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At least 289 records · Page 16Linked to original sources
PubMed1982

A new method for colony formation in vitro by leukemic cells in acute nonlymphocytic leukemia with PHA-stimulated lymphocyte conditioned medium as stimulating factor.

A new agar culture system to clone specifically acute nonlymphocytic leukemia (ANLL) is described, which consists of two phases: an initial liquid phase in the presence of PHA-stimulated lymphocyte conditioned medium (PHA-LCM) and a semi-solid phase. By preincubation of leukemic cells with PHA-LCM in a liquid phase, leukemic cells were found to be more sensitive to forming colonies and clusters in agar medium. In addition, this PHA-two step assay could eliminate colony formation by T cell progenitors. The leukemic origin of the colonies was proven by electron-microscopic analysis, which demonstrated the presence of nuclear blebs and abnormal perinuclear fibril formation. Morphological studies also indicated myeloid differentiation of the cells in the leukemic colonies, although considerable variation was observed among patients. The wide range of linearity between the number of cells plated and the number of colonies grown permits quantitative assay of colony-forming leukemic cells (L-CFU). This assay should be valuable for studies of chemotherapy, growth regulation, and differentiation of L-CFU.

Acute Disease↗
PubMed1980

A PMN-derived factor that enhances DNA-synthesis in PHA or antigen-stimulated lymphocytes.

The DNA synthesis potentiation of a mouse polymorphonuclear leukocyte (PMN) factor was studied by using phytohemagglutinin (PHA)-stimulated syngeneic thymocytes. The partially purified PMN factor was nonmitogenic and exhibited maximum potentiation of thymocytes when added within 3 hr of PHA stimulation. PHA-stimulated, but not nonstimulated thymocytes absorbed PMN potentiation factor. 125I-labeled factor binding to stimulated thymocytes was observed. Our results suggest that an acceptor site for PMN factor may be generated on the thymocyte surface only after PHA stimulation.

Absorption↗
PubMed1980

Regulatory role of humoral factors released by macrophages in reactivity of rat lymphocytes in vitro. III. Humoral factors released by lymph node macrophages regulating lymphocyte response to PHA.

Lymph node macrophages, derived from normal rats, generated in vitro factors enhancing or suppressing syngeneic lymph node lymphocyte stimulation in response to PHA and reconstituting the PHA-dependent response of lymphocytes deprived of native macrophages. The occurrence of one of the opposite activities depended on the degree of lymphocyte stimulation by PHA. Moreover, the expression of suppressing or stimulating effect no lymphocyte response to PHA of the same supernatants, changed twice with their dilution, (suppression leads to enhancement leads to suppression) suggesting the existence of two distinct factors: suppressing and enhancing.

Adjuvants, Immunologic↗
PubMed1981

Rosette-increasing factor (RIF) generated in vivo following PHA skin test.

In phytohaemagglutinin (PHA) skin test-positive individuals, rosette-increasing factor (RIF), which augmented active E and EA rosette formation, appeared in the serum following the PHA skin testing. This factor was detectable 6 hr after the application of PHA and reached a peak at 14 hr. The appearance of the factor was closely related to the delayed cutaneous hypersensitivity. When mononuclear (MN) cells derived from individuals exhibiting delayed cutaneous reaction were further cultured without the addition of PHA, a similar property was found in the supernatants. Fractionation of the cells by E rosetting revealed this factor to be the product of T lymphocytes. Moreover, despite a lower percentage in contaminated T lymphocytes, a higher RIF activity was observed in the supernatants of the nylon-wool-retained population. The production of RIF was completely inhibited by cytochalasin B, but was not affected by colchicine. Experiments utilizing cycloheximide revealed that new protein synthesis was only necessary for early activation steps. RIF is a kind of lymphokine synthesized in the active immune process in vivo. Therefore, to assay RIF in vivo may provide a new method for investigating the cellular immune competence of the given patients.

Cells, Cultured↗
PubMed1980

In vitro effect of DDS on phytohaemagglutinin (PHA)--induced lymphocyte transformation.

