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PubMed2006

Temporal regulation of foregut development by HTZ-1/H2A.Z and PHA-4/FoxA.

The histone variant H2A.Z is evolutionarily conserved and plays an essential role in mice, Drosophila, and Tetrahymena. The essential function of H2A.Z is unknown, with some studies suggesting a role in transcriptional repression and others in activation. Here we show that Caenorhabditis elegans HTZ-1/H2A.Z and the remodeling complex MYS-1/ESA1-SSL-1/SWR1 synergize with the FoxA transcription factor PHA-4 to coordinate temporal gene expression during foregut development. We observe dramatic genetic interactions between pha-4 and htz-1, mys-1, and ssl-1. A survey of transcription factors reveals that this interaction is specific, and thus pha-4 is acutely sensitive to reductions in these three proteins. Using a nuclear spot assay to visualize HTZ-1 in living embryos as organogenesis proceeds, we show that HTZ-1 is recruited to foregut promoters at the time of transcriptional onset, and this recruitment requires PHA-4. Loss of htz-1 by RNAi is lethal and leads to delayed expression of a subset of foregut genes. Thus, the effects of PHA-4 on temporal regulation can be explained in part by recruitment of HTZ-1 to target promoters. We suggest PHA-4 and HTZ-1 coordinate temporal gene expression by modulating the chromatin environment.

Adenosine Triphosphatases↗
PubMed1992

Effect of H37Ra on PHA-induced migratory inhibition of leucocytes in patient of tuberculosis.

Eighty nine cases of active pulmonary tuberculosis were classified into four stages depending on the clinical extent of disease. Leucocyte migration inhibition test (LMIT) was done using mitogen phytohaemagglutinin (PHA) and H37Ra specific antigen. Normal PHA responses were observed in all clinical stages while specific immune response in relation to inhibition of leucocyte migration decreased from stage I to stage IV. Effect of H37Ra on PHA induced release of lymphokine was studied by mixing the two and comparing the percentage migratory inhibition of the mixture with that of PHA alone. Increased or decreased values with mixture compared to those with PHA alone were regarded as enhancement or inhibitory effect respectively. Median percentage effect of H37Ra on PHA induced migratory inhibition was found to be inhibitory in first three stages and controls while enhancement effect was observed in stage IV. A total of 22 cases in all stages showed enhancement effect while 48 cases showed inhibitory effect. The possible mechanism of enhancement or inhibitory effect are discussed.

Antigens, Bacterial↗
PubMed1978

Dissociation of the mitogenic and rosette-forming effects of PHA to stimulators of cyclic AMP levels.

The mitogenic and the spontaneous E-rosette stimulating effects of phytohaemagglutinin (PHA) were studied by examining their responses to an elevation of intracellular cAMP induced by pretreatment with aminophylline, adrenaline or PGE1 or by adding exogenous cAMP. Elevation of the intracellular cAMP levels of lymphocytes was found inhibitory to the mitogenic effect of PHA, while leaving the increased early and stable spontaneous rosette formation unaffected. It also failed to depress the PHA-induced slight increase in the proportion of total spontaneous E-rosette-forming lymphocytes. The responses of the two PHA effects under study to the elevation of the cAMP level are thus dissociated, suggesting that the two effects represent two different manifestations of lymphocyte activation, though these manifestations may not be independent of each other. It has been confirmed that PHA produces a shift primarily within the T-cell population toward the active and the stable E-rosette-forming subpopulation, while the increase in the total E-rosette-forming lymphocyte population is but slight. This may possibly involve the activation of the T-derived "null" cells unable to bind sheep red blood cells without PHA treatment.

Adult↗
PubMed1977

Ther persistent PHA-responsive population in the mouse thymus. i. Characterization of the population.

Using an in vitro culture technique, mouse thymus graft cells were co-cultured with peripheral blood lymphocytes in the presence of phytohaemagglutinin (PHA). The persistent PHA-responsive thymus graft population (Elliott, 1973) was shown to be able to response to other T-cell mitogens (Con A, pokeweed mitogen, staphylococcal enterotoxin B), but not to E. coli lipopolysaccharide a known B-cell mitogen. The percentage of persistent PHA-responsive cells did not alter during 5 days in culture and was relatively unaffected by either hydrocortisone or anti-lymphocyte serum treatment in vitro. In allogeneic thymus grafts (AKR leads to CBA), persistent PHA-responsive cells could be demonstrated, which were destroyed when incubated with CBA anti theta AKR serum and complement. When thymus graft cells were allowed to sediment on a 0.2-2 per cent BSA gradient, it was seen that the PHA-responsive population sedimented faster than the bulk of thymus graft cells. Some separation could be obtained on this gradient between the persistent and non-persistent PHA-responsive cell populations.

