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PubMed1983

Mouse alloantibodies capable of blocking cytotoxic T cell function. IV. Comparative analysis of the blocking effects of anti-Lyt-2 and anti-H-2 antibodies on allogeneic and PHA-mediated killing.

Blocking of cell-mediated lympholysis (CML) by anti-Lyt-2 antibodies was compared with that by anti-H-2 antibodies which most likely inhibit CML by blocking antigen recognition by cytotoxic T lymphocytes (CTLs). Both antibodies were shown to inhibit the early Mg2+-dependent process of killing. Moreover, the anti-H-2-sensitive event was found to be reversible by the antibody as was the case with the anti-Lyt-2-sensitive event, suggesting that the two antibodies block the same event taking place during the Mg2+-dependent stage. Both types of antibody were also shown to be capable of inhibiting the phytohemagglutinin (PHA)-mediated non-specific killing activity of CTLs. However, in the case of anti-Lyt-2 antibodies, available monoclonal antibodies failed to inhibit PHA-mediated killing whereas conventional antisera did. The results thus suggest multiplicity and heterogeneity of Lyt-2 determinants or the existence of multiple products of Lyt-2-linked genes. In addition, an anti-H-2 antiserum also exerted a specific inhibitory effect on PHA-mediated killing. Thus there appears to be a general requirement for involvement of the Lyt-2 molecules on CTLs and major histocompatibility complex (MHC) products on the target cells. The implications of these observations are discussed.

Animals↗
PubMed1978

Use of an antiserum specific for human T lymphocytes as a diagnostic reagent: comparison with E rosette formation and mitogenic response to PHA.

An antiserum with specificity for human T lymphocytes was evaluated as a diagnostic reagent in the clinical immunology laboratory. The antiserum was used in indirect immunofluorescence to detect T cells in blood samples from normal controls and patients with various disorders involving the immune system. The same samples were also examined using two established tests for T cells, the rosette reaction with sheep red blood cells (E rosette assay) and the proliferative response to phytohaemagglutinin (PHA). The antiserum and the rosette assay detected approximately the same numbers of T cells in controls, but the antiserum indicated T cell deficiency in several patients who appeared normal by rosetting or PHA reactivity. PHA stimulation in autologous plasma was depressed in a further group of patients who had normal T cell numbers by the other two tests. In vitro experiments with normal lymphocytes indicated that the E-rosette receptor was distinct from determinants detected by the T-cell specific antiserum. The immunofluorescence test with the anti-T serum provides an additional assay which appears to be more sensitive than the other two tests in detecting certain cases of T lymphocyte deficiency.

Antibody Specificity↗
PubMed1992

Immunosuppressive property of a very high purity antihaemophilic preparation: a low molecular weight component inhibits an early step of PHA induced cell activation.

Immune deficiency has been reported in haemophiliac patients receiving antihaemophilic factor VIII preparations, but the mechanisms involved in the immunosuppression are not fully understood. By using the proliferative response of peripheral blood mononuclear cells to phytohaemagglutinin (PHA) as a test system, we investigated the inhibitory influence of a very high purity antihaemophilic factor (AHF) preparation on T cell proliferation and on T lymphocyte activation molecules. We observed that this preparation reduced significantly the PHA-induced mononuclear cell proliferation, independently of the monocyte concentration. The AHF preparation did not act through a cytotoxic mechanism or a steric hindrance of PHA. The AHF preparation had no effect on the immediate expression of T lymphocyte activation molecules such as CD54 (ICAM-1). In contrast, the very high purity AHF reduced the induced expression of two early T cell activation molecules: CD25 (interleukin-2 receptor) and CD71 (transferrin receptor). The very high purity AHF also had the capacity to inhibit the up-regulation of two late activation antigens, CD38 and CD11a/CD18, and to inhibit the induced expression of HLA-DR molecule, defined also as a late T cell activation molecule. The CD45R expression level, used as a control marker, was not changed after AHF exposure. The very high purity AHF therefore influenced an early step of cell proliferation. We have also shown that the immunoregulatory properties of the preparation were not restricted to the factor VIII itself, but resulted from the presence of dialysable and low molecular weight components in the preparation.

Antigens, CD↗
PubMed1982

Mechanisms of phytohaemagglutinin (PHA) stimulation of normal human lymphocytes: 'trigger' 'push' or both?

