IL 2 could not prevent inhibition completely. Intact AC but not IL 1 also reduced anti-CD3-mediated inhibition of PHA responsiveness, whereas the addition of both IL 2 and AC largely prevented inhibition. Thus, anti-CD3 in the absence of adequate AC signals exerted a number of distinct inhibitory effects on mitogen-induced T cell activation. These results suggest that the CD3 molecular complex may play a role in regulating T cell responsiveness after engagement of the T cell receptor by a number of mechanisms, some of which involve inhibition of IL 2 production.

Alteration of antigenic expression of human myeloid precursor cells (CFU-GM) in long-term cultures

Comparison of myelopoiesis in continuous human bone marrow and cord blood cultures was performed. The presence of major histocompatibility complex (MHC) antigens on myeloid precursors (CFU-GM) from cord blood was confirmed using monoclonal antibodies (MoAb) defining class I (i.e., anti- β 2-microglobulin (B 2, MG 1) anti-HLA-A,B heavy chain (W6/32), and anti-HLA locus B(4E), and class II (HLA-DR (7,2)) antigens. The effect of long-term culture on the expression of these antigens was investigated. Our studies indicate that following prolonged in vitro culture there is a decrease in the expression of MHC antigens on CFU-GM. Our observations provide a rationale for considering the use of cord blood leukocytes subjected to long-term culture as an alternative to allogeneic hematopoietic cell transplantation.

Novel method for detecting micro-organisms in blood cultures.

A method for detecting the growth of micro-organisms in blood culture by a visual signal is described. The system utilises a single blood culture medium that has been specifically formulated to support growth of aerobic, anaerobic, and microaerophilic micro-organisms. The system is based on the principle that when micro-organisms grow in the medium in a sealed bottle their metabolic products create positive pressure. This positive pressure displaces the infected blood and broth into an upper chamber, which acts as a visual signal of microbial activity. All the test micro-organisms, when inoculated at less than 20 colony forming units into simulated human blood cultures, gave a positive signal.

Differences in the induction of SCEs between human whole blood cultures and purified lymphocyte cultures and the effect of an S9 mix

Studies for SCE induction are frequently performed on human blood cultures. Either whole blood cultures (WBC) or purified lymphocyte cultures (PLC) are employed. However, it has been shown that fundamental differences with respect to metabolic activity exist between these two systems. In order to further characterize the whole blood culture and the purified lymphocyte culture, differently acting substances were studied comparatively with and without an Aroclor-1254-induced S9 mix. Treatment with ethyl methanesulfonate (EMS), a direct mutagen, produced distinct SCE induction in both systems. Cyclophosphamide (CP) and benzo[ a]pyrene (BP), two indirect mutagens, also led to a significant increase of SCEs both in WBC and PLC without S9 mix. Only with CP was this effect more pronounced after addition of S9 mix. Sodium selenite (Na 2SeO 3), which induced SCEs in WBC, did not show this effect in the PLC. After S9 mix was added to purified lymphocytes, an increase of SCEs by sodium selenite was observed as in WBC. H 2O 2, a radical former, led to SCE induction in purified lymphocytes but not in the whole blood culture. By adding S9 mix, a distinct reduction of the SCEs induced by H 2O 2 was established. These results show that human lymphocytes can metabolize indirect mutagens and that it should be kept in mind when using S9 mix that, besides mixed-function oxygenases, it also contains enzymes which influence the SCE-inducing effects of substances.

Use of Directigen and acridine orange for rapid identification of human blood culture isolates.

Of 7,871 blood cultures from hospital patients, 22 yielded growth of Streptococcus pneumoniae or Haemophilus influenzae type b. The identities of 19 (86%) of these 22 strains could be verified after 18 to 24 h of incubation by application of the Directigen meningitis test kit to the unheated, uncentrifuged supernatant from the cultures; thus, the turnaround time for these cultures was halved. Growth in 16 (72%) of the Directigen-positive cultures was detected by visual inspection, and that in 3 (14%) of the cultures was detected by acridine orange staining. Growth in the three remaining bottles (14%) was detected by blind subculturing after 18 to 24 h or incubation and, therefore, was delayed by 24 h. The systematic application the acridine orange stain was helpful in 40 (44%) of 91 cases for which macroscopic inspection failed to reveal growth after 24 h of incubation.