Animals↗
PubMed1988

[Effect of the tumor promoter TPA on the distribution of PHA-stimulated human lymphocytes by cell cycle phases].

Patterns of the cell cycle distribution in human peripheral blood lymphocytes, stimulated by PHA alone and PHA plus 12-o-tetradecanoylphorbol-13-acetate (TPA), were studied using DNA cytometry in different times after PHA stimulation. In the first period (nearly 3 days after PHA stimulation) TPA induces no significant differences in the characters under consideration, but in the later period, when the proliferation of the cultures stimulated by PHA alone is reducing, in other cultures stimulated by PHA plus TPA the percentage of cells in S-phase does not reduce, whereas the percentage of cells in G2-phase is rising, which may suggest that this phase is blocked. Concurrently the tetraploid cells are appearing. Accumulation of cells in G2-phase can be overcome by the application of chlorpromazine, which is known to inhibit the membrane-associated protein kinase C.

Cell Cycle↗
PubMed1988

In myelodysplastic syndromes progression to leukemia is directly related to PHA dependency for colony formation and independent of in vitro maturation capacity.

In an agar-liquid double-layer colony assay in which myeloid leukemia colony-forming cells require the presence of both the lectin PHA and CSF for in vitro proliferation, colony formation of bone marrow cells derived from patients with a myelodysplastic syndrome (MDS) was studied. In five of 14 MDS and all five leukemic transformed MDS cases, colony formation was found to require both PHA and CSF. Three of these five PHA-dependent MDS cases progressed to overt leukemia within 1 year, one progressed from RA to RAEB, and one patient received AML chemotherapy. PHA-dependent colony formation was associated with higher bone marrow blast counts, but not directly to FAB type or cytogenetic abnormalities. In nine other MDS cases only CSF was required for colony formation. In these PHA-independent cases the course of the disease was stable during the observation time (5-17 months). Two types of colonies were observed in this in vitro system: colonies adherent and colonies nonadherent to the agar underlayer. The former consisted of terminally differentiated myeloid cells, and the latter comprised immature cells. This suggests that the percentage of adherent colonies formed in vitro may be used as a measure for the maturation defect in MDS. However, no correlation was found between the percentage of adherent colonies and progression to leukemia of the MDS cases. Our findings suggest that the dependency on PHA for in vitro colony formation of colony-forming cells in MDS is predictive for the progression to leukemia. However, the in vitro differentiation capacity has no apparent prognostic significance.

Adolescent↗
PubMed1977

I. Increase in bone marrow CFU-S and CFU-C after in vivo phytohaemagglutinin (PHA) administration in mice.

An absolute increase in bone marrow CFU-S was observed within 4 hours after intraperitoneal administration of 3 mg PHA-M or 250 microgram PHA-P. At that time, there was no increase in spleen CFU-S, while a pronounced decrease in the number of circulating CFU-S was noted. A two to fourfold increase in bone marrow CFU-C was found within 12 hours after intraperitoneal administration of a similar dose of PHA-M or PHA-P. Addition of PHA-M or PHA-P at small concentrations resulted in a marked inhibition of granulocytic colony formation whereas macrophage colony growth became depressed only at very high concentrations.

Animals↗
PubMed1978

The persistent PHA-responsive population in the mouse thymus. I. Characterization of the population.

