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PubMed1976

Significance of PHA induced clonogenic cells in chronic myeloid leukemia and early acute myeloid leukemia.

A technique for cloning myeloblastic leukemic cells in semi-solid agar, after prior PHA stimulation, was studied in all phases of chronic myeloid leukemia. Phytohemagglutinin responsive cells were found in blast crisis and accelerated phase. In a small number of patients in the benign phase of their disease, colony growth with PHA was detected. A group of patients with early acute leukemia was also examined by this technique. The incidence of PHA colonies appeared to correlate with the progression of the disease. The potential application of this assay for early detection of leukemic clones and possible prediction of the rapidity of progression of the leukemic process is discussed.

Bone Marrow↗
PubMed1982

Effect of arabinosyl cytosine on the level of DNA polymerase and thymidine kinase activity in PHA-stimulated human tonsillar lymphocytes.

The effect of arabinosyl cytosine (ara-C) was studied on the uptake, phosphorylation and incorporation of 3H-thymidine in human tonsillar lymphocyte cultures is described along with its effect on the level of DNA polymerase and thymidine Kinase activities induced by phytohaemagglutinin (PHA). Freshly isolated tonsillar lymphocytes are stimulated cells with a remarkably high activity of DNA polymerase alpha and thymidine kinase. During in vitro culture, these stimulated cells are transformed to the resting state with low DNA polymerase and thymidine kinase activity. However, a new DNA synthesising cycle can be induced by PHA with maximum at 48 h. 10(-6) M ara-C inhibited the incorporation of 3H-thymidine by 90-95%. This inhibition may be reversed by rinsing the cells. The inhibition of the transport of 3H-thymidine seems to be only a consequence of the inhibitory effect of ara-C on the DNA polymerisation reaction, because at 10 degrees C, where DNA synthesis was arrested, ara-C does not influence the uptake and the phosphorylation of 3H-thymidine. Ara-C (10(-6) M) abolished also the PHA induced elevation of DNA polymerase alpha and thymidine kinase activities without influencing protein synthesis of the cell. This supports a coordinated regulation mechanism between DNA synthesis and the synthesis of enzymes involved in DNA replication.

Cells, Cultured↗
PubMed1991

Ultrastructure of giant and small thalamic terminals of cortical origin: a study of the projections from the barrel cortex in mice using Phaseolus vulgaris leuco-agglutinin (PHA-L).

By means of tracing with the lectin Phaseolus-vulgaris leucoagglutinin (PHA-L), we examined in the thalamus of the mouse, the axon terminals of fibers originating in the barrel cortex. Vibratome sections of the brain were subjected to PHA-L immunocytochemistry and processed for light and electron microscopy. We observed small (0.5-0.8 microns in diameter) varicosities of labeled fibers in the nucleus ventrobasalis (VB) and the nucleus posterior (PO) as well as labeled giant terminals (3-5 microns in diameter) in PO. The analysis involved examination of serial sections and computer-aided reconstruction of several terminals. The small varicosities in VB appear to be small axon terminals forming distinct asymmetric synapses with small dendritic profiles. Some labeled terminals are apposed to, but not synaptically related with, the cell bodies of neurons in VB that are retrogradely labeled with PHA-L. The small varicosities seen with the light microscope in PO are terminals forming asymmetric synapses with dendritic shafts. The giant terminals in PO appear as large, vesicle-filled profiles forming part of synaptic glomeruli, i.e. complexes of one corticothalamic terminal engulfing several excrescences of a single dendrite. A giant terminal forms several asymmetric synapses (about 8) with these excrescences, as well as numerous (up to 15) puncta adhaerentia. The glomeruli are enveloped in glial lamellae, and they are often found at the bifurcations of primary dendritic segments. We suggest that the small terminals in VB are in the service of feedback signalling from the barrel cortex to its principal thalamic relay nucleus; the functional importance of this projection may reside in increased spatio-temporal discrimination. We interpret the giant terminals in PO as elements serving feed-forward processing, allowing the barrel cortex to influence, via PO, parts of the motor pathway modulating the animal's ongoing behavior.

