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PubMed1975

Studies on the subpopulation and function of peripheral lymphocytes, and inhibitor to PHA stimulation existing in the serum of patients with liver disease.

In our recent report we observed the abnormal response to PHA stimulation in the peripheral lymphocytes of patients with chronic liver diseases. The present report concerns extensive studies of this earlier report; analysis of the subpopulation and function of peripheral lymphocytes in patients with liver diseases. PHA responsiveness in acute hepatitis was reduced in the acute phase and improved during the convalescent stage. In chronic liver disease, half of the patients showed a decrease in T-cell population as well as a reduced response to PHA stimulation. These phenomena were observed more frequently in HBs-antigen positive patients than in HBs-antigen negative cases. Furthermore, we found the existence of an inhibitor to PHA response in the serum of 8 cases among 19 patients with various liver diseases. In patients with such a serum inhibitor, the reactivity of the lymphocyte itself to PHA was considerably higher but the T-cell population was much more decreased than that of inhibitor negative cases.

Acute Disease↗
PubMed1989

Calcitriol inhibits the PHA-induced production of IL-2 and IFN-gamma and the proliferation of human peripheral blood leukocytes while enhancing the surface expression of HLA class II molecules.

1 alpha-dihydroxivitamin D3 [calcitriol; 1,25-(OH)2D3], the most biologically active metabolite of vitamin D3, exerts several effects on peripheral blood mononuclear cells (PBMC). We report here the effects of calcitriol on PBMC proliferation and on the expression of some lymphocyte surface differentiation markers, as well as its action on lymphokine production. Calcitriol inhibited the proliferation of PHA-activated PBMC in a dose-dependent manner, with peak activity at 10(-8) M. Exposure of PHA-stimulated PBMC to 10(-8) M calcitriol for 3 days tended to increase the percent of CD4- and CD8-positive cells, though statistical significance was not reached. A more striking effect of calcitriol was seen on the expression of the non-polymorphic determinants of HLA class II DR molecules; in cultures stimulated with PHA for 3 or 4 days; 10(-8) calcitriol doubled the percent of DR-positive cells as compared to controls treated with PHA alone. This activity peaked at 10(-9) M, a supra-physiologic dose. After 3 days in culture, 10(-8) M calcitriol strongly inhibited the production of both IL-2 and IFN-gamma. This effect was evident at different PHA concentrations (0.5, 1.5 and 3.0 micrograms/ml), and almost disappeared at 10(-10) M. These results underline the immunoregulatory role of calcitriol, but well defined experimental models in vitro are needed for elucidating the relevance of this compound in physiology and, possibly, in therapeutics.

Antigens, Differentiation↗
PubMed2000

Biosynthesis of poly(3-hydroxybutyrate-co-3-hydroxyalkanoates) by recombinant bacteria expressing the PHA synthase gene phaC1 from Pseudomonas sp. 61-3.

Pseudomonas sp. 61-3 accumulated a blend of poly(3-hydroxybutyrate) [P(3HB)] homopolymer and a random copolymer consisting of 3-hydroxyalkanoate (3HA) units of 4-12 carbon atoms. The genes encoding beta-ketothiolase (PhbA(Re)) and NADPH-dependent acetoacetyl-CoA reductase (PhbB(Re)) from Ralstonia eutropha were expressed under the control of promoters for Pseudomonas sp. 61-3 pha locus or R. eutropha phb operon together with phaC1(Ps) gene (PHA synthase 1 gene) from Pseudomonas sp. 61-3 in PHA-negative mutants P. putida GPp104 and R. eutropha PHB(-4) to produce copolyesters [P(3HB-co-3HA)] consisting of 3HB and medium-chain-length 3HA units of 6-12 carbon atoms. The introduction of the three genes into GPp104 strain conferred the ability to synthesize P(3HB-co-3HA) with relatively high 3HB compositions (up to 49 mol%) from gluconate and alkanoates, although 3HB units were not incorporated at all or at a very low fraction (3 mol%) into copolyesters by the strain carrying phaC1Ps gene only. In addition, recombinant strains of R. eutropha PHB(-4) produced P(3HB-co-3HA) with higher 3HB fractions from alkanoates and plant oils than those from recombinant GPp104 strains. One of the recombinant strains, R. eutropha PHB(-4)/ pJKSc46-pha, in which all the genes introduced were expressed under the control of the native promoter for Pseudomonas sp. 61-3 pha locus, accumulated P(3HB-co-3HA) copolyester with a very high 3HB fraction (85 mol%) from palm oil. The nuclear magnetic resonance analyses showed that the copolyesters obtained here were random copolymers of 3HB and 3HA units.