The growth in volume of human peripheral blood lymphocytes after stimulation with various concentrations of PHA was measured with an electronic particle counter. The percentage of growing cells and averaged values describing their growth rates during the elapsed period of culture were estimated by fitting to the observed data the volume distributions derived from a mathematical model. With sub-optimal doses, the percentage of cells stimulated , and their incremental growth rate, increased with increasing dose of PHA, but the time-course of recruitment into the G1-phase was similar with all PHA concentrations studied. The results provide strong support for the 'trigger' hypothesis that there is a distribution of stimulation thresholds within the lymphocyte population: consequently, increasing mitogen concentration will be expected to result in increased numbers of responding cells within the suboptimal concentration range.

Adult↗
PubMed1992

Altered proliferative kinetics in PHA-activated human T-lymphocytes treated with the anti-HLA class I monoclonal antibody 01.65.

Anti-HLA class I monoclonal antibody (mAb) 01.65 inhibited phytohaemagglutinin (PHA)-induced human lymphocyte proliferation. The inhibitory effect was inversely correlated to the strength of the proliferative response. It was increased when lymphocytes were stimulated with suboptimal doses of PHA but it disappeared with supraoptimal doses. Proliferation inhibition was achieved by prolonging the cell cycle time and by slowing down its recruitment rate. The former effect was not restricted to the G1-phase but also included the S phase. These results support the idea that HLA class I molecules are important in the PHA-induced proliferation of human T-lymphocytes.

Antibodies, Monoclonal↗
PubMed1975

Deficient lymphoid cell-mediated, PHA-induced cytotoxicity in rheumatoid arthritis patients.

Lymphoid cells from patients with rheumatoid arthritis were compared with those from healthy blood donors and from nonrheumatoid arthritis patients for the ability to manifest in vitro cytotoxicity against target cells in the presence of phytohemagglutinin (PHA) or anti-target cell antibodies. The PHA-induced cytotoxicity in the rheumatoid patient group was significantly lower than that of the blood donors (P less than 0.01) and of the nonrheumatoid patients (P less than 0.05). The rheumatoid arthritis patients appeared to fall into two groups, one with normal and one with distinctly subnormal PHA-induced cytotoxicity. No obvious differences were observed betes, or the proportion in peripheral blood of T lymphocytes (E-RFC) or Fc-receptor-bearing lymphocytes (EA-RFC). There were no significant differences between the groups with regard to antibody-dependent cytotoxicity.

Antibodies, Anti-Idiotypic↗
PubMed1999

Co-expression of 3-ketoacyl-ACP reductase and polyhydroxyalkanoate synthase genes induces PHA production in Escherichia coli HB101 strain.

The Escherichia coli 3-ketoacyl-ACP reductase gene (fabGEc) was cloned using a PCR technique to investigate the metabolic link between fatty acid metabolism and polyhydroxyalkanoate (PHA) production. Three plasmids respectively harboring fabGEc and the poly-3-hydroxyalkanoate synthesis genes phaCAc and phaC1Ps from Aeromonas caviae and Pseudomonas sp. 61-3 respectively were constructed and introduced into E. coli HB101 strain. On a two-stage cultivation using dodecanoate as the sole carbon source, recombinant E. coli HB101 strains harboring fabGEc and phaC genes accumulated PHA copolymers (about 8 wt% of dry cell weight) consisting of several (R)-3-hydroxyalkanoate units of C4, C6, C8, and C10. It has been suggested that overexpression of the fabGEc gene leads to the supply of (R)-3-hydroxyacyl-CoA for PHA synthesis via fatty acid degradation.

3-Oxoacyl-(Acyl-Carrier-Protein) Reductase↗
PubMed1987

Isoprinosine enhances PHA responses and has potential effect on natural killer cell (NK) activity of uremic patients in vitro.

We studied the effect of an immuno-stimulating agent, isoprinosine, which is being marketed as an antiviral drug, on some immune functions in vitro in 14 uremic patients treated by hemodialysis and in 10 healthy controls. PHA and PPD induced stimulations of DNA synthesis and interleukin-2 (Il-2) production and NK cell activity were measured from peripheral blood mononuclear cells cultured with and without isoprinosine. PHA responses were enhanced by isoprinosine (100 micrograms/ml) both in the patient group (p less than 0.001) and in the control group (p less than 0.05) but PPD responses in neither. The enhancement of PHA responses was not due to an increased production of Il-2(T-cell growth factor). Isoprinosine augmented NK activity in those patients whose NK activity was initially low. No enhancement could be seen in the controls or in those patients whose NK activity was comparable to that of the controls. Our results motivate a clinical study with isoprinosine in uremic patients at the risk of virus infection.