Animals↗
PubMed1980

Altered sensitivity to cholchicine and PHA in human cultured cells.

PHA-stimulated lymphocytes cultivated from a pair of human monozygotic twins yielded mostly tetraploid cells when colchicine was not used to arrest the metaphases. The rate of tetraploidy was also enhanced by colchicine in fibroblasts cultured without PHA. In in situ condition, larger than usual cells were observed. Other defects found in parental lymphocyte cultures included C-anaphase cells and increased cell aggregation. These results suggest a membrane mutation resulting in hypersensitivity to PHA and variant response to colchicine.

Cell Aggregation↗
PubMed1977

Comparison of RNA and DNA synthesis, spontaneous and PHa induced, between blood lymphoid cells of newborn infants, older infants, and adults. A study of scintillation counting and by autoradiography.

Spontaneous and PHA induced RNA and DNA synthesis was measured qualitatively by autoradiography and quantitatively by scintillation counting in blood lymphoic cells of newborn infants, older infants, and adults. Spontaneous RNA synthesis was found in transitional cells, large phagocytic lymphoid cells and lymphocytes. Transitional cells and phagocytic lymphoid cells also synthesized DNA spontaneously. Quantitatively, spontaneous RNA synthesis was active in both newborn infants and in adults, but significantly less so in older infants. PHA stimulation for 18 h increased RNA synthesis significantly in blood lymphoid cells of newborn infants and adults, and to a much lesser but still significant degree in older infants. Spontaneous DNA synthesis was significantly greater in newborn infants than in older infants and adults. PHA stimulation for 18 h had no effect on thymidine incorporation in any of the groups studied.

Adult↗
PubMed1978

[PHA- and PWM-stimulation in acute leukaemia (author's transl)].

Peripheral blood cells from patients with acute leukaemia were stimulated in RPMI-medium with Phytohaemagglutinin (PHA) and/or Pokeweed-Mitogen (PWM). Samples of the cultures were taken at 3, 5 and 7 day intervals for cytological characterization. From 119 cultures from 65 patients 76 cultures from 47 patients could be analysed. Cultures stimulated with PHA and/or PWM showed in 43,4% a higher stimulation rate in comparison with cultures from normal persons. These findings are discussed in relation to the increased agar-colony growth growth of leukaemic cells after in vitro PHA-stimulation and transformation of blast cells.

Humans↗
PubMed2006

PHA synthase engineering toward superbiocatalysts for custom-made biopolymers.

Poly-3-hydroxyalkanoates [P(3HA)s] are biologically produced polyesters that have attracted much attention as biodegradable polymers that can be produced from biorenewable resources. These polymers have many attractive properties for use as bulk commodity plastics, fishing lines, and medical uses that are dependent on the repeating unit structures. Despite the readily apparent benefits of using P(3HA)s as replacements for petrochemical-derived plastics, the use and distribution of P(3HA)s have been limited by their cost of production. This problem is currently being addressed by the engineering of enzymes involved in the production of P(3HA)s. Polyhydroxyalkanoate (PHA) synthase (PhaC) enzymes, which catalyze the polymerization of 3-hydroxyacyl-CoA monomers to P(3HA)s, were subjected to various forms of protein engineering to improve the enzyme activity or substrate specificity. This review covers the recent history of PHA synthase engineering and also summarizes studies that have utilized engineered PHA synthases.

Acyltransferases↗
PubMed2006

The major population of PHA-stimulated PBMC infected by R5 or X4 HIV variants after a single cycle of infection is predominantly composed of CD45RO+CD4+ T lymphocytes.