Acyltransferases↗
PubMed1978

PHA-induced cytotoxicity of human lymphocytes against adherent hela cells.

The conditions for a phytohaemagglutinin(PHA)-induced cytotoxicity test of human peripheral blood lymphocytes were investigated. [3H]thymidine prelabelled HeLa cells were used as target cells. Stimulation with 10 microliter PHA/ml during 24 h gave the best measure of lymphocyte cytotoxic capacity. Supernatants of PHA-activated lymphocytes showed no cytotoxicity against adherent HeLa cells. Mitomycin treatment did not influence cytotoxic capacity. Removal of phagocytizing mononuclear cells reduced spontaneous cytotoxicity, but increased PHA-induced cytotoxicity. Adherent cells showed high spontaneous cytotoxicity, with little increase on addition of PHA. The method was evaluated for clinical applicability by testing mononuclear cells from 19 normal subjects and purified lymphocytes from 15 normal subjects. Purified lymphocytes showed a higher PHA-induced cytotoxicity with a smaller variation and greater ratio dependent increase in cytotoxicity than unseparated mononuclear cells. Results with fresh purified lymphocytes were reproducible.

Cell Adhesion↗
PubMedDate not supplied

Age-related refractoriness of PHA-induced lymphocyte transformation. I. Comparable sensitivity of spleen cells from young and old mice to culture conditions.

The decline in phytohemagglutinin (PHA) responsiveness of spleen cells from aged mice was studied in an effort to determine the nature of the depressed PHA reactivity. The reduced response of DNA synthesis (thymidine incorporation) to PHA stimulation could not be attributed to a decreased viability of spleen lymphocytes from old mice in in vitro culture conditions, to different PHA dose requirements, or to longer periods of culture time for old mouse spleen cells to reach maximal activity. Culture of cells for 24-48 h prior to addition of PHA gave patterns of thymidine incorporation similar to those of freshly prepared spleen cells. The elimination of red blood cells by a brief hypotonic shock significantly increased thymidine incorporation in spleen lymphocytes from both yound and old mice.

Age Factors↗
PubMed1987

An economic anterograde axonal tracing method using Phaseolus vulgaris agglutinin (PHA) P-form.

Phaseolus vulgaris leucoagglutinin (PHA-L) has been demonstrated to be an excellent neuroanatomical anterograde tracer. So far there are relatively few applications of this technique, mainly due to the cost of the lectin. Instead of PHA-L, we have successfully used PHA-P, which is a crude and inexpensive form of PHA-L. The sensitivity of the present method, examined in the rat striatonigral pathway, is as high as that of the conventional method. Furthermore, a large amount of PHA-P, injected into the cat eye, demonstrated the retinofugal projection in detail.

Animals↗
PubMed1994

A diet containing the lectin phytohaemagglutinin (PHA) slows down the proliferation of Krebs II cell tumours in mice.

Mice injected intraperitoneally with Krebs II cells and then fed on a diet containing the lectin phytohaemagglutinin (PHA) developed ascites tumours more slowly than mice fed on a control diet. After an 8-day period following injection the number of cells recovered from mice maintained on the PHA diet was half that from those fed the control diet. A switch of diet from control to PHA on day 4 after injection resulted in a large decrease in number of tumour cells recovered. Mice injected s.c. also developed tumours at later times when fed on the PHA diet. A quantitative of ribosomes in polysome-containing fractions showed no major differences in protein synthesis in control mice and those fed the PHA diet.

Animals↗
PubMed2004

The coordinate regulation of pharyngeal development in C. elegans by lin-35/Rb, pha-1, and ubc-18.

Organ development is a complex process involving the coordination of cell proliferation, differentiation, and morphogenetic events. Using a screen to identify genes that function coordinately with lin-35/Rb during animal development, we have isolated a weak loss-of-function (LOF) mutation in pha-1. lin-35; pha-1 double mutants are defective at an early step in pharyngeal morphogenesis leading to an abnormal pharyngeal architecture. pha-1 is also synthetically lethal with other class B synthetic multivulval (SynMuv) genes including the C. elegans E2F homolog, efl-1. Reporter analyses indicate that pha-1 is broadly expressed during embryonic development and that its functions reside in the cytoplasm. We also provide genetic and phenotypic evidence to support the model that PHA-1, a novel protein, and UBC-18, a ubiquitin-conjugating enzyme that we have previously shown to function with lin-35 during pharyngeal development, act in parallel pathways to regulate the activity of a common cellular target.

