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Low in vitro response to PPD and PHA in lymphocytes from BCG-induced pleurisy in guinea pigs.

In order to study any correlation between functional properties of lymphocytes in BCG-induced pleural exudation and the development of the pleurisy a previously described experimental model was used. This model with a duration of effusion of more than 17 days has characteristic stages. From the third day and onwards there are lymphocytes in sufficient amount for in vitro cultures. Proliferation of lymphocytes from the fluid was measured as uptake of 14C-thymidine. The response of the lymphocytes to PPD tuberculin and to phytohemagglutinin (PHA) was studied, and their spontaneous activity was measured. Comparisons were made with lymphocytes from regional lymph nodes. Pleural lymphocytes sampled on the third post-induction day did not respond to PPD or PHA stimulation. In later stages, pleural lymphocytes were stimulated by PPD to approximately the same degree as the lymph node lymphocytes. The response to PHA was weak at all stages of pleurisy, though in later stages there were some cases with high values. Variations in activation ability, related to disease staging, were demonstrated. However, low activities, and variability of the responses, without concomitant variations in disease, speak against a connection between the course of disease and functional status of the lymphocytes as measured in this study.

Animals↗

Growth characteristics of PHA-induced colonies in primary and secondary agar culture.

PHA-induced colonies were obtained from peripheral blood mononuclear cells (MC) grown in agar-medium. When the colonies were harvested from mass cultures, pooled as single cell suspensions and plated again in presence of PHA, they failed to generate new colonies unless they were seeded on an underlayer containing uncultured blood MC. Cytogenetic studies indicate that most secondary colonies were derived from primary colonies. Autologous as well as heterologous feeder cells were able to promote the growth of secondary colonies. No granulocyte (G) or macrophage (M) colony formation was observed in secondary cultures. These experiments show that the progenitors of PHA-induced colonies differ from G or M CFCs and that they are still detected in these colonies which contain 82 +/- 12% T-cells. In contrast, colony formation requires the presence of factor(s) provided by cooperating cells (CC) which are no longer detected in primary colonies and this is associated with a depletion in non-T elements from the initial MC population.

Cells, Cultured↗

Developmental profile of chicken splenic lymphocyte responsiveness to Con A and PHA and studies on chicken splenic and bone marrow cells capable of inhibiting mitogen-stimulated blastogenic responses of adult splenic lymphocytes.

Embryonic and newly hatched chicken splenic lymphocytes were analyzed for their ability to respond to 2 mitogens, Con A and PHA. Acquisitions of the ability to respond to both mitogens began at hatch and responses to both mitogens reached normal adult blastogenic response levels by 11 days post-hatch. Splenic lymphocytes in the developing embryo and newly hatched chicken were found to be capable of inhibiting the Con A- and PHA-mediated blastogenic responses of both syngeneic and allogeneic adult splenic lymphocytes. The ability to suppress adult responses was first observed on E12, reached a maximum of suppressive ability on E18, and the suppressive ability was gradually lost with development such that no suppressive ability was exhibited after H11. Ability to suppress adult splenic lymphocyte proliferation to mitogens was found to not be a unique property of splenic lymphocytes but was also exhibited by bone marrow cells. The ability of bone marrow cells to suppress adult splenic lymphocyte proliferation to Con A and PHA gradually decreased with chicken aging but the suppressive ability was never lost and adult bone marrow cells were found to exhibit significant degrees of suppressive ability. The mechanism of suppression by the 1-day-hatched splenic lymphocytes was found to be cell-to-cell contact mediated and did not require any newly synthesized H1 splenic products, since x-ray killed cells were able to suppress as well as live cells.

Aging↗

[Role of serum factors in decreasing the reaction of peripheral blood lymphocytes in schizophrenia to PHA stimulation].

The blood serum of schizophrenic patients and three of its studied fractions (gamma-globulins and remaining after elimination of gamma-globulins and ultrafiltrate protein fractions) inhibit the lymphocyte response of normals to PHA stimulation (an increase of the DNA content in cells). An insignificant inhibitor effect in the blood serum of normals was seen only in the protein fraction. The PHA precipitating activity of the blood serum in schizophrenic patients which is mainly connected with its protein fraction is increased by 30%. However, there are no correlations between this blood serum activity and its capability to inhibit the response of lymphocytes to PHA stimulation.