PHA-stimulated peripheral blood mononuclear cells (PBMCs) are widely used for investigating replication and neutralization of HIV primary isolates in vitro. The objective of this study was to identify the T lymphocyte subset(s) that are found infected after one replication cycle by either R5- or X4-HIV-1 variants in PHA-stimulated PBMCs from healthy donors. Infected T lymphocytes were detected by intracellular p24 staining and characterized by cell surface immunophenotyping using flow cytometry. The predominant lymphocyte subset expressing p24 after 24 h of infection with either R5 or X4 HIV-1 strains was found to exhibit mainly the memory CD45RO phenotype, a greater percentage of CD62L(+)CD45RO(+) central memory T lymphocytes was infected with X4 HIV strains. Although some CD45RA(+) lymphocytes were also infected, these cells co-expressed CD45RO(+). The proportion of lymphocytes expressing CD4 and CD4/CD45RO decreased by 20% after 24 h of infection. A 2-fold decrease of CD4(+)CD8(+) T lymphocytes could also be recorded, even though this subset accounted for less than 5% of total lymphocytes in control cultures. Moreover, CD4(+)CD8(+) T cells further decreased by 90% after 4 days of infection, a time at which they scored p24(+). Therefore, our results indicate that the in vitro infection system of PHA-stimulated PBMC utilized in neutralization assays provides an appropriate model for the study of infected CD45RO(+) lymphocytes but not CD45RA(+) lymphocytes.

CD4-Positive T-Lymphocytes↗
PubMed1986

Phytohemagglutinin (PHA) activated human T-lymphocytes: concomitant appearance of insulin binding, degradation and insulin-mediated activation of pyruvate dehydrogenase (PDH).

Binding and degradation of A14125I-Insulin as well as the effect of insulin on pyruvate dehydrogenase (PDH) activation were studied in non-stimulated and phytohemagglutinin (PHA)-stimulated thymic-derived lymphocytes (T-lymphocytes) of man under varying conditions of time, temperature, and cell concentration. The nonstimulated viable T-lymphocytes exhibited neither binding, degradation, nor PDH activation in response to insulin. With PHA stimulation, a time and temperature-dependent binding was noted in T-lymphocytes which paralleled the appearance of cell-associated insulin degrading activity. Concomitant with the emergence of insulin binding and degrading activities in these cells, PDH activation was observed which was responsive to as little as 5.0 microU/ml of insulin. We conclude that in PHA-activated T-lymphocytes of man the process of insulin binding and degradation is closely related to insulin sensitive activation of PDH. These activated cells may serve as a useful model in which to study insulin binding and processing, as well as effects of insulin on postreceptor events.

Enzyme Activation↗
PubMed1988

The efferent projections of the rat lateral habenular nucleus revealed by the PHA-L anterograde tracing method.

The efferent connections of the rat lateral habenular nucleus (LHb) were demonstrated using anterograde transport of the lectin Phaseolus vulgaris leucoagglutinin (PHA-L). Following PHA-L injections into the LHb, neuronal somata located in the lateral two thirds of the LHb were labeled with PHA-L. Individual axonal fibers and terminal specializations were clearly visible. This permitted detailed mapping of both efferent fiber pathways and terminal distributions. Previous reports on fiber pathways were substantially confirmed and several new findings were revealed. (1) Major rostrally oriented fibers enter the medial forebrain bundle via 3 routes which initially branch from the fasciculus retroflexus: the mediodorsal thalamic nucleus and ventromedial thalamic nucleus; the zona incerta and fields of Forel; and the ventral tegmental area of Tsai. (2) A major decussation to the contralateral thalamic nuclei occurs in the central medial thalamic nucleus. (3) Caudally directed fibers follow two courses: one to the deep mesencephalic nucleus, central grey, dorsal raphe nucleus and the deep layers of the superior colliculus; and the other to the median raphe nucleus, oral pontine reticular formation and raphe pontis nucleus. The present results offer more detailed information concerning the dorsal diencephalic system.

Animals↗
PubMed1984

Heterogeneous accessory cell requirement for human peripheral blood T lymphocyte activation by PHA into IL-2-responsive colony-forming cells.

Mitogen-driven T cell proliferation in liquid culture requires accessory cells that cooperate in interleukin 2 production. We have investigated the accessory cell requirement for human lymphocyte colony formation under PHA stimulation. Semisolid medium limits cell-to-cell contact emphasizing the role of cooperating cells both in growth factor production and in triggering events. Culturing at high T cell density demonstrates that accessory cells can be substituted for colony formation by exogenous IL-2. Culturing at low T cell density in the presence of IL-2 also demonstrates that accessory cells are required for activation of a subset of progenitors into IL-2 responsive colony-forming cells. Consequently, T colony progenitors, contained in the E-rosetting cell fraction of peripheral blood, are heterogeneous in their triggering signals: a minor subset is directly inducible by PHA, and a major subset is inducible by PHA in the presence of accessory cells. We found that monocytes and some leukemic B cells support effective accessory function in both colony growth factor production and colony progenitor sensitization.