Using an in vitro culture technique, mouse thymus graft cells were co-cultured with peripheral blood lymphocytes in the presence of phytohaemagglutinin (PHA). The persistent PHA-responsive thymus graft population (Elliott, 1973) was shown to be able to respond to other T-cell mitogens (Con A, pokeweed mitogen, staphylococcal enterotoxin B), but not to E. coli lipopolysaccharide a known B-cell mitogen. The percentage of persistent PHA-responsive cells did not alter during 5 days in culture and was relatively unaffected by either hydrocortisone or anti-lymphocyte serum treatment in vivo. In allogeneic thymus grafts (AKR leads to CBA), persistent PHA-responsive cells could be demonstrated, which were destroyed when incubated with CBA anti thetaAKR serum and complement. When thymus graft cells were allowed to sediment on a 0.2--2 per cent BSA gradient, it was seen that the PHA-responsive population sedimented faster than the bulk of thymus graft cells. Some separation could be obtained on this gradient between the persistent and non-persistent PHA-responsive cell populations.

Animals↗
PubMed1983

Induction of phenotypic differentiation, interleukin 2 production, and PHA responsiveness of "immature" human thymocytes by interleukin 1 and phorbol ester.

Small human thymocytes (ST) representing 70% of the thymocytes were isolated according to size by centrifugal elutriation. Although these ST contained approximately 30% PNA-cells, they failed to respond to lectins, indicating the existence of a PNA-ST subset that can be considered to belong to the "immature" thymocyte population. The ST were induced to proliferate if, in addition to PHA, IL 1-containing supernatants of highly purified monocyte cultures or 12-O-tetradecanoyl-phorbol-13-acetate (TPA) were present. The incubation of the ST for 90 hr with TPA or IL 1 in the absence of PHA resulted in a strong reduction in the percentage of cells reacting with the immature thymocyte markers TdT and PNA. In addition, the OKT6+ cells were partially reduced after incubation with IL 1. Concomitantly, an increase in the percentage of cells reacting with the mature T cell markers OKT1 and OKT3 was observed, whereas HLA antigens became strongly expressed on all ST. Although IL 1 or TPA were unable to induce proliferation of the ST, these substances induced IL 2 production by these cells. These shifts to cells with more "mature" phenotypes that are able to produce IL 2 were not observed if the ST were incubated with PHA or culture medium only. The responder capacity of the ST to PHA plus TPA was not significantly affected by the depletion of the more "mature" OKT3+ and OKT1+ cells. In addition, in this situation OKT1+, OKT3+, OKT6- cells were found to be generated from OKT1-, OKT3-, OKT6+ cells. Therefore, it could be excluded that the proliferative responses were due to a selective expansion of a preexisting mature T cell population. Our results indicate that TPA mimics IL 1 in the induction of differentiation of the ST to a stage in which subpopulations of these cells are able to produce IL 2 and to respond to PHA. Because only the proliferating ST were found to react with a monoclonal antibody, which is thought to be directed at the IL 2 receptor (anti-Tac), our data suggest that PHA is required for the induction of expression of receptors for IL 2 in those ST subpopulations that are able to proliferate in the presence of IL 2 generated in situ.

Antigens, Differentiation, T-Lymphocyte↗
PubMed1981

Early biochemical events associated with lymphocyte activation in ageing. I. Evidence that Ca2+ dependent processes induced by PHA are impaired.

The requirement for Ca2+, a divalent ion which plays a fundamental role in cell activation, has been analysed in cultures of PHA-stimulated human peripheral blood lymphocytes (PHA-PBL) from adult (range: 20-35 years) and old (over 70 years) subjects. For this purpose, increasing concentrations of Ca2+ chelators (EGTA and EDTA) were added to cultures in order to compare the effect of progressive extracellular Ca2+ (Ca2+EC) depletion on [3H]-Tdr incorporation by PHA-PBL. Kinetic analysis showed that Ca2+EC requirement was restricted to the first 24 h after culture initiation. At optimal doses of PHA, the PHA-PBL from old subjects were more sensitive than those from adult subjects to increasing concentrations of both chelators. They also required larger amounts of Ca2+ supplements to restore their normal response after total inhibition by EGTA. Furthermore, the PHA-PBL from the elderly were hypersensitive to verapamil (Isoptin), a drug which instigates a reversible inhibition of Ca2+-dependent processes associated with lymphocyte transformation, by a quite similar reaction to that induced by chelators. We conclude that the Ca2+-dependent processes in lymphocyte activation are impaired with ageing. Following further experiments and recent work suggesting that lymphocytes need more than one signal to proliferate, the authors speculate on a deficiency of a late activation signal requiring cell-cell interactions in the elderly.

