PHA-stimulated Human Peripheral Blood Research Articles

Changes in Serum and Cell Homeostasis under Irradiation: Factors of Distant Action

Extracellular DNA-containing substances can be used as a marker of the balance between two processes: elimination of cells with critical number of lesions and repair of genetic material due to the work of adaptive systems. The idea of using this criterion was inspired by the results of measuring exDNA in blood plasma of chronically irradiated individuals. Here we used material obtained after gamma-irradiation (total dose 0.5 Gy) and subsequent culturing of PHA-stimulated human peripheral blood lymphocytes. The culture medium and exDNA isolated by various methods were treated with DNase and pronase. The concentration of free exDNA isolated by phenol extraction was an individual sign. The culture medium supernatant contained considerable amounts of exDNA in the form of nucleoprotein; cleavage of the protein component of this nucleoprotein considerably increased the size of fragments (from 1 to 20 kb) and reduced their resistance to DNase.

Immunosuppressive effects of Cucurbitacin B on human peripheral blood lymphocytes.

This study investigates the immunomodulating effects of cucurbitacin B, a Thai herbal plant; isolated from Trichosanthes cucumerima L., on PHA-stimulated human Peripheral Blood Mononuclear Cells (PBMCs). Stimulated PBMCs were cultured in the presence or absence of cucurbitacin B and then markers of cell activation were measured. The results showed that 0.5 and 0.25 µg/ml of cucurbitacin B significantly inhibited the expression of CD69+CD3+, and CD25+CD3+ whereas no significant inhibition from 0.05 µg/ml of cucurbitacin B was detected. The secretion of TNF-a from stimulated PBMC was significantly inhibited by cucurbitacin B. After 24 h of incubation, the total Cytotoxic Lymphocyte (CTL) activity was significantly reduced by 0.5 µg/ml of cucurbitacin B when compared to the control group (9.83 ± 0.95 and 40.63 ± 2.93, P = 0.0033) or compared with Phytohemagglutinin (PHA) stimulation (9.83 ± 0.95 and 70.09 ± 3.35, P < 0.0001). A similar pattern of CTL activity was found after 48 and 72 h of incubation. In conclusion, cucurbitacin B inhibited PBMC activation by PHA. This study provides evidences that cucurbitacin B could be used as immunosuppressive agent. Key words: Cucurbitacin B, Phytohemagglutinin stimulated peripheral blood mononuclear cell, cytotoxic lymphocyte function, CD69.

Effects of prenatal morphine exposure on behavioral responses to LPS in rats

Phytohemagglutinin (PHA) and concanavalin A (Con A)-induced blastogenesis of peripheral blood lymphocytes was examined in heat-stressed pre- and postpartal sheep. The peak responses of lymphocytes to PHA and Con A in heat-stressed sheep revealed significant reduction before and after parturition compared with those in the corresponding control animals kept under thermoneutral conditions. Furthermore, the effect of serum from control or heat-stressed sheep on PHA-induced lymphocyte blastogenesis was examined. Supplementation of serum from heat-stressed sheep significantly suppressed the blastogenesis of lymphocytes obtained from healthy sheep, bovine, and human donors. Unlike dexamethasone, heat-stressed sheep serum did not inhibit IL-2 production by PHA-stimulated human peripheral blood lymphocytes. These results indicate that the immunosuppression of heat-stressed sheep is in part mediated by serum factor(s) that can modulate T-cell function in a species nonspecific manner.

Highly concentrated urine-purified Tac peptide fails to inhibit IL-2-dependent cell proliferation in vitro

Tac peptide, i.e., the p55 chain of the human interleukin-2 receptor (IL-2R) complex, is detectable as a soluble form (sIL-2R) in normal sera and, at increased levels, in patients with different diseases. Since several immunological abnormalities are observed in most conditions associated with an increase in sIL-2R levels, a down-regulatory effect on IL-2-dependent functions has been postulated as a consequence of binding and functional block of IL-2 by the excess of sIL-2R. To test this hypothesis, we purified sIL-2R from the urine of a patient with hairy cell leukemia and investigated the possible inhibitory effect of this peptide on the in vitro IL-2-induced cell proliferation. The urine-purified molecule was detectable by the specific immunoassay utilized to measure the serum Tac peptide and was constituted by a single polypeptide of about 50 kDa which was able to bind IL-2. Experiments performed with the IL-2-dependent murine CTLL-2 cell line and with PHA-stimulated human peripheral blood mononuclear cells showed that the purified sIL-2R at concentrations up to about 300 n M was unable to block IL-2-dependent cell proliferation. According to these data, which can be explained by the low affinity for IL-2 of the p55 IL-2R chain, it seems unlikely that in vivo the soluble Tac peptide can exert a down regulatory effect on IL-2-induced phenomena through a functional block of IL-2.