Adjuvants, Immunologic↗
PubMed1979

Quantitation of lymphocyte response to PHA by flow cytofluorometry. III. Heterogeneity of induction period.

Early events in phytohaemagglutinin (PHA) stimulation of mouse splenocytes have been quantitated by using flow cytometry and supravital staining with acridine orange (AO). Increasing percentages of single cells with increased metachromatic (red) AO staining were demonstrated in cultures stimulated by PHA for up to 24 hr. These differences in staining could be eliminated by fixation with 1:1 ethanol/acetone before staining. Stimulated cells showed an increase in nonspecific esterase activity as measured by flow cytometry after supravital staining with fluorescein diacetate (FDA). The data reported show a heterogeneity in the per cell response of mouse splenocytes to PHA. The relationship between these data and the mechanism of mitogen stimulation is discussed.

Acridine Orange↗
PubMed1981

PHA and PPD reactivity of lymphocytes in pleural effusions.

The functional properties of lymphocytes in pleural fluid were studied in 23 patients admitted to the hospital for the diagnostic evaluation of a unilateral or bilateral pleural effusion. The in vitro reactivities of the patients' pleural fluid lymphocytes and peripheral blood lymphocytes to phytohemagglutinin (PHA) and to purified protein derivative (PPD) were compared. In most patients with pleural effusion, the function of pleural fluid lymphocytes was intact and comparable to that of lymphocytes from peripheral blood. The PHA- and PPD-stimulated 3H-thymidine incorporation by peripheral blood lymphocytes did not differ significantly from that by pleural fluid lymphocytes in any of the patient groups. Similarly, the comparison of PHA- and PPD-stimulated 3H-thymidine uptake by peripheral blood or pleural fluid lymphocytes from patients with pleural effusions of various causes revealed no significant differences. In vitro reactivity to PPD by peripheral blood and pleural fluid lymphocytes correlated positively with the in vivo intradermal reactivity to PPD.

Adult↗
PubMed1975

Inhibitor of PHA-induced lymphocyte blastformation generated from maternal lymphocytes during early pregnancy.

The supernatant of phytohemagglutinin (PHA)-stimulated lymphocyte culture derived from pregnant women in the first two trimesters inhibited DNA synthesis of normal human lymphocytes after PHA induction. This soluble factor was heat-labile and the same factor could not be found in the supernatant generated from lymphocytes of pregnant women in 3rd trimester as well as from those of the non-pregnant. In contrast, the heatstable factor in the supernatant generated from lymphocytes of non-pregnant women in all trimesters contained an stimulating factor on the DNA synthesis of normal human lymphocytes after PHA induction. An endogenous alternation in lymphocyte competence or an alternation in subpopulation of lymphocytes was suggested at the early stage of pregnancy.

Female↗
PubMed1985

The effect of angiotensin II on human mononuclear cell reactivity: suppression of PHA-P-induced thymidine incorporation.

Recent evidence suggests that angiotensin II may participate in the regulation of inflammation. We therefore studied the effects of angiotension II on human peripheral blood mononuclear cell reactivity. Peripheral blood mononuclear cell suspensions stimulated with PHA-P revealed decreased thymidine incorporation when simultaneously cultured for 48 or 72 hours with angiotensin II. Experiments were next conducted to determine whether angiotensin II acted directly on lymphocytes. Complete monocyte depletion (greater than 99.5%) resulted in low PHA-P-induced reactivity which was still inhibited by the addition of angiotensin II. Purified (greater than 99.5%) lymphocytes incubated with angiotensin II prior to reconstitution of mixed mononuclear cell suspensions led to reduced PHA-P-stimulated thymidine incorporation. These findings suggest that angiotensin II can effect mononuclear cell uptake possibly through actions on the lymphocyte population.

Adult↗
PubMed1979

In vitro effect of an acute nonspecific inflammatory exudate on tritiated-thymidine incorporation by unstimulated and PHA-stimulated spleen cells.