Cells, Cultured↗

Differential modification of radiation damage in 5-bromo-2-deoxy-uridine sensitized human glioma cells and PHA-stimulated peripheral leukocytes by 2-deoxy-D-glucose.

Effects of 5-bromo-2-deoxy-uridine (BrdU) and 2-deoxy-D-glucose (2-DG) on 60-Co-gamma-ray induced damage were studied in monolayer cultures of glioma (BMG-1) cells, and PHA-stimulated peripheral leukocytes from normal donors. Micronuclei formation was used as an index of cytogenetic damage. BrdU and 2-DG treatments did not induce micronuclei formation in unirradiated cultures. Presence of BrdU (0.8 microM) for more than one cell cycle (24 hr) significantly increased gamma-ray (1-4 Gy) induced micronuclei formation in exponentially growing BMG-1 cells. Incubation of irradiated cells under sub-optimal growth conditions (DMEM with 1% serum) for 3 hr, instead of growth medium, significantly decreased micronuclei formation. Post-irradiation presence of 2-DG (5 mM; 3 hr, in DMEM + 1% serum) significantly increased radiation damage. In BrdU sensitized cells also, 2-DG significantly increased radiation damage further. In PHA-stimulated leukocytes from normal donors, 2-DG (5mM, equimolar with glucose; for 2 hr) did not increase gamma-ray (2-Gy, 42 hr after PHA-stimulation) induced micronuclei formation. Pre-irradiation presence of BrdU (1.6 microM) significantly increased micronuclei. On the contrary, 2-DG treatment reduced radiation induced micronuclei formation in BrdU sensitized leukocyte cultures. These results suggest that (i) radiation induced lesions leading to micronuclei formation in proliferating tumour cells, are, at least, partly repairable; (ii) combination of 2-DG could reduce BrdU doses required for radiosensitization of proliferating tumour cells; and (iii) 2-DG could differentially increase radiation damage in BrdU sensitized proliferating tumour cells, while reducing manifestation of damage in normal proliferating cells.

Adult↗

Biological effects of Escherichia coli lipopolysaccharide (LPS) in vivo. II. Selection in the mouse thymus of PHA- and con A-responsive cells.

The effect of LPS injection on mouse thymocytes was examined by in vitro stimulation with PHA and Con A. Thymocytes from LPS treated mice were found more responsive than normal thymocytes to PHA and Con A. The increase of responsiveness was different for PHA and Con A, and dependent on the dose of LPS injected. This dose dependency suggests that LPS in vivo modifies the thymocyte population by progressively eliminating T1 cells and selecting T2 cells. These effects seem to be mediated by corticosteroids because they were prevented by adrenalectomy, a finding in agreement with the notion that the injection of cortisone induces similar cell changes in the thymus.

Adrenalectomy↗

Inhibition of protein kinase C in PHA-activated PBMC treated with anti-HLA class I monoclonal antibody is associated to decreased proliferation and expression of cell cycle related genes.

Treatment of PHA-activated PBMC with anti-HLA class I monoclonal antibody (mAb 01.65) shows: 1) depletion of particulate protein Kinase C (PKC) and partial reduction of cytosolic PKC after only 10 min.; 2) inhibition of tritiated thymidine (3H-Td) incorporation; 3) slowing down of cell cycle; 4) reduced expression of four cell cycle related genes. These findings suggest that the depletion of PKC is reflected on the cell cycle progression and expression of cell cycle related genes. We studied, in PHA-activated PBMC cultures, the effect of N-N-Staurosporine (StSp) acting as PKC inhibitor at nanomolar concentrations, alone and combined with mAb 01.65. StSp, inhibits the proliferative response of PHA stimulated PBMC in a competitive fashion with mAb 01.65. We report here that StSp alone and combined with mAb 01.65 affects also the expression of c-myc and cdc2 and the membrane expression of two proliferation markers: IL-2R (CD25) and TfR (CD71).

Alkaloids↗

Sequential proliferation induced in human peripheral blood lymphocytes by mitogen. II. Suppression by PHA-activated cells.