A study to find out the in vitro effect of diamino diphenyl sulphone (DDS) on phytohaemagglutinin (PHA)-induced lymphocyte transformation was carried out in three phases using a wide range of DDS concentrations. Lymphocytes from healthy volunteers were investigated. Volunteers were divided into three groups to conduct the study in three phases. In each phase in addition to 0.02 ml of PHA, 4 different concentrations of DDS were added per 10(6) lymphocytes in tissue culture system. A statistically significant depression (P less than 0.05) in the per cent of blast cell formation induced by PHA was observed in the cultures with all the concentrations of DDS except with the lowest concentration (0.01 microgram) of DDS. While the depression observed in the first and the third phase was found to be dose dependent, no significant correlation was noted between the DDS concentration and depression in PHA induced blastogenesis in the second phase. The significance of these observations are discussed.

Adult↗
PubMed1994

Enhancement of haemopoietic stem cell adherence to bone-marrow derived stroma by phyto-haemagglutinin treated leucocyte conditioned medium (PHA-LCM).

Stem cell adhesion to bone-marrow derived stroma, plays a crucial role in haemopoiesis. However, there is very little information as to the nature of the adhesion molecule. In this paper we have shown that human bone-marrow derived stroma can be established in tissue culture. This stroma is able to adhere human bone-marrow mononuclear cells including the multipotent stem cell, viz. CFU-GEMM. Their adherence increases when the stroma is treated with lymphokines in the form of PHA-treated leucocyte conditioned medium (PHA-LCM). Triton X-100 extracts of the untreated and PHA-LCM treated stroma were analysed on single dimension PAGE. It was observed that PHA-LCM treated stromal extracts showed two extra bands and an increase in the density of a band of approximately 14 kDa. Whether these changes have anything to do with the increased adhesion of stem cell is not yet known.

Bone Marrow↗
PubMedDate not supplied

Adoptive immunotherapy of advanced renal cell cancer using PHA-stimulated autologous lymphocytes.

Patients with advanced renal cell carcinoma, previously failed maximal treatment with standard chemo-hormonal-radiation therapies, were treated with plant lectin phytohemagglutinin (PHA)-stimulated autologous peripheral blood lymphocytes in a 10-year study with a 16-year follow up period. In a phase I-II setting, 52 patients were given subcutaneously 40-80 x 10(6) PHA-stimulated lymphocytes weekly for 3 weeks and then escalated to a maximum number of 80 x 10(9) lymphocytes over the next 9 weeks at 3 week intervals. In vitro blastogenesis under study conditions (10 micrograms/ml PHA for 72 hr) measured by [3H]thymidine uptake was optimal with lymphocyte stimulating indexes approaching 300. Lymphocytes obtained from patients with breast cancer, melanoma and renal cell carcinoma responded to PHA similarly to those from normal volunteers. All patients that responded developed erythematous reactions at the sites of injection; malaise, joint paint and chill-fever for 24-48 hr. The patients that responded the best were those with at least 1 positive reaction out of 4 skin tests (tuberculosis, yeast, dermatophytin, mumps) prior to therapy. All toxicity was transient and did not exceed Grade I based on criteria of the Southwest Oncology Group. The majority of patients developed a lymphopenia in the first 24 hr followed by a lymphocytosis 48-72 hr later. For some patients the lymphocytosis was as much as 30% atypical lymphocytes. Of 41 evaluable patients, there were 5 complete responses, 8 partial responses, 3 stable diseases, and 25 progressive disease. The overall response rate was 32% and the median survival was 2.8 years.(ABSTRACT TRUNCATED AT 250 WORDS)

Carcinoma, Renal Cell↗
PubMedDate not supplied

The early effect of specific immunotherapy on lymphocyte response to PHA and allergens in atopic patients with allergic rhinitis.