Animals↗
PubMed1976

Demonstration of the production of human thyroid-stimulating immunoglobulins (HTSI) by Graves' lymphocytes cultured in vitro with phytohaemagglutinin (PHA).

The lymphocytes from patients with Graves' disease or from healthy subjects have been cultured in vitro either alone or with phytohemagglutinin (PHA). After six days the culture supernatants have been assayed for their human thyroid-stimulating activity by measuring increases in adenosine 3',5' monophosphate (cyclic AMP) in human thyroid slices with which the supernatants have been incubated. Significant levels of human thyroid stimulator activity were found in the culture in which Graves' lymphocytes were cultured with PHA. This activity has been abolished by precipitation of the IgG from the culture supernatant with goat antihuman IgG serum. In contrast, when Graves' lymphocytes were cultured alone, or when control lymphocytes were cultured either alone or with PHA, there was no overall significant production of human thyroid-stimulating immunoglobulin (HTSI). It is concluded that Graves' lymphocytes can be stimulated by PHA to produce HTSI in vitro. Since PHA is known to stimulate only the T lymphocytes, which do not themselves elaborate immunoglobulins as the B lymphocytes do, the above observations indicate a cooperation between T and B lymphocytes in the production of HTSI, at least in this system.

Cells, Cultured↗
PubMedDate not supplied

Characterisation of serotonin transport mechanisms in rainbow trout peripheral blood lymphocytes: role in PHA-induced lymphoproliferation.

In this study, we investigated the serotonin transport mechanisms in rainbow trout (Oncorhynchus mykiss) peripheral blood Lymphocytes. We have observed that the transport of serotonin is a membrane transport process that have the properties of a secondary active transport system. The binding isotherm of [3H]-paroxetine, a serotonin transport blocker, demonstrated a high-affinity binding site with a positive type of cooperativity, Hill coefficient being higher than unity. Known specific inhibitors of the mammalian serotonin transporter significantly inhibited the uptake process in fish lymphocytes. In order to demonstrate the physiological relevance of the serotonin transporter in T-cell activation, we conducted experiments on lymphocytes activated or not by phytohemagglutinin (PHA), a T-cell mitogen. We have observed that addition of PHA for 24hrs, increased the Vmax but not the Km of this transporter. Serotonin uptake inhibitors diminished the PHA-activated proliferation of fish lymphocytes. The intracellular concentrations of cAMP were found to regulate the serotonin uptake and the PHA-stimulated proliferation as the agents known to augment cAMP stimulated serotonin uptake, and inhibited the lymphoproliferation. Inhibitory effects of increased cAMP on the proliferation were reversed by the addition of the nanomolar concentrations of 8-OH-DPAT, a 5-HT1A receptor agonist which is known to diminish the intracellular cAMP concentrations, suggesting that serotonin also regulates PHA-induced proliferation via 5-HT1A membrane receptors in an autocrine manner. These results all together demonstrate that fish lymphocytes possess an active serotonin transporter that is implicated in the proliferation of these immunocompetent cells.

Animals↗
PubMed1998

Formation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) by PHA synthase from Ralstonia eutropha.

The acetoacetyl-CoA reductase and the polyhydroxyalkanoate (PHA) synthase from Ralstonia eutropha (formerly Alcaligenes eutrophus) were expressed in Escherichia coli, Klebsiella aerogenes, and PHA-negative mutants of R. eutropha and Pseudomonas putida. While expression in E. coli strains resulted in the accumulation of poly(3-hydroxybutyrate) [PHB], strains of R. eutropha, P. putida and K. aerogenes accumulated poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) [poly(3HB-co-3HHx)] when even chain fatty acids were provided as carbon source, and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [poly(3HB-co-3HV)] when odd chain fatty acids were provided as carbon source. This suggests that fatty acid degradation can be directly accessed employing only the acetoacetyl-CoA reductase and the PHA synthase. This is also the first proof that the PHA synthase from R. eutropha can incorporate 3-hydroxyhexanoate (3HHx) into PHA and has, therefore, a broader substrate specificity than previously described.

3-Hydroxybutyric Acid↗
PubMed2001

Low-dose intragastric administration of Phaseolus vulgaris agglutinin (PHA) does not induce immunoglobulin E (IgE) production in Sprague-Dawley rats.

Native Phaseolus vulgaris agglutinin (PHA) poses a potential health threat, when ingested with improperly cooked red kidney beans. Since PHA triggers human basophilic granulocytes in culture to rapidly release considerable amounts of interleukin-(IL-)4 and IL-13, key cytokines for inducing immunoglobulin E (IgE) production, the question was addressed whether this lectin can evoke in vivo IgE production. IgE-low-responder (Sprague-Dawley) rats received PHA (6 mg/rat/day) intragastrically by gavage over a period of 10 days. Up to day 35, there was no IgE induction regardless of whether the animals were boostered subcutaneously with PHA or not, indicating that PHA cannot be regarded as a general IgE inducer in rats.