Human peripheral blood cells from cultures stimulated by phytohemagglutinin (PHA) for 3 days suppressed continued proliferation when added to feeder layer cultures of autologous lymphocytes previously stimulated with mitogen. Proliferation 1 day after the addition of inhibitory cells was reduced 50 to 60% as measured by tritiated thymidine (3HTdR) uptake and mitotic index. It is suggested that a similar suppressive phenomenon may be acting in most standard mitogen-stimulated cultures to limit cell division after only a few days of culture. Suppression of continued lymphocyte proliferation in the feeder layer cultures by cells from PHA-stimulated cultures was not due to cell crowding, carry-over of mitogen, or nutrient depletion; nor was it an artifact of thymidine incorporation. Apparent suppression of proliferation by an extract of these cells, however, was artifactual; the extract inhibited 3HTdR uptake but did not reduce mitotic activity. It is not known whether the inhibition exerted by the PHA-activated cells is mediate through a discrete subpopulation of "suppressor cells" or represents a regulatory function of any mitotic or post-mitotic cell, or both.

Cell Division↗

The effect of X-irradiation on phytohemagglutinin (PHA)-stimulated rat lymphocytes.

The effect of X-irradiation on PHA-stimulated rat lymphocytes has been investigated. The animals were exposed to doses of 100 to 800 R. Blood obtained by cardiac puncture was defibrinated and the lymphocytes sedimented by Dextraven. Lymphocytes were separated from supernatant and cultivated for three days in standard medium supplemented with rat serum and PHA. It was found that irradiation reduces the percentage of blast cells in function of the doses of irradiation. This reduction is accompained by an increased number of macrophage-like cells. The labelling index with tritiated thymidine in PHA-transformed lymphocytes was also reduced following irradiation.

Animals↗

[The effect of ascorbic acid (vitamin C) on the proliferation of human lymphocytes stimulated with phytohemagglutinin (PHA)].

The lymphocytes (in whole blood) from 15 human patients were stimulated with phytohaemagglutinin (PHA)--40 micrograms/ml in standard 199 medium supplemented with ascorbic acid (AsA). As against the standard medium, the lymphocyte proliferation in AsA-added medium represented, on the average, 183 and 330% with the 40 and 80 micrograms/ml doses respectively. No mitotic activity (SI approximately 1) was recorded with the 160 micrograms/ml dose (two samples). When no PHA was present, the AsA addition did not induce lymphocyte proliferation. The amplification of the lymphocytic mitogenesis, as an AsA effect, was present irrespective of the SI value of the variants stimulated with PHA in standard medium, without exceeding 500%.

Adult↗

Response of human lymphocytes to PHA and Con A, dependent on and regulated by THF, a thymic hormone.

The response of human lymphocytes to T lectins was shown to be dependent on and regulated by THF (thymus humoral factor). When human lymphocytes were stimulated with T lectins in the presence of dialyzed human plasma (DHP) which probably contains a low thymic hormone concentration, we observed a reduced response to PHA and Con A, compared to that observed in the presence of whole human plasma (WHP). This reduced reactivity to T lectins in the presence of DHP was restored by the addition of plasma dialyzates or THF. On the contrary, addition of THF to cultures in the presence of WHP caused a significant reduction in reactivity to T lectins, suggesting that enhancement or reduction of the response of human lymphocytes to T lectins by THF depends on the endogenous content of the thymic hormone in the plasma. From the present data it is suggested that the response of human cells to PHA behaves differently from that to Con A, as reflected in their dependence on THF. Although the response of human cells to Con A is increased by THF only in the presence of DHP, the response to PHA is also enhanced by THF in the presence of low WHP concentrations of even in the absence of WHP.

Concanavalin A↗

Projections from the lateral vestibular nucleus to the upper cervical spinal cord of the cat: A correlative light and electron microscopic study of axon terminals stained with PHA-L.

