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In vitro DNA synthesis by bone marrow cells and PHA-stimulated lymphocytes. Suppression by nonradioactive thymidine of the incorporation of 3H-deoxyuridine into DNA: enhancement of incorporation when inadequate vitamin B12 or folate is corrected.

Previous studies demonstrated that excess deoxyuridine (dU) added to short-term bone marrow and PHA-stimulated lymphocyte cultures, blocks the incorporation of radioactive thymidine into DNA via the salvage pathway. In the current study, we investigated the effects of added thymidine (TdR) in varying concentrations (10(-6) to 1 mumol) on the incorporation of 3H-dU into thymine-DNA, i.e. we executed 'thymidine suppression tests.' Increasing concentrations of exogenous TdR caused progressive inhibition of 3H-dU incorporation into DNA, and decreasing 3H-dU incorporation was parallelled by increasing incorporation of added 14C-TdR. These findings demonstrate reciprocity of the salvage and the de novo pathways of thymine-DNA synthesis, presumably mediated by thymidine-triphosphate (dTTP), the common end product of both pathways, via feedback inhibition. In patients with folate and/or vitamin B12 deficiency, the addition of appropriate vitamins to marrow and lymphocyte cultures enhanced the incorporation of 3H-dU into DNA. As predicted, this was not observed in normal subjects. The enhancing effect of these vitamins on in vitro incorporation of 3H-dU into DNA by deficient cell systems was similar to their correcting effect on abnormal dU suppression. These findings support the theoretical concept that the dU suppression test defines biochemical megaloblastosis due to deficiency of folate and vitamin B12.

Bone Marrow↗

Evidence that soluble products released by PHA-stimulated human lymphoid cells activate immunosuppressive monocytes.

Soluble mediators, lymphokines, released by stimulated lymphoid cells can modify immunological responses in several ways. In this investigation we have examined whether the supernatants of phytohaemagglutinin (PHA)-activated human lymphocytes (active SUPs) contain factors that can suppress proliferative responses of lymphocytes in vitro. The results have shown that crude preparations of peripheral lymphoid cells incubated for 24 h in active SUPs can suppress the responses of cocultured autologous lymphocytes to PPD tuberculin in vitro. Their suppressive activity was not abolished by mitomycin treatment. Some reduction of phytomitogen responses was also noted. Maximal suppressive activity was obtained within 24 h of incubation in active SUPs and it could not be induced in cell preparations depleted of monocytes-macrophages. Similar results were obtained by treating lymphoid cells with lipopolysaccharide from Escherichia coli, which is a known activator of monocytes. These results thus show that lymphokines released by stimulated lymphoid cells can activate monocytes-macrophages in such a way that they become immunosuppressive.

Cell Division↗

A novel arachidonic acid metabolite with stimulatory effect on PHA-induced mitogenesis of lymphocytes.

The isolation of a novel arachidonic acid (Aa) metabolite from the supernatant of unstimulated human cord blood mononuclear leucocytes is reported. The metabolite, arbitrarily named 'compound 4' is neither a known lipoxygenase nor a cyclooxygenase product. 'Compound 4' was added to PHA-stimulated peripheral blood mononuclear leucocytes (PBML) from healthy blood donors, from mothers at term and from patients with immunodeficiency. 'Compound 4' induced an increase in the 3H-TdR incorporation by the maternal PBML and by the PBML from patients with various immunodeficiencies such as Wiskott-Aldrich syndrome and common variable immunodeficiency, whereas it had no effect on the proliferation of PBML from blood donors.

Arachidonic Acid↗

Demonstration of platelet electrophoretic mobility slowing factor (PSF) in supernatants of human lymphocyte cultures stimulated by phytohaemagglutinin (PHA).

It has been demonstrated that human peripheral lymphocytes, stimulated with PHA, form or release a factor that slows platelet electrophoretic mobility (PEM). The factor is chromatographed on Ultrogel AC a 44, has a molecular weight of approximately 45,000, is stable on heating at 56 degrees C for 30 min, but is inactivated by heating at 80 degrees C for 10 min. In addition, it is distinct from leukocyte inhibiting factor, which elutes in the range of BSA (mol. wt. 69,000). The authors conclude, in accordance with previous investigations, that cellular immunity is involved in thrombotic processes.

Blood Platelets↗

PHA-induced interferon in multiple sclerosis: association between gamma interferon and clinical and genetical variables.