Animals↗
PubMed2001

Melatonin inhibits PHA-stimulated chicken lymphocyte proliferation in vitro.

Many studies have shown that melatonin plays a fundamental role in neuroimmunomodulation. There are known differences between mammals and birds in immunomodulatory function of melatonin exerted in vivo. In present study the effect of exogenous melatonin on chicken lymphoid cell proliferation in vitro was examined. Melatonin alone (10(-10)M-10(-5)M) did not exert any effect on the proliferation of the chicken thymocytes, splenocytes and lymphocytes from the bursa of Fabricius. On the other hand, melatonin addition strongly inhibited the proliferation of PHA-activated thymocytes and splenocytes from young chickens. The effect of melatonin was hormone- and mitogen-(PHA) dose-dependent; the most pronounced effect was obtained at low PHA and melatonin concentrations i.e., 1.6-3.125 microg/100 microl of PHA and 10(-10) M-10(-7) M melatonin, respectively. Prior immunization of chickens with sheep red blood cells (SRBC) inhibited the proliferative response of splenocytes to the same dose of PHA and, consequently, the effect of melatonin addition was not observed. The results reported demonstrate a direct inhibitory effect of melatonin on in vitro activated chicken lymphocytes.

Animals↗
PubMed1997

PHA synthase activity controls the molecular weight and polydispersity of polyhydroxybutyrate in vivo.

A synthetic operon for polyhydroxyalkanoate (PHA) biosynthesis designed to yield high levels of PHA synthase activity in vivo was constructed by positioning a genetic fragment encoding beta-ketothiolase and acetoacetyl-CoA reductase behind a modified synthase gene containing an Escherichia coli promoter and ribosome binding site. Plasmids containing the synthetic operon and the native Alcaligenes eutrophus PHA operon were transformed into E. coli DH5 alpha and analyzed for polyhydroxybutyrate production. The molecular weight of polymer isolated from recombinant E. coli containing the modified synthase construct, determined by multiangle light scattering, was lower than that of the polymer from E. coli containing the native A. eutrophus operon. A further decrease in polyester molecular weight was observed with increased induction of the PHA biosynthetic genes in the synthetic operon. Comparison of the enzyme activity levels of PHA biosynthetic enzymes in a strain encoding the native operon with a strain possessing the synthetic operon indicates that the amount of polyhydroxyalkanoate synthase in a host organism plays a key role in controlling the molecular weight and the polydispersity of polymer.

Acyltransferases↗
PubMed1999

Effect of insulin-like growth factor 1 on PHA-stimulated cord blood mononuclear cell telomerase activity.

Telomerase may contribute to the capacity for cell replication by compensating for the loss of telomere length. Exploring the use of biological modifiers in increasing cellular replicative potential through telomerase activity may be useful for in vitro expansion of haemopoietic stem cells for transplantation or lymphoid cells for adoptive immunotherapy. In this study we showed for the first time that insulin-like growth factor 1 (IGF-1) modulated telomerase activity in human cord blood mononuclear cells (MNC) and some of the known functional determinants of telomerase activity. We found that cord blood MNC expressed constitutively a low level of telomerase activity and human telomerase reverse transcriptase (hTRT) mRNA, and a high level of human telomerase RNA component (hTR) and telomerase-associated protein-1 (TP1) mRNA. Interestingly, IGF-I alone did not increase the telomerase activity of cord blood MNC but could enhance the PHA-induced increase in telomerase activity. These alterations in telomerase activity were not completely in phase with those of proliferation response. On the other hand, IGF-I did not alter hTRT mRNA expression but enhanced the PHA-induced increase in hTRT whereas TP1 mRNA expression was stimulated by either IGF-I or PHA but showed no additive increase when stimulated by both IGF-1 and PHA. Neither IGF-1 nor PHA altered hTR expression. Finally, the temporal dynamics of hTRT mRNA expression and telomerase activity in cord blood MNC over 5 d in culture were not totally concordant. suggesting that key factors other than hTRT were involved in regulating telomerase activity in cord blood MNC. The modulatory effect of IGF-1 on telomerase activity supports its potential role in increasing replicative potential of cord blood lymphoid cells or haemopoietic stem cells.

Cell Division↗
PubMed2001

PHA-induced IL-12R beta(2) mRNA expression in atopic and non-atopic children.