In the first part of this study, peripheral lymphocyte subpopulations and their proliferative response to PHA and allergens were investigated in the 30 patients with allergic rhinitis compared to 20 healthy non-atopic individuals. Data obtained employing a PHA-induced lymphoproliferative response assay revealed that the allergic rhinitis generated significantly less activity than did the normal control group. Significantly decreased ration of CD4+/CD8+ T cells was noted in the patients with allergic rhinitis. Mean values of stimulation indices by allergen extracts were higher in the patients sensitive to same antigen than others especially in concentration of 1000 SQU/ml. Stimulation of active lymphocytes revealed no statistically significant group differences between allergens. In the second part of the study, the early effect of immunotherapy on T cell subsets and lymphocyte proliferative response to PHA and allergens were examined in the peripheral blood lymphocytes of patients. A significant increase in PHA-induced and in allergen induced lymphoproliferative response were observed in all patients after sixth months of immunotherapy. It is concluded that there may be an association between allergic rhinitis and deficiency of circulating CD4+ cells but further studies are required to substantiate this hypothesis.

Adult↗
PubMed1980

PHA and ConA lymphocyte response in normal, hyperplastic and neoplastic human thymus: morphologic and functional correlations.

In the present work the in vitro response to PHA and ConA was determined for thymus lymphocytes from normal, hyperplastic, and neoplastic conditions as well as for peripheral blood lymphocytes from normal control donors. Our investigation has revealed these results: (1) lymphocytes from normal thymus are less responsive to both mitogens than lymphocytes from peripheral blood and abnormal thymus. (2) Lymphocytes from thymic benign lymphoid hyperplasia show an increased response only to PHA regardless of the extent of hyperplasia. (3) Lymphocytes from thymic neoplasia display an increased response to both mitogens regardless of the histologic pattern of the tumor studied. (4) Thymic lymphocytes from patients with myasthenia gravis have a mitogenic response higher than that from thymic lymphocytes from nonmyasthenic subjects. (5) In patients with myasthenia gravis, lymphocytes from neoplastic thymus show a response to PHA higher than that observed for lymphocytes from hyperplastic thymus. It seems clear that two conditions maximally enhance mitogenic response of thymus lymphocytes: thymomas and myasthenia.

Adolescent↗
PubMed1990

Postnatal development of crossed and uncrossed corticorubral projections in kitten: a PHA-L study.

Morphological changes in individual corticorubral fibers and the pattern of crossed and uncrossed corticorubral projections were studied during the postnatal development of cats in order to understand cellular mechanisms for restriction of corticorubral projections with development. The anterograde tracer Phaseolus vulgaris leucoagglutinin (PHA-L) was injected into restricted areas of the pericruciate cortex in kittens and PHA-L-labeled axons in the red nucleus were examined at postnatal days (PND) 7-73. In accordance with our previous study (Murakami and Higashi, Brain Res. 1988; 447:98-108), a crossed corticorubral projection was observed in addition to the uncrossed one in every experimental animal. During the early period of development (PND 7-8), swellings of irregular shape were observed along the entire course of the axons and they were often interconnected with extremely fine axonal segments. These axons bifurcated only infrequently and often ended as growth cones. These features were common to both uncrossed and crossed corticorubral axons. At later stages of development (PND 28 or later), the total number of swellings decreased and axonal swellings with smooth contours became dominant. A quantitative examination of axonal branches indicated that axons on the ipsilateral side branch occurred more frequently at later stages of development. However, there was no substantial change in branching frequency for the crossed corticorubral fibers during development. In parallel with morphological changes in individual axons, the crossed projection that was initially relatively abundant was reduced during development. Since a PHA-L injection can be confined to a small region of cortex, topographic projections can easily be detected. At PND 7-8 there was no well-defined topographic order in the ipsilateral corticorubral projection. Adult-like topography was first discernible at PND 13. These observations suggest that the unilateral uncrossed corticorubral projection in the adult cat is achieved at least in part by the formation of axonal arbors in the uncrossed projection. This was accompanied by the failure of crossed fibers to form complex arbors. It is possible that a similar mechanism also operates in the formation of topographic maps.

Aging↗
PubMed1991

Medial terminal nucleus terminals in the nucleus of the optic tract contain GABA: an electron microscopical study with immunocytochemical double labeling of GABA and PHA-L.