Gamma interferon (INF-gamma) production, after PHA stimulation of peripheral blood mononuclear cells, from multiple sclerosis (MS) patients with the acute remitting and chronic progressive forms, in attack and remission phases, and from normal controls, was studied by immunoradiometric assay. MS patients in all these 4 clinical states of disease produced less INF-gamma (log value range from 2.55 to 2.65). MNC from the total MS patients produced significantly low levels of INF-gamma compared to the control group (log values 2.60 vs. 2.82; P = 0.001). No association between the interferon production and antigens at any HLA locus (A, B, C, Dw and Bf) was found. There was no correlation between IFN-gamma production and age, sex, duration of disease, or disability index. However there was a slight tendency to negative correlation with the progression index of the disease. The results suggest that this lower IFN-gamma production in MS may be secondary to the disease, and the primary defect may be a severe reduction of the essential lymphocyte populations required for an effective lymphokine cascade to produce the normal immune response against infection.

Adult↗

Three-dimensional reconstructions of nucleolus-organizing regions in PHA-stimulated human lymphocytes.

Ultrastructure and three-dimensional distribution of nucleolus-organizing regions have been studied on ultrathin serial sections of PHA-stimulated human lymphocytes. During the 48 hr of activation the size of fibrillar centers (FCs) decreased from 0.6-0.9 microns to 0.2-0.3 microns and the number of FCs increased rapidly from one to 75-107 per cell. The number of fibrillar complexes (i.e. associations of a different number of FCs connected by the dense fibrillar component) also increased but did not reach the maximum number of nucleolar organizers presented here. Three-dimensional computer reconstructions of fibrillar complexes showed that lymphocyte activation was accompanied by early (2-4 hr) changes in the shape of the primary fibrillar center. Invagination of the dense fibrillar component on its surface occurred and division into two or more smaller FCs followed. Gradually, the typical structure of the nucleolus with several fibrillar complexes and many FCs was formed. These results confirm the hypothesis of fibrillar complex-nucleolar organizer correlation published recently.

Humans↗

The response to phytohaemagglutinin (PHA) of lymphocytes from cancer patients.

The uptake of tritiated thymidine by lymphocytes from 100 cancer patients and 31 normal subjects was tested in the presence and absence of phytohaemagglutinin (PHA) in a medium containing autologous or homologous serum. Significant differences were found between normal subjects and cancer patients in both the time course and degree of uptake. Serum from normal individuals restored the response of lymphocytes from cancer patients towards normality whilst serum from other cancer patients had no effect. Serum from cancer patients impaired the response of normal lymphocytes.

Breast Neoplasms↗

Insoluble PHA- A B-cell mitogen in man?

Blood lymphocytes of normal individuals and of patients with chronic lymphocytic leukemia were evaluated for their blastogenic response to insoluble PHA. In at least preferential stimulation of normal B-lymphocytes was observed, whereas leukemic lymphocytes were almost insensitive. This result supports the hypothesis of a functionally defective B-lymphocyte population in chronic lymphocytic leukemia.

B-Lymphocytes↗

A T cell model for age-related loss of homeostasis: identification of the cell type responsible for age-related delay in recovery of the PHA-induced mitogenic response from radiation injury.

Immunologic recovery from radiation injury was used to assess age-related loss of immunologic homeostasis. Mice were exposed to 500 rad to determine whether macrophages, T helper cells and/or T responder cells were responsible for the age-related delay in recovery of PHA-induced mitogenesis. Recovery of macrophages was determined by facilitation of macrophage-deficient cultures, recovery of T helper cells by interleukin 2 production, and recovery of T responder cells by incorporation of tritiated thymidine. The results showed that in old mice macrophages are resistant to radiation and aging, T helper cells recover completely from radiation, and recovery is incomplete for T responder cells.

Aging↗

Presence of basophils in PHA-reacted skin sites of mice. An electron microscopic study.

Subcutaneous injections of the lymphocyte mitogen PHA in mice induced a local reaction which was delayed in time course and characterized histologically by dermal infiltration with blood granulocytes. Basophils were identified in the skin sections by electron microscopy as their granules differ from those of other blood granulocytes and tissue mast cells.

Animals↗

Gamma-ray- and fission neutron-induced micronuclei in PHA stimulated and unstimulated human lymphocytes.

Two groups of normal human blood cells, one stimulated with phytohaemagglutinin (PHA) for 24 hr (G1-S phase of the cell cycle) and one unstimulated (G0 phase), were irradiated with 60Co gamma rays or 252Cf radiation. A comparison of radiation-induced micronucleus frequencies showed that the high-dose-rate gamma rays were more effective in inducing micronuclei than low-dose-rate gamma rays. In the cells exposed to low-dose-rate irradiation, there was little difference between the frequency of micronuclei in the G0 phase and the G1-S phase. However, cells in the G1-S phase were more sensitive than G0-phase cells to high-dose-rate gamma rays. The relative biological effectiveness of 252Cf neutron irradiation measured in micronucleus assays was consistent with the value obtained for the lethal effect of 252Cf on cultured cells.

Adult↗

Effect of morphine on PHA-stimulated human lymphocyte transformation.