EFFECTS OF DILTIAZEM UPON METABOLISM AND IMMUNOSUPPRESSIVE ACTION OF CYCLOSPORINE IN KIDNEY GRAFT RECIPIENTS

It is widely believed that calcium antagonists such as diltiazem exert immunosuppressive effects in kidney graft recipients--however, the mechanism is unclear. In a randomized controlled trial, kidney graft recipients who received diltiazem during transplantation and for an average of 12 months thereafter experienced significantly fewer rejection episodes than patients treated with cyclosporine and steroids alone. Furthermore, 1-year (97% vs. 85%) and 4-year (80% vs. 70%) graft survival rates were higher in diltiazem-treated patients, but the difference was not statistically significant. In vitro, diltiazem had little immunosuppressive activity. Concentrations of diltiazem which blocked the proliferation of PHA-stimulated human peripheral blood mononuclear cells, or prevented activation-associated accumulation of interleukin-2 mRNA, or p50- and p70-IL-2 receptor mRNA exceeded pharmacological concentrations by more than 100-fold. Both, CsA and high doses of diltiazem caused an increase of IL-6 mRNA. In contrast to these findings, the IL-6 plasma concentrations were comparable in both groups, whereas the serum concentration of soluble IL-2 receptors was decreased in patients treated with diltiazem. Administration of diltiazem caused an alteration of CsA metabolism. The whole-blood concentration of CsA metabolite 17 was significantly increased in diltiazem-treated patients, resulting in a five-times-higher concentration of this metabolite in the cellular blood compartment compared with the parent drug. Changes in metabolites 1, 8, and 18 levels were less pronounced. Although direct immunosuppressive properties of diltiazem are unlikely, diltiazem could support immunosuppression by altering CsA metabolism, and promoting accumulation of certain metabolites.

Effects of asbestos on the cell cycle of PHA-stimulated human peripheral blood lymphocytes.

It is well known that persons exposed to asbestos display systemic immunological alterations: impaired lymphocyte response to PHA, the appearance of autoantibodies and elevation of the serum immunoglobulin level. The authors intend in this report to determine whether asbestos fibres (crocidolite, chrysotile and amosite) have any effect on the cell cycle of human lymphocytes after PHA stimulation. Peripheral blood mononuclear (PBM) cells were incubated with 10 micrograms/ml PHA for 2 days. After PHA stimulation, a decrease in the percentage of cells in the G0 phase and an increase in that of cells in the G1A, G1B and S phases was observed. When asbestos fibres or titanium dioxide (TiO2) was added to the culture dish at the beginning of the experiment, the progression of the cell cycle was inhibited only by asbestos fibres, in which case the percentage of cells in the G0 phase was significantly increased, while those of cells in the G1B and S phases were significantly decreased. The experiment for determining the critical period for inhibition of blastogenic response revealed that no inhibition was demonstrable when crocidolite fibre was added at 24 hr after PHA stimulation. Although the mechanisms of asbestos-fibre-mediated suppression remain to be clarified, these results showed that asbestos fibres act at an early stage (G0 phase) of the cell cycle and suppress the PHA stimulation of PBM cells.

Suppressed peripheral blood lymphocyte blastogenesis in pre- and postpartal sheep by chronic heat-stress, and suppressive property of heat-stressed sheep serum on lymphocytes

Phytohemagglutinin (PHA) and concanavalin A (Con A)-induced blastogenesis of peripheral blood lymphocytes was examined in heat-stressed pre- and postpartal sheep. The peak responses of lymphocytes to PHA and Con A in heat-stressed sheep revealed significant reduction before and after parturition compared with those in the corresponding control animals kept under thermoneutral conditions. Furthermore, the effect of serum from control or heat-stressed sheep on PHA-induced lymphocyte blastogenesis was examined. Supplementation of serum from heat-stressed sheep significantly suppressed the blastogenesis of lymphocytes obtained from healthy sheep, bovine, and human donors. Unlike dexamethasone, heat-stressed sheep serum did not inhibit IL-2 production by PHA-stimulated human peripheral blood lymphocytes. These results indicate that the immunosuppression of heat-stressed sheep is in part mediated by serum factor(s) that can modulate T-cell function in a species nonspecific manner.