B-Lymphocytes↗
PubMed1986

T cells from patients with chronic liver diseases: abnormalities in PHA-induced expression of HLA class II antigens and in autologous mixed-lymphocyte reactions.

The expression of HLA Class II antigens by resting and phytohemagglutinin (PHA)-activated T cells and their functional properties in autologous mixed-lymphocyte reactions (MLR) were investigated in patients with chronic active hepatitis, with alcoholic cirrhosis, and with primary biliary cirrhosis. In all groups of patients the percentage of resting T cells expressing HLA Class II antigens was significantly higher than that in controls. The percentage of T cells which acquired HLA Class II antigens following PHA stimulation was reduced in patients with chronic active hepatitis, serum hepatitis B surface antigen (HBsAg) positive, and in those with alcoholic cirrhosis, HBsAg negative, although the level of [3H] thymidine incorporation was within normal limits. The degree of proliferation in autologous MLR with PHA-T cells was significantly reduced in patients with chronic active hepatitis, HBsAb positive, and in those with alcoholic cirrhosis, HBsAg positive. A reduced proliferation was also detected in autologous MLR with non-T cells, in patients with chronic active hepatitis, HBsAg positive. The abnormalities of autologous MLR are selective, since the proliferative and stimulatory activities of cells from patients with chronic liver diseases in allogeneic MLR were within normal ranges. The immunoregulatory role of HLA Class II antigens and of autologous MLR suggests that the abnormalities we have identified may play a role in the immunological dysfunctions underlying chronic liver diseases.

Adult↗
PubMed1989

PHA activation of encephalitogenic T cells: in vitro line selection overcomes splenic suppression.

In this study we compared myelin basic protein (MBP) and phytohemagglutinin (PHA) for their ability to induce proliferation and experimental autoimmune encephalomyelitis (EAE) transfer activity in mixed cell cultures obtained from spleen and lymph nodes versus highly selected MBP-specific T cell lines and clones. Established MBP-specific cells derived initially from immune lymph nodes attained both proliferative and EAE-transfer activities after in vitro activation with either MBP or PHA. In contrast, PHA was unable to induce immune spleen cells to transfer EAE, in spite of its potent mitogenic activity. On the basis of these results, we evaluated the in vitro proliferation and differentiation responses of MBP-specific T cells during the line selection process using cells derived from both immune lymph node and immune spleen. During the initial selection process with MBP, proliferation of MBP-specific T cell precursors from immunized spleen populations was reduced relative to lymph node cells. After antigen-dependent selection the encephalitogenic cells from either organ exhibited identical in vitro response characteristics. Freshly isolated immune spleen cells were potent suppressors of MBP-specific T cell proliferation suggesting that the in vitro differences between the two organs was due to splenic suppression of the encephalitogenic cells.

Animals↗
PubMed1990

Inhibition by anti-HLA class I mAb of IL-2 and IL-2 receptor synthesis in lymphocytes stimulated with PHA-P.

Inhibition by anti-HLA Class I monoclonal antibody (mAb) Q6/64 of phytohemagglutinin (PHA)-P-induced peripheral blood mononuclear cell (PMBC) proliferation is associated with a reduction of Tac expression and interleukin 2 (IL-2) secretion. To analyze the mechanism(s) underlying the latter phenomena, the Tac gene and IL-2 gene transcription was analyzed by a nuclear transcription assay. No synthesis of Tac and IL-2 mRNA was detected in PBMC stimulated with PHA-P in the presence of mAb Q6/64. In conjunction with our recently published data, these results indicate that the blocking by anti-HLA Class I mAb of PHA-P-induced PBMC proliferation reflects an inhibitory effect within the signal transduction pathway leading to transcriptional activation of IL-2 and IL-2 receptor genes.