Adult↗
PubMed1982

Acute myeloid leukemia colony growth in vitro: differences of colony-forming cells in PHA-supplemented and standard leukocyte feeder cultures.

Bone marrow or blood of patients with acute myeloid leukemia was subjected to cell separation and the cells investigated for in vitro colony growth. Discontinuous albumin density gradient centrifugation and depletion of E-rosette-forming cells resulted in purified fractions of acute myeloid leukemia cells. From these fractions, growth of large leukemic colonies was obtained in the PHA-leukocyte feeder (PHA-LF) colony technique in 12 of 14 patients. The standard double agar layer techniques with a leukocyte feeder for granulocyte-macrophage colony forming cells (GM-CFC) supported colony formation in only four cases. The PHA-LF leukemic colony-forming cells (CFC) were found to be of low buoyant density (always less than or equal to 1.062 g.ml-1) when compared to normal marrow GM-CFC (peak at 1.065 g.ml-1). The density profile of PHA-LF CFC paralleled the distribution of the nucleated cells in 8 cases, but in 4 patients, the cFC peak was found at a distinctly lower density; this suggested that a specific leukemic subpopulation had a colony-forming capacity. In three of the four patients with colony growth in the double layer agar technique, it was evident that these CFC had density properties different from those of PHA-LF CFC. These findings suggest that cells giving rise to large colonies in the PHA-LF and double layer agar assays represent distinct leukemic subpopulations.

Adult↗
PubMed1981

T cell growth factor-enhanced PHA response of human thymus cells: requirement for T3+ cells.

Human thymocytes normally proliferate poorly in PHA-stimulated cultures, as measured by thymidine uptake. Their response increases when supernatant factors from mitogen-stimulated human lymphocyte cultures (SUP) are added. The cells that proliferate in PHA + SUP-stimulated cultures continue to divide if they are resuspended with fresh SUP every 3 to 4 days. PHA-stimulated thymocytes are deficient in the production of the T cell growth factor interleukin 2 (IL 2). The phenotype of thymocytes prior to stimulation was 90% T6+, 80% T4+, 83% T8+, and 12% T3+, whereas that of the cells after 3 days in culture with PHA + SUP was 1% T6+, 18% T4+, 66% T8+, and 80% T3+. Lysis of T3+ cells from the thymocyte preparation before culture greatly reduced the proliferative response to PHA + SUP. This suggests that the PHA-induced proliferation of more mature thymus cells is restricted by a lack of essential growth factors (either IL 1 or IL 2) and that the co-stimulating activity of SUP on human cells is predominantly on thymus cells that already are T3+.

Antibodies, Monoclonal↗
PubMed1975

Humoral and cell-mediated immune response in patients with malignant melanoma. I. In vitro lymphocyte reactivity to PHA and antigens following immunization.

In vitro lymphocyte function was evaluated in 61 patients with different clinical stages of malignant melanoma. Thirty-one of these patients had localized disease, 13 regional metastases, 10 distant lymph node or skin metastases, and 7 visceral metastases. Following immunization, in vitro lymphocyte reactivity to three antigens (diphtheria toxoid, tetanus toxoid and alpha-hemocyanin of Helix pomatia) was studied in the presence of autologous serum, in addition to lymphocyte reactivity to phytohemagglutinin (PHA). The relationship of these tests with the clinical stage and the subsequent course of the disease in a 6 months' observation period was determined. The patients with visceral metastases (7) had a lowered lymphocyte reactivity to PHA compared with controls and the patients with other stages, while they also had a low reactivity to the test antigens (only significantly lowered compared with patients with localized disease). All these patients showed tumor progression or died from metastatic disease. Between the other stages (54 patients) there was no difference in lymphocyte reactivity to the test antigens or PHA. No correlation between lymphocyte reactivity to PHA and the subsequent course of the disease could be demonstrated in these 54 patients. However, lymphocyte reactivity to the test antigens following immunization showed a definite correlation with the subsequent course. Sixty-four percent (9/14) of patients without any lymphocyte reactivity to the three antigens showed tumor recurrence or progression, against 3% (1/40) of patients with positive lymphocyte reactivity to one, two, or three antigens. A suppressive effect of autologous serum on lymphocyte reactivity could be found only in 1 of 20 patients with a low reactivity to PHA or antigens. It is concluded that defects in lymphocyte function are related to subsequent tumor growth in patients with malignant melanoma.