Identification of Pure and Mixed Basophil Colonies in Culture of Human Peripheral Blood and Marrow Cells

We present a colony assay system that allows in situ identification of human basophil/mast cell (basophil) colonies. In methylcellulose culture, in the presence of phytohemagglutinin-leukocyte conditioned media (PHA-LCM), human peripheral blood and bone marrow cells form colonies that can be distinguished by their unique morphological characteristics. Pure basophil colonies are diffuse, small colonies containing small, round, highly refractile cells. These characteristics of the constituent cells led us to the observation that a significant number of basophils are found in combination with eosinophils. The mixed eosinophil/basophil colonies have the distinctive elements of pure eosinophil and pure basophil colonies. Usually, these are diffuse colonies with compact clusters of slightly larger, darker-appearing cells. We also found colonies that contained basophils and neutrophils/monocytes, but this type could not be consistently identified by in situ morphology. Cytochemical analysis confirmed the metachromatic nature of the granules in the basophils. The presence of IgE receptors on the cells was documented by indirect immunofluorescent staining after passive sensitization with purified human IgE. Peripheral blood cells from six healthy volunteers formed 5.7 +/- 1.0 (mean +/- SEM) pure colonies in 2 X 10(5) cells. Cultures of bone marrow cells from patients with various types of anemia had 9.0 +/- 1.5 colonies in 10(5) cells. This is the first description of a colony assay system for in situ identification of a pure population of basophilic granulocytes.

Identification of pure and mixed basophil colonies in culture of human peripheral blood and marrow cells

We present a colony assay system that allows in situ identification of human basophil/mast cell (basophil) colonies. In methylcellulose culture, in the presence of phytohemagglutinin-leukocyte conditioned media (PHA-LCM), human peripheral blood and bone marrow cells form colonies that can be distinguished by their unique morphological characteristics. Pure basophil colonies are diffuse, small colonies containing small, round, highly refractile cells. These characteristics of the constituent cells led us to the observation that a significant number of basophils are found in combination with eosinophils. The mixed eosinophil/basophil colonies have the distinctive elements of pure eosinophil and pure basophil colonies. Usually, these are diffuse colonies with compact clusters of slightly larger, darker-appearing cells. We also found colonies that contained basophils and neutrophils/monocytes, but this type could not be consistently identified by in situ morphology. Cytochemical analysis confirmed the metachromatic nature of the granules in the basophils. The presence of IgE receptors on the cells was documented by indirect immunofluorescent staining after passive sensitization with purified human IgE. Peripheral blood cells from six healthy volunteers formed 5.7 +/- 1.0 (mean +/- SEM) pure colonies in 2 X 10(5) cells. Cultures of bone marrow cells from patients with various types of anemia had 9.0 +/- 1.5 colonies in 10(5) cells. This is the first description of a colony assay system for in situ identification of a pure population of basophilic granulocytes.

Changes in normal polymorphonuclear leucocyte motility after ingestion of IgG aggregates.

Changes in normal polymorphonuclear leucocyte (PMN) motility after membrane-binding and internalisation of IgG aggregates (a model of soluble immune complexes) have been studied by the micropore filter assay. The results have confirmed that IgG aggregates stimulate as well as inhibit PMN chemotaxis. These effects are dependent on the size and concentration of the IgG aggregates in solution as well as the length of time of incubation. Stimulated chemotaxis was observed in a small subset of the whole PMN population which was apparent only when cell distribution through the filters was analysed. These results indicate the need for caution when drawing conclusions about PMN function from results obtained by these assay techniques.