Vestibulospinal axon collaterals in C1 and C2 were stained following injections of Phaseolus vulgaris leucoagglutinin (PHA-L) into the lateral vestibular nucleus (LVN). The distribution and geometry of collaterals within three regions of the ventral horn were determined at the light microscopic level. These processes were subsequently examined at the electron microscopic level to define the relationship between their ultrastructural characteristics and their geometry and location. All round or elliptical varicosities, whose diameters exceeded the diameter of the adjacent axon shaft by a factor of two, as measured at the light microscopic level, contained synaptic vesicles and contacted dendrites or somata. These varicosities accounted for 82% of labelled axon terminals found at the electron microscopic level. Thus, axon terminals stained with PHA-L can be identified reliably at the light microscopic level, but synaptic density will be slightly underestimated. One-hundred and thirty-eight axon terminals were classified as excitatory or inhibitory on the basis of well-established morphological criteria (e.g., vesicle shape). Placed in the context of previous physiological observations describing the excitatory or inhibitory actions of medial and lateral vestibulospinal tract (MVST and LVST) neurons, our results suggest that projections from the LVN to the ipsilateral ventral horn originate primarily from the LVST. These connections are excitatory. Ipsilateral connections via the MVST are inhibitory and are largely confined to a region near the border of laminae VII and VIII. Most axon terminals in the contralateral ventral horn were inhibitory. This result indicates that the LVN is the source of a specific subset of crossed MVST axons with inputs from the posterior semicircular canal.

Animals↗

Depressed PHA transformation and acute leukemia in macroglobulinemia.

Two out of fifty-three patients with macroglobulinemia developed acute leukemia following chemotherapy. The Phytohemagglutinin (PHA) Transformation Index performed prior to the appearance of acute leukemia was found to be markedly depressed in these two patients in comparison with ten other patients with macroglobulinemia. In addition, a clone with monosomy 7 was detected in one of the currently reported patients when the leukemic process became apparent. The cytogenetic analysis was normal in the same patient 48 months earlier, when macroglobulinemia was diagnosed. The low PHA Transformation Index and the chromosomal hypodiploidy are of interest and their clinical significance merits further investigation.

Adolescent↗

Comparison of the response to PHA and suppressor macrophage function in different age groups of cancer patients and healthy controls.

The mitogenic response of peripheral blood lymphocytes was examined in 37 healthy individuals and in 44 patients with various malignant neoplasms. Tested individuals were divided into three major age groups (20-40; 40-60; and over 60 years of age). There was a significant decrease in Phytohemagglutinin (PHA) response with age in the normal controls, but no significant change with age was observed in the cancer patients. Comparison between the various age groups of cancer patients and their corresponding normal controls did not reveal a significant difference in mitogenic response of the young and middle age groups. However, the older cancer patients responded significantly better compared with their corresponding older normal controls. The reduced responsiveness of older normal controls was partially explained by a high activity of suppressor macrophages observed in this group. Reduction of the proportion of phagocytic cells improved their response to PHA. On the other hand, the activity of suppressor macrophages in the older cancer patients was much less prominent. These observations emphasize the importance of appropriate age grouping when immune function tests in cancer patients are compared with those performed in normal individuals and suggest the possibility of higher mitogen responsiveness in certain age groups of cancer patients.

Adult↗

PHA-induced cellular cytotoxicity. Inhibition by a nonimmunoglobulin factor present in sera from patients with active systemic lupus erythematosus.

Phytohemagglutinin-induced cellular cytotoxicity by normal mononuclear cells (MNC) is inhibited by serum of patients with active untreated systemic lupus erythematosus (SLE) but not by that of patients with untreated inactive SLE or rheumatoid arthritis. Three-hour preincubation of normal MNC with SLE sera suffices to inhibit throughout the assay. Preincubation with phytohemagglutinin (PHA) before addition of sera does not prevent the inhibition. DEAE-cellulose chromatography, Sephadex G-200 fractionation and immunoelectrophoretic analysis have shown the nonimmunoglobulin nature of this factor. It is thermostable and not affected by deoxyribonuclease or ribonuclease treatment. The mitogen-induced cellular cytotoxicity-inhibiting factor seems to be independent of other serum factors capable of blocking PHA-induced blastogenic transformation.

Arthritis, Rheumatoid↗

Three-peaked chemiluminescent response of human peripheral blood leukocytes following stimulation with phytohaemagglutinin (PHA).