Adult↗
PubMed1993

Cerebellar nuclear projections from the basilar pontine nuclei and nucleus reticularis tegmenti pontis as demonstrated with PHA-L tracing in the rat.

Small iontophoretic placements of the orthogradely transported axonal tracer Phaseolus vulgaris leucoagglutinin (PHA-L) were made in portions of the basilar pontine nuclei (BPN) or nucleus reticularis tegmenti pontis (NRTP) to determine if these cell groups provide projections to the cerebellar nuclei (CN) in the rat and if so, to visualize the morphology of the axons and terminals and illustrate any topographical organization in this system. Axons that originated from BPN or NRTP neurons and contained PHA-L were visualized by an immunohistochemical procedure that involved sequential incubation of tissue sections with goat anti-PHA-L antibody, biotinylated rabbit anti-goat immunoglobulin, and a biotin-avidin-peroxidase conjugate. Following injections of PHA-L restricted to ventral and medial portions of the BPN, labeled fibers were observed in the brachium pontis, the white matter dorsal to the CN, and to a lesser extent, in the white matter of the parafloccular stalk. Labeled preterminal axons entered the CN and gave rise to beaded axons that arborized primarily within dorsal portions of the lateral, interposed, and medial cerebellar nuclei. Injections of PHA-L restricted to either lateral portions of the BPN or ventrolateral regions of NRTP produced labeled fibers in the cerebellum that most frequently involved the parafloccular stalk and ventral portions of the CN. In contrast, dorsomedial NRTP injections resulted in the presence of labeled fibers both in the dorsal cerebellar white matter and the parafloccular stalk as well as dorsal and ventral portions of the CN. With the exception of the rostral and medial territory of interpositus anterior which received very sparse input, all portions of each CN subdivision seemed to exhibit some degree of terminal labeling. The density of labeled axon terminals in the CN appeared to be somewhat greater in the NRTP-injected cases compared to BPN-injected animals. These observations indicate that in the rat, both the BPN and NRTP contain neurons whose axons distribute to the CN. It is likely that most of the axons which project to the CN are collaterals of fibers that continue into the cerebellar cortex as mossy fibers but confirmation of this point must await further investigation. In light of the extensive projections from the cerebral cortex to the BPN and NRTP, this axonal system provides the cerebral cortex with a relatively direct route of access to the CN via one synapse in the BPN or NRTP.

Animals↗
PubMed1988

Cyclosporin A does not inhibit the PHA-stimulated increase in intracellular Ca2+ concentration but inhibits the increase in E-rosette receptor (CD2) expression and appearance of interleukin-2 receptors (CD25).

The immunosuppressive drug cyclosporin A (CsA) inhibits mixed lymphocyte responses, blocks the generation of cytotoxic T lymphocytes, and inhibits the T lymphocyte proliferative response stimulated by polyclonal activators such as phytohemagglutinin (PHA). Nevertheless, there have been contradictory reports attempting to explain the mechanism(s) for this immunosuppressive activity. In the current studies, human peripheral blood mononuclear cells (PBM) were stimulated with PHA in the presence or absence of CsA. Flow cytometric examination of PBM loaded with the Ca2+-sensitive dye Indo-1 showed that concentrations of CsA sufficient to inhibit 90-100% of tritiated thymidine incorporation had no effect on the PHA-stimulated increase in the intracellular Ca2+ concentration ([Ca2+]i). Likewise, inhibitory amounts of CsA had virtually no effect on the increase in cell volume that occurs during T lymphocyte activation. These results were not altered by pretreating the PBM with CsA for 30 min at 37 degrees C prior to adding the PHA. On the other hand, inhibitory concentrations of CsA prevented the expression of receptors for T cell growth factor (interleukin-2, IL-2), as measured by monoclonal antibodies to CD25 after 16-24-hr incubation. In like manner, CsA also prevented the increase in the expression of the E-rosette receptor (CD2) on these same cells. If cultures containing PHA and inhibitory amounts of CsA were incubated for 40-72 h, there was partial recovery both of proliferative activity and of the expression of CD25 and CD2. Thus, CsA does not appear to affect the initial activation signal(s), but does interfere with one or more subsequent events necessary to initiate the appearance of "activation antigens."