The effect of an acute nonspecific inflammatory exudate with mitogenic activity for macrophages in culture has been tested on the spontaneous and PHA-induced DNA synthesis by spleen cells in vitro. Stimulatory effect of this exudate was observed on the spontaneous DNA synthesis which was detectable over a range of 1:4 to 1:4096 concentrations. After optimal PHA stimulation, an inhibition of mitogen-induced DNA synthesis was observed when the cells were exposed to the highest concentrations (up to 1:128) of the exudate. Thereafter, the phenomenon could be reversed and the stimulation was maximal at the concentration of 1:2048. When a suboptimal dose of PHA was used, the stimulatory effect was more pronounced and detected from 1:8 up to 1:4096 concentrations.

Animals↗
PubMed1992

[Blastogenesis of splenic lymphocytes to specific antigens and PHA in Paragonimus westermani infected mice].

Paragonimus westermani is a common fluke in Korea. The present study aimed to observe the cell mediated immune response in experimental paragonimiasis of mice. The mouse (BALB/c) was orally inoculated with 40 metacercariae of P. westermani from Cambaroides similis. During the infection (1, 2, 4, 6 weeks) of mouse, blastogenic response of splenic lymphocytes to P. westermani adult antigen, metacercaria antigen, and PHA were observed. Sera from infected and noninfected mice added to normal mouse splenic lymphocytes with or without PHA. The blastogenic response of splenic lymphocytes to PHA was reduced after 1 week of infection. However after 6 weeks of infection, the response was restored to the control level. The blastogenic response of splenic lymphocytes to P. westermani adult or metacercaria antigen increased significantly on 1 week after infection, and maintained up to 6 weeks after infection. The response of non-infected mice was suppressed by addition of the infected mouse serum. The present results suggested that cellular immunity was involved in P. westermani infected mice and that P. westermani anti-serum inhibited proliferation of T lymphocytes.

Animals↗
PubMed1976

Synergistic effect of cortisol and prostaglandin E2 on the PHA response. Relation to immunosuppression induced by trauma.

Surgical and thermal trauma in man are followed by depressed immunological responses in vivo and reduced lymphocyte reactivity in vitro. The possibility that these are related to trauma-induced rises in tissue levels of cortisol and prostaglandins was examined by studying the effect of a wide range of concentrations of cortisol and prostaglandin E2 (PGE2), separately and together on the phytohaemagglutinin (PHA) response of human peripheral blood lymphocytes. These effects were plotted on two-dimensional dose:effect graphs; the shapes of the curves connecting combinations of equal effect (isoboles) showed that these agents acted with marked synergy in suppressing the response, provided they were present while the response was taking place. Synergy was also shown by using a simple equation relating the concentrations of the agents producing a given effect when used in combination to the concentrations needed to produce the same effect when used separately. Cortisol at concentrations reached in the peripheral blood after trauma in man (1-4 X 10(-6)M) and PGE2 at concentrations to be expected in traumatized tissues (up to 4 X 10(-7)M) each suppressed the response only slightly. The former reduced the response to 0-7 of controls and the latter 0-5 (means of seven subjects). When both were present together at these concentrations, the response was markedly depressed (mean 0-06, range 0-02--0-13 of controls). However, when lymphocytes were incubated at 37 degrees C with cortisol and PGE2 for 20 hr and then washed before exposure to PHA, the response was not inhibited, even by substantially higher concentrations than the above, and was usually moderately enhanced. Therefore, these in vitro experiments do not explain the depressed PHA response observed in peripheral blood lymphocytes after trauma. It is possible, however, that raised cortisol and prostaglandin levels depress the reactivity of lymphocytes while they remain in the traumatized region and its lymph drainage area.

Adult↗
PubMed1999

Morphology of single pontine reticulospinal axons in the lumbar enlargement of the cat: a study using the anterograde tracer PHA-L.