Human laminin a chain (LAMA) gene: Chromosomal mapping to locus 18p11.3

Laminin, an integral component of basement membranes, consists of three subunit polypeptides, A, B1, and B2 chains. We have recently isolated cDNAs corresponding to human laminin A chain. These cDNAs were utilized for chromosomal in situ hybridizations to establish the genomic location of the laminin A chain gene. Metaphase chromosomes of PHA-stimulated human peripheral blood leukocytes were examined by in situ hybridization with 3H-labeled cDNAs, and the chromosomes were identified by R-banding (fluorochrome-photolysis-Giemsa method). The results indicated that the human laminin A chain is at locus 18p11.3. Since human laminin B1 and B2 chain genes have been previously mapped to chromosomes 7 and 1, respectively, the results indicate that genes encoding human laminin chains reside in separate chromosomes.

Interferon-beta 2 (BSF-2) mRNA is expressed in human monocytes.

We previously have reported the presence of interferon-beta 2 (IFN-beta 2) mRNA in PHA-stimulated human peripheral blood leukocytes (PBL), as well as in nonstimulated cells, although at a lower level. The IFN-beta 2 cloned from a leukocyte library appeared to be similar to that of the fibroblast IFN-beta 2 gene first described in fibroblasts. To assess the nature of the cell population in which the synthesis of IFN-beta 2 takes place, PBL were fractionated in adherent and nonadherent cells. The antiviral activity of the culture supernatants of adherent cells was characterized as the IFN-beta type by neutralization with polyclonal antibodies raised against purified fibroblast IFN-beta 2. IFN-beta 2 mRNA was observed in enriched monocyte populations and accumulated very rapidly, peaking at 2.5 h. RNA extracted from these cultures encoded in a reticulocyte lysate a protein immunoprecipitated by the anti-IFN-beta 2 antiserum. In addition, IFN-beta 2 secreted in monocyte supernatants also was immunoprecipitated by the specific antiserum and was able to compete with the fibroblast IFN-beta 2, suggesting a strong similarity between the fibroblast and monocyte proteins.

Spurious Detection of Terminal Deoxynucleotidyl Transferase in Phytohemagglutinin-Stimulated Lymphocytes

Expression of terminal transferase (TdT) is believed to be restricted to primitive lymphoid cells; recently, however, indirect immunofluorescent (IF) assays have been used to demonstrate the apparent presence of TdT in phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes and in various nonlymphoid malignancies. Using an IF assay, we found that a heteroantiserum to TdT reacted with cultured and PHA-stimulated human peripheral blood mononuclear cells, but we were unable to confirm the presence of TdT in these cells using immunoblotting and biochemical assays. We conclude that the IF results are spurious and most likely represent recognition by the heteroantiserum of inducible protein(s) other than TdT.

Spurious detection of terminal deoxynucleotidyl transferase in phytohemagglutinin-stimulated lymphocytes

Expression of terminal transferase (TdT) is believed to be restricted to primitive lymphoid cells; recently, however, indirect immunofluorescent (IF) assays have been used to demonstrate the apparent presence of TdT in phytohemagglutinin (PHA)-stimulated peripheral blood lymphocytes and in various nonlymphoid malignancies. Using an IF assay, we found that a heteroantiserum to TdT reacted with cultured and PHA-stimulated human peripheral blood mononuclear cells, but we were unable to confirm the presence of TdT in these cells using immunoblotting and biochemical assays. We conclude that the IF results are spurious and most likely represent recognition by the heteroantiserum of inducible protein(s) other than TdT.

Dose relationships between different effects of aphidicolin in JU56 cells

Some effects of aphidicolin have been investigated in relationship to dose, in a permanent cell line, JU56. Inhibition of semi-conservative DNA synthesis occurred at concentrations greater than 3 × 10 −7 M. In this respect the cells were about as sensitive as L1210 and HeLa cells, and more than 10-fold more sensitive than PHA-stimulated human peripheral blood leucocytes. Delay of progress of cells through G 2 occurred at concentrations which inhibited synthesis to about 2% of control levels. Chromatid aberrations appeared in cells at concentrations which decreased synthesis to 5%. Synergism with X-rays in the production of chromatid aberrations occurred at doses which reduced semi-conservative synthesis to 40% of control levels. Isochromatid aberrations appeared in cells continuously exposed to aphidicolin in G 2 at concentrations which reduced synthesis to 5% of control units.