Antigens, Surface↗
PubMed1980

Total and exchangeable calcium in lymphocytes: effects of PHA and A23187.

Calcium has been suggested as an internal second messenger when lymphocytes are stimulated by mitogens to enter the cell cycle. We have assessed the effect of 2 lymphocyte stimulants, the plant lectin phytohemagglutinin (PHA) and the calcium ionophore A23187, on human lymphocyte nucleic acid synthesis, total cell calcium content, and 45Ca labeling. We have used an ultrasensitive method for the measurement of total cell calcium in the same samples used for radiolabeling. Mitogenic concentrations of A23187 (approximately .25 mumole/liter) caused an increase in both total cell calcium and 45Ca labeling. These increases were almost completely blocked by inhibitors of mitochondrial respiration, suggesting that the calcium increment after ionophore treatment was located in the mitochondria. In contrast, total cell calcium was not altered at optimal mitogenic PHA concentrations (0.1 microgram/ml and above). However, at the minimum PHA concentrations that caused stimulation (0.025 to 0.1 microgram/ml), the dose response of 45Ca uptake was very similar to that of DNA synthesis. Importantly, we could not stimulate DNA synthesis with PHA without increasing lymphocyte 45Ca labeling. Thus, an increase in total cell calcium is not essential for mitogenesis; however, an increase in 45Ca exchange is closely associated with the mitogenic effects of A23187 and PHA.

Anti-Bacterial Agents↗
PubMed1980

Enhancement of lymphocyte response to PHA by lysosomal enzymes from polymorphonuclear leukocytes of RA joint fluid. I. Biological effect on T lymphocyte function.

The effect of polymorphonuclear leukocyte (PMN) granule lysates obtained from joint fluid of RA on the in vitro DNA synthesis of PHA-stimulated autologous lymphocytes from joint fluid was studied. Lymphocytes were cultured for 3 days with or without PMN lysates in 2 ml of RPMI-1640 supplemented with 10% heat-inactivated fetal calf serum (FCS). The lymphocytes were stimulated with phytohemagglutinin (PHA-M). The DNA synthesis was measured by counting the [3H]thymidine incorporation. Lymphocytes from RA joint fluid stimulated with PHA-M showed 19,466+/-987 cpm (mean+/-SE) per 10(6) cells in the absence of PMN lysates. Upon addition PMN lysates to the PHA-stimulated lymphocytes, the maximum in vitro DNA synthesis increased to 44,877+/-1338 cpm. The enhancing effect of PMN lysates was abolished by plasma inhibitors or by passage through a column of protease inhibitor (Trasylol). It was concluded, therefore, that the enhancing effect of PMN lysates on PHA-stimulated lymphocytes may be associated with lysosomal proteases. Based on experiments using separated T and B lymphocytes, the enhancing effect of PMN lysates was considered to result from the activation of T lymphocytes. The results obtained in the present study suggest an important role for lysosomal proteases in the perpetuation of rheumatoid synovitis.

ABO Blood-Group System↗
PubMed1980

Immunomorphologic study of regional lymph nodes in cancer: response of regional lymph node cells from gastric and colorectal cancer to PHA stimulation.

To obtain information regarding the immunologic capacity of uninvolved regional lymph nodes (RLNs) draining tumor, the in vitro response of regional lymph node cells (RLNCs) to PHA was investigated in lymph nodes from 55 patients with gastric or colorectal cancer, and gastric ulcer. Evaluation of data relative to cancer progress demonstrated that, in gastric cancer patients, the response of stimulated RLNCs from patients with middle stage carcinoma was significantly higher compared with those of cells from patients with early or late stage carcinoma, such being similar to the response of RLNCs from control patients with gastric ulcer. The results from colorectal cancer patients were also similar to those from patients with gastric cancer. Our results suggest that RLNs without metastasis contain cells capable of responding to PHA despite the presence of growing tumors in these cancer patients. In the results of PHA stimulation in relation to the lymph node morphology, we observed in both groups of patients with gastric or colorectal cancer that, regardless of the extent of the cancer lymph nodes with lymphocyte predominance there was a high PHA response while lymph nodes with lymphocyte depletion and unstimulated nodes exhibited a low PHA response. These results show a close relationship between this lymph node morphology and immunologic status of nodes.

Adult↗