In this study the medial terminal nucleus (MTN) projection to the nucleus of the optic tract (NOT) was investigated in pigmented rats at the light and electron microscopical levels with a new combination of techniques. MTN terminals were anterogradely labeled with Phaseolus vulgaris-leucoagglutinin (PHA-L). Preembedding immunocytochemistry, followed by gold intensification, was used to visualize PHA-L. Postembedding immunocytochemistry with 15 nm immunogold particles was carried out to demonstrate the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). Both PHA-L and GABA labeling can be easily discriminated at the electron microscopical level even when present in the same neuronal profiles. Light microscopically MTN-NOT fibers proved to have several branches with many varicosities. MTN terminals were found concentrated in terminal fields. Electron microscopically, it was shown that MTN boutons display characteristics resembling F-type terminals, i.e., terminals with dark mitochondria, pleomorphic vesicles, and symmetrical synapses. All NOT afferents originating from the MTN contained GABA and made multiple contacts exclusively with GABA negative NOT somata and dendrites. These results indicate the existence of a strong and direct inhibitory input onto GABA negative projection neurons in the NOT. This substantiates earlier physiological and morphological reports. It was further demonstrated that the location and organization of MTN terminals in the neuropil differ from that of the retinal input: MTN terminals are largely separated from retinal terminals. MTN terminal fields contain large amounts of GABA positive F terminals in contrast to retinal terminal areas. MTN terminals take part in irregularly shaped agglomerations of terminals, which contain many F terminals and dendritic processes and are surrounded by a glial sheet. Retinal terminals are found grouped together in small circular arrangements contacting a central dendrite.

Animals↗
PubMed1994

Associational and commissural afferents of parvalbumin-immunoreactive neurons in the rat hippocampus: a combined immunocytochemical and PHA-L study.

Nonpyramidal neurons containing the calcium-binding protein parvalbumin (PV) are one of the inhibitory elements of the hippocampal network. Previous studies have indicated that they are involved in septohippocampal disinhibitory circuits. This study analyzes the commissural and ipsilateral associational afferents of parvalbumin neurons. Injections of the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L) into the hilus of the fascia dentata labeled numerous axons in the molecular layer that established synaptic contacts with parvalbumin-immunoreactive neurons on both the injection and the contralateral side. Mossy fibers, labeled by injections into the granule cell layer, terminated on parvalbumin neurons in the hilus and in CA3. Injections of PHA-L into CA3 resulted in a dense labeling of fibers in the hilus and in CA3, CA2, and CA1 on both the injection and the contralateral side. In all these hippocampal fields, PHA-L-labeled fibers established asymmetric contacts with PV-immunoreactive, presumably GABAergic, inhibitory neurons. These observations indicate that parvalbumin-immunoreactive inhibitory neurons in the hippocampus are targets of presumably excitatory associational and commissural projections and suggest that they are involved in feed-forward and feed-back circuits.

Animals↗
PubMed1990

Effect of interleukin-2 on cell proliferation, sister-chromatid exchange induction, and nuclear stress protein phosphorylation in PHA-stimulated Fischer 344 rat spleen lymphocytes: modulation by 2-mercaptoethanol.

The effect of interleukin-2 (IL-2) on cell proliferation, sister-chromatid exchange (SCE) frequency, and the phosphorylation of nuclear stress proteins was evaluated in phytohemagglutinin (PHA)-stimulated spleen lymphocytes isolated from Fischer 344 rats. In addition, the ability of 2-mercaptoethanol (2-ME) to modulate the induction of these biological responses was characterized. Cell proliferation, as measured by the mitotic index, increased significantly (P less than .003) from a range of 3-4% in PHA-stimulated cultures to a range of 8-11% in PHA-stimulated cultures exposed to IL-2. The average generation time (AGT) did not respond to IL-2 in a concentration-dependent manner and decreased significantly (P less than .05) when 20 microM 2-ME was included with IL-2 in the culture medium. The number of SCE increased significantly (P less than .004) from control frequencies, which ranged from 13.1 to 15.6 SCE per cell, to frequencies of 18.5 to 21.5 SCE per cell as the concentration of IL-2 in the culture medium increased to 50 half-maximal units per ml. A reduction in SCE frequency was observed when cells were cultured with 20 microM 2-ME and IL-2 compared to IL-2 alone. Three nuclear proteins, with relative molecular masses of approximately 13,000-18,000, 20,000, and 80,000, were phosphorylated in IL-2-exposed G1-phase nuclei. Elicitation of these nuclear proteins in IL-2-exposed cells was not affected by exposure to 2-ME.