Luminol-enhanced chemiluminescence (CL) was used to examine the response of various leukocyte populations following stimulation with a crude extract of Phaseolus vulgaris, namely phytohaemagglutinin (PHA-C). Populations stimulated included a human peripheral mixed leukocyte preparation (MLP), and purified preparations of lymphocytes, monocytes and polymorphonuclear leukocytes (PMNL). Mouse peritoneal exudate cells and the lymphocytic cells lines Molt #4 and Daudi were also stimulated. Following stimulation, a characteristic three-peaked chemiluminescent response was obtained from the MLP population. Little or no response was obtained from the purified lymphocytes. Monocytes produced a sharp peak corresponding to the second peak of the MLP response and PMNL produced a broad peak corresponding to the third peak of the MLP response. Mouse peritoneal exudate cells containing lymphocytes and monocytes/macrophages showed a two-peaked stimulation which corresponded to the first two peaks of the MLP response. Molt #4 and Daudi showed no chemiluminescence if stimulated individually, but if added to a MLP substantial enhancement of the first and second peaks was observed. These results indicate some form of lymphocyte/monocyte interaction leading to enhanced CL following PHA-C stimulation.

Animals↗

Nigropedunculopontine projection in the rat: an anterograde tracing study with phaseolus vulgaris-leucoagglutinin (PHA-L).

The termination of the substantia nigra pars reticulata efferents in the nucleus tegmenti pedunculopontinus was studied in the rat by using the anterograde tracer Phaseolus vulgaris-leucoagglutinin (PHA-L). Both large and small injections of PHA-L in various portions of the substantia nigra pars reticulata labeled varicose fibers in the ipsilateral and contralateral nucleus tegmenti pedunculopontinus, subnucleus dissipatus as well as in the ipsilateral nucleus tegmenti pedunculopontinus, subnucleus compactus. However, the bulk of the nigral fibers appeared to terminate in the medial two-thirds of the ipsilateral subnucleus dissipatus of the pedunculopontine nucleus and exhibited a discrete dorsoventral topographical pattern. The terminal plexus displayed patches of uneven density, which was partly due to the numerous fiber bundles passing through the pedunculopontine nucleus, but also to an obvious preference of nigral fibers for some cells. Electron microscopic examination confirmed that nearly all of the varicosities observed in the light microscope contained synaptic vesicles and represented either terminal boutons or boutons en passant. The labeled boutons were elongated (average length: 1.5 microns) and consistently contained a prominent group of mitochondria. The results suggest that the nigral input to the nucleus tegmenti pedunculopontinus may be directed toward specific subpopulation(s) of pedunculopontine neurons and may influence not only cells in the subnucleus dissipatus, but also in the subnucleus compactus.

Animals↗

Projections of the tectospinal tract to the upper cervical spinal cord of the cat: a study with the anterograde tracer PHA-L.

The goal of the present experiments was to re-examine the spinal projections of neurons in the superior colliculus (SC) of the cat by taking advantage of the high sensitivity of the anterograde tracer, phaseolus vulgaris leucoagglutinin (PHA-L). In seven experiments, multiple injections of PHA-L into different regions of the SC labelled a total of 172 axons in the predorsal bundle; yet only 11 tectospinal tract (TST) axons were found in the upper cervical spinal cord. Collaterals emerging from these axons were rare and arose exclusively from TST axons with a diameter of less than 1 micron. Individual collaterals had different termination zones: some terminated in the lateral part of lamina V and VI after taking a dorsolateral course through lamina VII and VIII; others terminated in the medial part of lamina VII. One collateral terminated within lamina IX and the ventral part of lamina VIII. The combined termination of all collaterals was densest in lamina VII and dorsal lamina VIII. A small number of boutons were also found in the lateral parts of laminae V and VI, and in lamina IX and immediately adjacent regions in lamina VIII. Compared to axons belonging to other spinal descending systems, individual TST axons give rise to much simpler intraspinal collaterals with relatively few boutons. This feature, together with the relative paucity of TST axons, suggests that direct connections from the SC to neurons in the upper cervical spinal cord are sparse. Furthermore, our results are consistent with electrophysiological studies that show that few, if any, neck motoneurons receive monosynaptic connections from TST neurons. Projections to neck motoneurons must therefore involve a relay, either through other descending pathways, such as the reticulospinal system, or via local segmental interneurons.

Animals↗