The fine morphology of single pontine reticulospinal axons in the lumbar enlargement was investigated by using an anterograde Phaseolus vulgaris-leucoagglutinin (PHA-L) tracing technique. Localized injections of PHA-L were made into the nuclei reticularis pontis oralis and caudalis in four cats. Following survival periods of 8-9 weeks, PHA-L-labeled axons were found throughout the lumbar enlargement from segments L4 to S2, in which the diameter of labeled axons was 0.6-2.5 microns. From serial transverse sections (50 microns), trajectories of 21 single pontine reticulospinal axons were traced in continuity over distances of 18.9-36.3 mm, corresponding to three to six segments, respectively. All the identified axons gave off multiple (two to nine) axon collaterals along their courses, with mean intercollateral distances of approximately 5-6 mm. Detailed reconstruction of the collateral arborization in the lumbar enlargement showed a high degree of similarity to that of single axons in the cervical enlargement previously reported (Matsuyama et al. [1997] J. Comp. Neurol. 377:234-250). First, axon collaterals arising from a majority (n = 18) of identified axons innervated the gray matter unilaterally, ipsilateral to the parent axons, whereas those from the remaining three axons innervated the gray matter bilaterally. Second, collateral projections terminated mainly in laminae VIII and VII, with the arborization field confined to a narrow rostrocaudal extent (< 1 mm). Third, the termination fields of axon collaterals arising from a given reticulospinal axon were similar at each segmental level and differed from one stem axon to another. These results suggest that the long descending pontine reticulospinal pathway is composed of different types of axons that may innervate the cervical and lumbar enlargements in continuity in a similar manner.

Animals↗
PubMed1976

[Local reaction to the intradermal injection of PHA in healthy persons and in chronic lympholeukosis patients].

Phytohemagglutinin (PHA) that stimulates the lymphocytes to blast transformation in conditions of cellular cultures provokes local skin reaction, when applied intradermally. At the beginning that reaction is manifested with hypermia at the place of the application and later an infiltration is formed. In healthy subjects the infliltration reaches maximal dimensions on the 48th to the 72nd hour post PHA injection. The presence of lymphocytes was established with the cytologic examination of the material from that infilitration at the 24th hour, lymphoblasts and hyperbasophilic blast cells at the 48th and 72nd hour and single reactive lymphocytes were observed at the 96th hour. The infiltration appears later in patients with quiet development of chronic leukosis, only after the 96th hour and is with smaller demensions. Negligible infiltration is observed in malignant leukosis, its progress in time does not differ from that in healthy subjects. However, skin reaction in intraderma injection of PHA gives a certain orientation about the immune organism reactivity and in case of 1 chronic leaukosis--about the form and severity of the disease.

Adult↗
PubMed2000

Different expression profiles of human cyclin B1 in normal PHA-stimulated T lymphocytes and leukemic T cells.

BACKGROUND: In a previous work, we demonstrated with flow cytometry (FCM) methods that accumulation of human cyclin B1 in leukemic cell lines begins during the G(1) phase of the cell cycle (Viallard et al. , Exp Cell Res 247:208-219, 1999). In the present study, FCM was used to compare the localization and the kinetic patterns of cyclin B1 expression in Jurkat leukemia cell line and phytohemagglutinin (PHA)-stimulated normal T lymphocytes. METHODS: Cell synchronization was performed in G(1) with sodium n-butyrate, at the G(1)/S transition with thymidine and at mitosis with colchicine. Cells (leukemic cell line Jurkat or PHA-stimulated human T-lymphocytes) were stained for DNA and cyclin B1 and analyzed by FCM. Western blotting was used to confirm certain results. RESULTS: Under asynchronous growing conditions and for both cell populations, cyclin B1 expression was essentially restricted to the G(2)/M transition, reaching its maximal level at mitosis. When the cells were synchronized at the G(1)/S boundary by thymidine or inside the G(1) phase by sodium n-butyrate, Jurkat cells accumulated cyclin B1 in both situations, whereas T lymphocytes expressed cyclin B1 only during the thymidine block. The cyclin B1 fluorescence kinetics of PHA-stimulated T lymphocytes was strictly similar when considering T lymphocytes blocked at the G(1)/S phase transition by thymidine and in exponentially growing conditions. These FCM results were confirmed by Western blotting. The detection of cyclin B1 by Western blot in cells sorted in the G(1) phase of the cell cycle showed that cyclin B1 was present in the G(1) phase in leukemic T cells but not in normal T lymphocytes. Cyclin B1 degradation was effective at mitosis, thus ruling out a defective cyclin B1 proteolysis. CONCLUSIONS: We found that the leukemic T cells behaved quite differently from the untransformed T lymphocytes. Our data support the notion that human cyclin B1 is present in the G(1) phase of the cell cycle in leukemic T cells but not in normal T lymphocytes. Therefore, the restriction point from which cyclin B1 can be detected is different in the two models studied. We hypothesize that after passage through a restriction point differing in T lymphocytes and in leukemic cells, the rate of cyclin B1 synthesis becomes constant in the S and G(2)/M phases and independent from the DNA replication cycle.

Blotting, Western↗