Animals↗
PubMed1991

Organization of CA1 projections to the subiculum: a PHA-L analysis in the rat.

The organization of CA1 projections to the rat subiculum was investigated with the anterograde tracer, Phaseolus vulgaris leucoagglutinin (PHA-L). Discrete iontophoretic injections of PHA-L were placed into various transverse positions of the CA1 field at different septotemporal levels of the hippocampus. The distribution of CA1 projections was observed in dissected and extended hippocampal preparations. CA1 cells located proximally in the field, i.e., close to the CA2 field, gave rise to projections that terminated in the distal third of the subiculum, i.e., close to the presubiculum. CA1 cells located distally in the field, i.e., close to the subiculum, gave rise to projections that terminated proximally in the subiculum, i.e., just across the CA1/subiculum border. CA1 cells in the middle of the field projected to a midtransverse portion of the subiculum. The same general pattern of projections was observed at all septotemporal levels of the hippocampus. Varicose fibers from the CA1 neurons terminated among the basal dendrites of the subicular pyramidal cells, within the pyramidal cell layer, and in the deep portion of the molecular layer. In addition to the CA1 to subiculum projections, the discrete PHA-L injections provided the opportunity of examining the extent of local and associational connections within CA1. In general, associational connections in CA1 are far less extensive than in CA3. CA1 is not entirely without local connections, however. CA1 cells located close to the subicular border, for example, originated axons that first innervated the proximal subiculum and then reentered the CA1 field at the interface between stratum radiatum and stratum lacunosum-moleculare. In most of the experimental cases, there were collaterals located in stratum oriens of CA1 that branched from the fibers directed toward the subiculum. Thus, the basal dendrites of CA1 cells may receive associational inputs. The organization of the CA1 projections to the subiculum is discussed in relation to the organization of CA3 projections to CA1 and the differential output of transverse regions of the subiculum. The possibility is raised that information may be "channeled" through the hippocampal formation via the transverse organization of these connections and ultimately distributed to different recipients of hippocampal efferent projections.

Afferent Pathways↗
PubMed1995

Retinoic acid and methotrexate specifically increase PHA-E-lectin binding to a 67-kDa glycoprotein in LA-N-1 human neuroblastoma cells.

Retinoic acid (RA) decreased growth and increased morphologic differentiation of human neuroblastoma LA-N-1 cells. These phenomena correlated with a specific enhancement of PHA-E lectin binding to a glycoprotein of MW 67 kDa (gp67). Gp67 was found susceptible to N-glycanase and displayed BSA binding by affinity chromatography analysis. The chemotherapeutic agent methotrexate (MTX) also reduced growth and induced differentiation of LA-N-1 cells. In addition, the cells responded to MTX as well as to doxorubicin by a marked increase in PHA-E binding to gp67. We conclude that reduced growth and induction of morphological differentiation of LA-N-1 cells correlates with increased binding of PHA-E to gp67.

Amidohydrolases↗
PubMed2003

Ribavirin inhibits DNA, RNA, and protein synthesis in PHA-stimulated human peripheral blood mononuclear cells: possible explanation for therapeutic efficacy in patients with chronic HCV infection.