Suppression of natural killer activity in human blood and bone marrow cultures by bone marrow-adherent OKM1-positive cells

Maintenance and regulation of natural killer (NK) cell activity in human bone marrow cultures were studied using K562 leukemia cells as targets. Culture of bone marrow cells in medium supporting long-term generation of myeloid cells resulted in a rapid loss of NK activity in 1–3 days. In contrast, antibody-dependent cytotoxicity to an NK-resistant tumor was maintained for more than 7 weeks. Horse serum, a component of the myelopoietic culture medium, was found to diminish NK cytotoxicity of blood and bone marrow cultures whereas hydrocortisone supplement did not. In addition, an adherent cell is present in bone marrow which greatly inhibits NK activity. Nonadherent bone marrow cells exhibited higher cytotoxicity than unfractionated cells at all days of culture; adherent cells were not cytotoxic to K562. Purified adherent marrow cells inhibited the cytotoxic capacity of nonadherent blood or marrow mononuclear cells during coculture. Indomethacin, an inhibitor of protaglandin synthesis, augmented levels of NK activity in cultures of bone marrow cells, indicating that macrophages may be suppressing this effector function via prostaglandins. Further identification of the adherent suppressor cells came from experiments in which suppression was prevented by treatment of the adherent cells with monoclonal OKM1 antibody plus complement. This study shows that bone marrow-adherent OKM1-positive cells, presumably macrophages, negatively regulate NK activity, and it defines conditions for analysis of the generation and/or positive regulation of NK cells in human bone marrow.

Maleic hydrazide induces sister-chromatid exchanges in mammalian cells in vitro

Maleic hydrazide (MH) in high concentrations (from 8 × 10 −3 M upward) induces SCEs in human blood cultures if it is added either for the entire culture period (72 h) or for the last 24 h. Parallel to the induction of SCEs, there is a delay in the proliferation of the cultures, as can be seen by the distribution of the first, second and third mitoses following the addition of BrdUrd. MH leads to a pronounced induction of SCEs in V79 cells (quadrupeling the control value) if added for 24 h simultaneously with BrdUrd. Treating the cells for 1 or 3 h prior to the addition of BrdUrd has no or little effect on the frequencies of SCEs.

Clastogenic interactions of γ radiation and caffeine in human peripheral blood cultures

In order to determine whether the micronucleus test could be used as a rapid assay for mutagenic interactions, we studied the effect of 50–800 R of γ radiation in combination with 10 −6–10 −3 M caffeine in cultured human lymphocytes, with two treatment protocols. In one protocol (T 0), whole blood was irradiated with 50–800 R of γ radiation, then stimulated with PHA and cultured for 72, 96 or 120 h in the presence or absence of caffeine. Under these conditions, γ radiation produced micronuclei in proportion to dose but post-treatment with 1 mM caffeine significantly decreased the number of micronuclei observed. The effect of caffeine was greater with the higher radiation doses and at earlier fixation times. Caffeine also decreased the mitotic index which, in turn, decreased the number of micronuclei observed; but caffeine post-treatment still had a significant effect even after mitotic activity was taken into account. In a second protocol (T 48), PHA-stimulated (actively cycling) cultures were irradiated 48 h after innoculation, then treated with caffeine, and fixed at 72 h post-innoculation (PI). With this protocol γ radiation produced more micronuclei than at T 0; this suggests that many of the cells damaged at T 0 are either lost or repaired. At T 48 1 mM caffeine significantly increased the number of micronuclei observed after γ radiation at all doses except 50 and 200 R. The mitotic index increased after 400–600 R, but only in the absence of caffeine. These results indicate that caffeine can have paradoxical effects on micronucleus frequency, apparently by potentiation of both the lethal (at T 0) and clastogenic (at T 48) effects of γ radiation. Whether the mechanisms of action at T 0 and T 48 are the same awaits further research. We have thus shown that in this system the micronucleus test is a valuable assay for mutagenic interactions and may also indicate repair and cell cycle sensitivity.

Cytogenetic effects of alachlor and mancozeb

The cytogenetic effects of 2 pesticides, alachlor and mancozeb, were determined in human lymphocytes in vitro and in rat bone-marrow cells in vivo. A dose-dependent increase of chromosomal aberration frequency was found, at concentrations of 1, 2, 4, 10, 20 and 40 μg/ml, for alachlor and mancozeb when administered during the last 24 h in 72-h human blood cultures. No increase in aberration yield as compared with that of controls was observed at the lowest concentrations of 1 and 2 μg/ml. A high level of heavily damaged cells, with despiralated, shattered and concomitantly separated chromatids was found at the highest concentration of 40 μg alachlor and mancozeb per ml. Doses of 1.25, 2.50 and 5 μg alachlor/g b.w. and of 2.5, 5 and 10 μg mancozeb/g b.w. were given to Wistar rats as a single injection for each dose. Bone-marrow cells for cytogenetic observations were gathered 24 h afterwards. All or almost all rats died within 2–4 h after the highest doses. Dose-related clastogenic effects were found for the 1.25 and 2.50 μg/g b.w. doses of alachlor and the 2.5 and 5 μg/g b.w. doses of mancozeb. Food containing alachlor and mancozeb, 200 ppm, was supplied to Wistar rats during 280 days at a daily intake of about 1.7 μg/g b.w. Chronic administration of mancozeb, but not alachlor, produced a significant level of chromosome damage.