Accessory cell requirement for activation antigen expression and cell cycle progression by human T lymphocytes.

Stringent accessory cell (AC) depletion by a three-step procedure--plastic adherence, nylon wool adherence, followed by simultaneous treatment with two anti-AC monoclonal antibodies + complement--has allowed the demonstration of several AC-dependent stages in the T cell activation pathway. Simultaneous analysis of DNA content and cell surface immunofluorescence (correlation of activation antigen expression with cell cycle position) or DNA and RNA content (cell cycle position) of cultured cells was accomplished by dual parameter flow cytometry. AC-depleted, PHA-stimulated human peripheral blood T lymphocytes (PBTL) failed to exhibit "early" indicators of activation, including increased RNA content, expression of three activation-associated cell surface proteins (IL 2 receptor, transferrin receptor, and 4F2 protein), and the production of IL 2. The AC-depleted PBTL that failed to express these "early" markers of activation also failed to progress into the "late" phase of activation, DNA synthesis. All indicators of PHA responsiveness were fully replenished upon addition of AC but were only reconstituted to intermediate levels by addition of excess quantities of either highly purified IL 1 or crude AC-conditioned medium with lymphocyte-activating factor activity. These data suggest that the AC membrane plays a key and as yet undefined role in the stimulation of T cells by PHA.

The role of cytoplasmic intermediates in IL 2-induced T cell growth.

In previous studies we showed that PHA-stimulated human peripheral blood lymphocytes contain an extractable cytoplasmic protein that is capable of inducing DNA synthesis in isolated quiescent nuclei. This factor, which we have called ADR (for activator of DNA replication), appears to represent an endogenous intracellular mitogen, or cytoplasmic transducer of extracellular mitogenic signals. In light of the known role of IL 2 in PHA-induced T cell mitogenesis, we continued these studies to investigate whether ADR may be involved in the mechanism of action of IL 2. Results from both dose-response and time-response studies strongly suggest a functional role for ADR in IL 2 mediated T cell growth. Furthermore, the absence of ADR in dexamethasone-treated, PHA-stimulated cells suggests that the induction of ADR is not an early event in T cell activation associated with the expression of IL 2 receptors; rather, the induction of ADR appears to be a later event resulting directly or indirectly from the binding of IL 2 to IL 2-responsive cells. In sum, our findings suggest a pathway for T cell growth in which extracellular exogenous mitogenic signals are transmitted to the nucleus first through an extracellular endogenous growth factor (IL 2) and finally through an intracellular, endogenous molecule (ADR) that acts at the level of the nucleus itself.

Early nuclear activation in PHA-stimulated human peripheral blood lymphocytes

Early nuclear activation in PHA-stimulated human peripheral blood lymphocytes

The Effect of Estrogen on the Incorporation of 3H-Thymidine by PHA-Stimulated Human Peripheral Blood Lymphocytes

Summary The incorporation of 3H-thymidine by PHA-stimulated human peripheral blood lymphocytes from healthy adult males was significantly (p 0.002) suppressed in the presence of diethylstilbestrol diphosphate. The observed suppression of blastogenesis in the presence of exogenous estrogen suggests that the palliative effects of estrogenic therapy in the clinical treatment of patients with hormonally-responsive tumors may be countered by its adverse effect of the host's cell-mediated immunologic responsiveness.

Effect of Lymphocyte Stimulants on Specific Antibody Synthesis in vitro

SINCE Nowell's original description of phytohaemagglutinin (PHA)-induced lymphocyte transformation1, there has been considerable controversy as to whether responses to this substance involve immunoglobulin and specific antibody synthesis. Several workers2–5, using a variety of techniques, claim to have demonstrated immunoglobulin synthesis by PHA-stimulated human peripheral blood lymphocyte cultures, but others have been unable to confirm these findings6–9. Recent studies with the pokeweed mitogen (PWM) suggest that responses to this stimulant differ morphologically from those to PHA (ref. 10) and that a sub-population of cells responding to PWM may synthesize immunoglobulin9,11.