The treatment of choice for patients infected chronically with HCV is the combination of IFN-alpha and ribavirin. Monotherapy with ribavirin leads to a clinical and histological improvement, but its exact mechanism of action is unknown. Therefore, the effect of ribavirin on synthesis of inflammatory cytokines and on apoptosis in stimulated peripheral blood mononuclear cells (PBMCs) was investigated. PBMCs were isolated from the blood of HCV infected patients and from healthy volunteers. The effect of ribavirin on IFN-gamma and IL-1beta release in the supernatant of unstimulated and phytohemagglutinin (PHA) stimulated PBMCs was investigated by enzyme linked immunosorbent assay (ELISA). The effect on total DNA, RNA, and protein synthesis was analyzed by measurement of 3H-thymidine, 3H-uridine and 3H-leucine incorporation into cellular macromolecules. Ribavirin led to a dose-dependent decrease of the IFN-gamma but an increase of IL-1beta release into the supernatant of PHA-stimulated PBMCs. At the same time, a dose-dependent decrease of total DNA, RNA, and protein synthesis in cultures of PHA-stimulated PBMCs was demonstrated. These effects could be compensated by the addition of equimolar amounts of guanosine. The rate of apoptotic CD45+ and CD14+ cells in PBMCs cultures increased in a dose-dependent manner. Our data suggest that ribavirin administration to chronically HCV-infected patients could lead to a decrease of the synthesis of proinflammatory cytokines (e.g., IFN-gamma) by an inhibition of total DNA-, RNA-, and protein-synthesis and by induction of apoptosis in the cells of the inflammatory infiltrate. Furthermore, ribavirin could influence the synthesis of viral particles in the hepatocytes.

Antimetabolites↗
PubMed1984

Self-renewal and maturation of immature T-cell progenitors within PHA-induced agar T-cell colonies.

T-cell colonies derived from peripheral blood mononuclear cells plated in agar with phytohemagglutinin (PHA) contain about 5-7% E-, OKT3-, OKT6-, TdT-, SIg- cells phenotypically similar to bone marrow precursors since they are also RFB1+, OKT10+, HLA-DR+, and PNA+. These cells can generate secondary E+, OKT3+ T-cell colonies when replated in the presence of both PHA and a colony-promoting activity distinct from the T-cell growth factor, Interleukin 2. These observations suggest that E-, OKT3- cells detected in primary PHA-induced T-cell colonies belong to a pool of prethymic cells which are able to mature into T-lymphocytes of T4 and T8 type in the absence of the thymic microenvironment.

Clone Cells↗
PubMed1998

Apoptotic killing of CD4+ T lymphocytes in HIV-1-infected PHA-stimulated PBL cultures is mediated by CD8+ LAK cells.

In vitro infection of PHA-stimulated, normal CD4+ human peripheral blood T lymphocytes (PBLs) with several HIV-1 isolates did not result in cytopathology, despite high levels of virus replication and the fact that some of these isolates were cytopathic in certain cell lines. In contrast, infection of unfractionated PBLs (containing CD8+ as well as CD4+ lymphocytes) with these isolates always resulted in death of the infected CD4+ T lymphocytes. It has been well documented that PHA stimulation and culture of PBLs in medium containing IL-2 generates lymphokine-activated killer (LAK) cell activity which can destroy many transformed cells and virus-infected normal cells. When CD8+ T lymphocytes from PHA-stimulated PBLs were added to HIV-1-infected purified CD4+ T lymphocytes, significant lysis occurred. This cytotoxicity was not MHC class I-restricted, and depletion of CD8+ T lymphocytes from unfractionated PBL cultures shortly after HIV infection largely abolished the killing of the infected CD4+ T lymphocytes. These results demonstrated that CD8+ LAK cells were killing the CD4+ T lymphocytes in unfractionated PBL cultures infected with these noncytopathic HIV-1 strains. Care is thus warranted when studying HIV cytopathology in unfractionated PBL cultures. Morphological and DNA gel electrophoretic analyses of HIV-infected CD4+ T lymphocytes being killed by CD8+ LAK cells demonstrated that apoptosis was the predominant mechanism of LAK cell-mediated killing. In contrast, necrosis was the major mechanism involved in killing of purified CD4+ T lymphocytes by HIV-1 strains which were directly cytopathic. These findings may explain some of the discrepancies in the literature concerning reports of either apoptotic or necrotic killing of cells by HIV in vitro. Moreover, these data strongly suggest that direct killing by replicating HIV-1 in vivo should reveal necrotic cells and immune effector cell killing should reveal apoptotic cells. Since the latter are much more frequently observed in vivo, perhaps immune effector-mediated depletion of CD4+ T lymphocytes is more important as a pathogenic mechanism.

Apoptosis↗