Antibodies, Monoclonal↗
PubMed1982

Effects of adenosine and deoxyadenosine on PHA-stimulation of lymphocytes of man, horse and pig.

1. Adenosine inhibits thymidine and uridine incorporation of PHA-stimulated lymphocytes of man and horse at concentrations higher than 50 and 10 microM, respectively. Deoxyadenosine is inhibitory at concentrations higher than 100 microM. Thymidine and uridine incorporation of porcine lymphocytes are elevated 5-7-fold by 25-100 microM adenosine, deoxyadenosine, inosine and hypoxanthine. Leucine incorporation of PHA-stimulated lymphocytes was affected by adenosine and deoxyadenosine in the same way, but to a lower extent. 2. Effects of adenosine and deoxyadenosine were more pronounced at shorter cultivation times. 3. EHNA potentiated the effects of adenosine and deoxyadenosine on human and equine lymphocytes. With human lymphocytes inhibition by deoxyadenosine and EHNA was higher than by adenosine and EHNA. With porcine lymphocytes only the combination of deoxyadenosine and EHNA was inhibitory. 4. Homocysteine potentiated the inhibition of thymidine incorporation by the combination of adenosine and deoxyadenosine with equine lymphocytes, but not the inhibition of adenosine or deoxyadenosine alone. 5. Adenosine suppressed the PHA-stimulated elevation of PRPP concentrations. With porcine lymphocytes PRPP remained at the level of 0 hr, while with equine lymphocytes PRPP concentration decreased to below that level. 6. The various effects of adenosine and deoxyadenosine on lymphocytes of man, horse and pig can partially be related to differences in adenosine and deoxyadenosine metabolism.

Adenine↗
PubMed1976

Lymphocyte activation. VIII. The application of a whole blood test to the quantitative analysis of PHA responsive T cells.

In diluted blood predominantly T cells responded to purified PHA (pure leucoagglutinin Wellcome). Red cells and non-T mononuclear cells increased PHA responsiveness of T cells at concentrations present in the peripheral blood. Addition of polymorphonuclear leucocytes was a very slight further advantage. The incorporation of [3H] thymidine reflected the numbers of T cells in culture, although not perfectly linearly, since counts increased when more T cells were added to the cultures. It was therefore concluded that in cultures of diluted whole blood non-specific amplification mechanisms were provided and responses reflected the performance of stimulated T lymphocytes. Since the method is simple and the number of technical variables is relatively small, it is possible to establish a "normal" range of PHA responsiveness expressed in absolute counts per minute, and thus directly compare results from different laboratories.

Blood↗
PubMed1980

PHA selection electrophoresis--a screening method for monomeric IgM.

Pentameric IgM is precipitable by PHA whereas monomeric IgM is not. During electrophoresis in PHA-containing agar gel pentameric IgM is selected by precipitation from concomitant monomeric IgM which may be visualized separately as a precipitation line with specific antibody. This antibody-like use of the lectin in PHA selection electrophoresis provides a quick and simple method of screening macroglobulinemic sera and other biological fluids for the presence of naturally occurring monomeric IgM subunits.

Absorption↗
PubMed1981

Removal of PHA from supernatants containing T-cell growth factor.

Supernatants of human PBL cultures stimulated with PHA contain a lymphokine named T-cell growth factor (TCGF). To remove the contaminant PHA from these supernatants, we have designed a simple and effective method based on the binding properties of this lectin to chicken red blood cells. This procedure completely removes the functional activities of a PHA solution, both in lectin-induced cellular cytotoxicity against erythroid targets and in DNA synthesis of fresh PBL. The TCGF activity remaining in the supernatants after absorption with CRBC is dependent on the cellular concentration employed in the preparation of the supernatants. The absorption procedure does not remove significant TCGF activity in the supernatants prepared at high cellular concentrations (5 X 10(6) cells/ml), whereas in those prepared at low cellular concentrations (1 X 10(6) cells/ml) a partial loss of TCGF activity is detected.

Animals↗