The effect of morphine on lymphocyte blastoid transformation induced by PHA was studied in vitro by observing the uptake of 3H-thymidine into nucleic acid of the lymphocytes. Morphine at the estimated clinical blood concentrations did not inhibit lymphocyte blastoid transformation.

Humans↗

Fusobacterium nucleatum T18 aggregates human mononuclear cells and inhibits their PHA-stimulated proliferation.

In previous studies Fusobacterium nucleatum has been shown to induce either stimulatory or inhibitory effects on human mononuclear cells. We examined the interaction of human mononuclear cells with human and cynomolgus monkey strains of F. nucleatum. Peripheral blood mononuclear cells (PBMCs) isolated from normal donors were aggregated in the presence of cells of F. nucleatum but not control bacteria. The aggregation of PBMCs and F. nucleatum T18 was inhibited by either L-arginine, L-lysine, or pretreatment of the bacterial cells with heat, but was unaffected by the presence of sugars or normal human serum. Strain T18 aggregated purified T-cells and monocytes at approximately equal concentrations. When F. nucleatum T18 was incubated with PHA-stimulated PBMCs, DNA synthesis in the PBMCs was significantly inhibited and detection of IL-2R alpha on the PBMCs was reduced. These studies indicate that F. nucleatum aggregates PBMCs, and that this interaction is associated with both an inhibition of PBMC proliferation and a decrease in IL-2 receptor expression. The ability of F. nucleatum to inhibit mononuclear cell proliferation may be significant in the pathogenesis of periodontal diseases.

Animals↗

Carbohydrate metabolism in PHA stimulated human peripheral blood lymphocytes.

Human peripheral blood lymphocytes (PBL) stimulated in vitro by phytohemoagglutinin (PHA) manifest augmented glycolysis and oxidation of glucose-1-14C, indicating an increased utilization of the pentose pathway. Lactic acid production, as index of increased glycolysis, follows the same kinetic of thymidine incorporation and can be easily quantitated by an enzymatic assay.

Carbon Dioxide↗

Survival of osteocompetent marrow cells in vitro and the effect of PHA-stimulation on osteoinduction in composite bone grafts.

The osteoinductive capacities of bone-marrow autografts, washed marrow-free bone allografts, and composite grafts of washed allogeneic bone impregnated with viable or killed autologous marrow cells have been compared in a heterotopic site in rabbits. Composite grafts (similar to 40 mg allogeneic bone) with viable marrow performed equally well as autografts, providing that these implants contained at least 50,000 marrow cells. Composite grafts with dead marrow were noninductive, and grafts with 10,000 viable marrow cells produced much less new bone than grafts with 50,000 cells. Postoperative treatment of host rabbits with PHA-P increased the osteoinductive capacity of composite grafts with initially subcritical numbers of marrow cells to control levels. These results indicate that the osteoinductive cells in composite grafts are mobilized from the marrow elements rather than from the undifferentiated cells in the host bed.

Animals↗

Differential projections to the intralaminar and gustatory thalamus from the parabrachial area: a PHA-L study in the rat.

The organization of projections from the parabrachial (PB) area to the ventral posterior parvicellular (VPpc) "gustatory" and intralaminar nuclei of the thalamus was studied in the rat by using microinjections of Phaseolus vulgaris leucoagglutinin (PHA-L), into subregions of the PB area. The present study is a follow-up of three former studies (Bernard et al. [1993] J. Comp. Neurol. 329:201-229; Aldén et al. [1994] J. Comp. Neurol. 341:289-314; Bester et al. [1997a] J. Comp. Neurol. 383:245-281) that examined PB projections onto the amygdala, the bed nucleus of the stria terminalis, and the hypothalamus. Our data showed that (1) the region centered in the internal lateral PB subnucleus projects densely with a bilateral and symmetric pattern to the caudal portion of the paracentral and, to a lesser extent, to the adjacent portion of the central and parafascicular medial thalamic nuclei; (2) the mesencephalic PB region centered in the ventral lateral subnucleus and scattered neurons in the subjacent brachium conjunctivum project primarily, although diffusely, to the central medial thalamic nucleus. The third region includes two subgroups: (3a) the medial subgroup, including the medial, the waist area, and the ventral lateral subnuclei of the pontine PB area, projects bilaterally but with a weak ipsilateral predominance to the VPpc, terminals bearing large varicosities. Additionally, a diffuse projection with small varicosities spreads in the area between the two VPpc nuclei and the central medial nucleus. (3b) The lateral subgroup, centered in the external medial subnucleus, projects with a contralateral predominance in the periphery of the VPpc nuclei, most terminals being located around the dorsomedial tip. It is suggested that the PB projections to the intralaminar nucleus could be involved in cortical limbic arousal processing in relation with nociceptive, (somatic, visceral, and intraoral) and gustatory aversive stimuli. The projection with large varicosities inside the VPpc could process gustatory discrimination.

Animals↗