Investigations into the production of acetate from ethanol by human blood and bone marrow cells in vitro.

The metabolism of ethanol to acetate and CO2 by suspension cultures of human blood and marrow cells has been investigated. The average rate of metabolism of ethanol by relatively pure preparations of monocytes plus lymphocytes, neutrophils and erythrocytes obtained from normal peripheral blood were, respectively, 12.58, 5.44 and 0.32 nmol/10(7) cells/h, when the concentration of ethanol in the culture was 2.63 mM. Under similar culture conditions, the average rate of metabolism of ethanol by suspension cultures of human marrow cells was 19.0 nmol/10(7) nucleated marrow cells/h. The metabolism of ethanol by nucleated marrow cells was only slightly inhibited by pyrazole and 3-amino-1,2,4-triazole indicating that it was largely independent of pyrazole-sensitive alcohol dehydrogenase and catalase. By contrast, the oxidation of ethanol by isolated rat hepatocytes was markedly inhibited by pyrazole and therefore appeared to be mainly dependent on alcohol dehydrogenase. It is concluded that bone marrow cells have a considerable capacity to metabolize ethanol and that the predominant biochemical pathway involved in this metabolism is different from that involved in rat hepatocytes.

Tumor promoter phorbol 12-myristate 13-acetate induces a clastogenic factor in human lymphocytes.

The mechanism of the clastogenic action--i.e., the ability to induce chromosomal aberrations--of the tumor promoter phorbol 12-myristate 13-acetate (PMA) was investigated. PMA at 10 and 100 ng/ml induced the formation of a low molecular weight (less than 10,000) clastogenic factor (CF) in phytohemagglutinin-stimulated human blood and lymphocyte cultures. Bovine erythrocyte Cu-Zn superoxide dismutase strongly inhibited PMA clastogenicity, both the formation of CF and the action of previously formed CF. The nonsteroidal anti-inflammatory agents indomethacin, imidazol, and 5,8,11,14-icosatetraynoic acid inhibited PMA clastogenicity and the clastogenic activity of previously formed CF. These results suggest that superoxide radicals and stimulation of the arachidonic acid cascade play a role in PMA-induced clastogenicity and the mechanism of action of the CF. The CF may relate the initial interaction of PMA with the cell membrane to the genome.

Clonai Origin of Human Erythro-Eosinophilic Colonies in Culture

We have observed the presence of erythropoietic bursts containing eosinophils and their precursors in methylcellulose culture of human peripheral blood and marrow nucleated cells in the presence of erythropoietin and medium conditioned by phytohemagglutinin-stimulated leukocytes (PHA-LCM). It was possible to identify these bursts (colonies) in situ in methylcellulose culture on the basis of their unique red and black colors. Transmission electron microscopy revealed that the constituent erythroid and eosinophilic cells lay intermixed with each other, and through close intercellular connections formed compact colonies and bursts consisting of several sub-colonies. Differential counts of individual erythro-eosinophil colonies (EEo colonies) revealed only a small percentage of blast cells in most of the colonies. Replating experiments of single EEo colonies yielded only eosinophilic colonies and clusters and erythroid colonies. The clonal nature of the EEo colonies was documented by analysis of Y-chromatin-positive cells in individual EEo colonies derived from cocultures of male and female peripheral blood mononuclear cells. Comparison of conditioned media indicated that PHA-LCM is the best stimulator for EEo colonies. These studies suggest that the differentiation capabilities of the progenitors for EEo colonies are restricted to erythroid and eosinophilic differentiation.