BACKGROUND: IL-12 is a strong inducer of Th1 responses. Stimulation via the CD2 receptor increases IFN-gamma production and enhances the responsiveness of activated T-cells to IL-12, possibly due to an up-regulation of the signal transducing beta(2) chain of the IL-12 receptor (IL-12R beta(2)). Atopic children have a reduced Th1-like immunity and a reduced CD2 expression. Our hypothesis is that atopic individuals have a reduced function of the CD2 pathway, causing reduced responsiveness to IL-12 and decreased IFN-gamma production. OBJECTIVE: The aim was to study the mRNA expression of the IL-12R beta(2) chain, after stimulation via the CD2 pathway in peripheral blood mononuclear cells (PBMC), of atopic and non-atopic children, and to investigate correlations to the production of Th1 and Th2 cytokines. MATERIALS AND METHODS: The study included 23 skin prick test positive, and 9 non-sensitized, 12-year-old children. PBMC were stimulated for 24 h with phytohemagglutinin (PHA) (2 microg/mL), which stimulates T cells through the CD2 pathway. Expression of IL-12R beta(2) mRNA was analysed by quantitative real time PCR and the cytokine production was detected with ELISA. RESULTS: Atopic and non-atopic children had similar baseline expression of IL-12R beta(2) mRNA, whereas PHA-induced IL-12R beta(2) mRNA expression was lower in atopic than in non-atopic children. The PHA-induced IL-12R beta(2) mRNA expression correlated well with the PHA-induced IFN-gamma production and with the IFN-gamma/IL-4 ratio. CONCLUSION: PBMC from atopic children expressed less IL-12R beta(2) mRNA than non-atopic children after stimulation via the CD2 pathway (PHA). This may indicate a reduced capacity to respond to Th1-inducing stimuli in atopic children.

Child↗
PubMed2001

Competition of IL-1 and IL-1ra determines lymphocyte response to delayed stimulation with PHA.

BACKGROUND: Human peripheral blood mononuclear cells (PBMC) left in microcultures for 24h without mitogen do not respond to subsequent stimulation with PHA. They regain reactivity if the native culture medium is absorbed with other party lymphocytes or partially replaced with the medium from a PHA-stimulated culture. The observations suggest that, during the incubation, some inhibitory agent had accumulated in the culture medium. AIM: The study was performed to determine the nature of the observed phenomenon in respect of the possible role of monocytes and their products IL-1 and IL-1 receptor antagonist (IL-1ra), and to test for immunodiagnostic purposes the significance of quantifying the lymphocyte response to delayed stimulation with PHA in patients suffering from inflammatory prosesses. METHODS: Lymphocyte response to delayed stimulation with PHA, calculated as the lymphocyte-monokine interaction (LM) index, was determined in the microcultures of PBMC isolated from the blood of healthy donors or of patients with acute tonsilitis. The values of LM indices were compared with the ratios of IL-1ra/IL-1beta concentration estimated by enzyme-linked immunosorbent assay method in the culture supernatants. The influences of exogenous IL-1beta, IL-1ra, anti-IL1ra antibodies and antibiotic cefaclor on the monokine concentrations and on the values of LM index were tested. RESULTS AND CONCLUSIONS: The results show that the level of lymphocyte response to delayed stimulation with PHA (LM index) is inversely proportional to the ratio of IL-1ra/IL-1beta concentration in the culture. The low LM values at high IL-1ra/IL-1beta ratios in PBMC cultures from healthy donors, reversed proportions found in patients' PBMC (acute tonsilitis), and the cefaclor-induced reduction of LM value with correlated increase of the IL-1ra/IL-1beta ratio suggest that the LM assay may prove to be useful for immunodiagnostic purposes.

Adult↗
PubMed1986

Regulation of human gamma-interferon and beta-interferon gene expression in PHA-activated lymphocytes.

Two interferon (IFN) messengers were synthesized in phytohemagglutinin (PHA)-activated lymphocytes: IFN-gamma mRNA and a messenger hybridizing with the IFN-beta 2 probe. They were induced rapidly and declined at a stage when overall RNA was still efficiently transcribed. The IFN-beta 2 mRNA (15S) present in the lymphocytes was slightly different from its fibroblastic counterpart (14S). The kinetics of the accumulation and decay of both lymphocyte IFN messengers differed when assessed by hybridization with the two IFN probes, IFN-gamma mRNA was not detected before mitogenic activation and accumulated for up to 15 h postactivation, while IFN-beta 2 mRNA accumulated even in the absence of PHA activation for up to 5 h, even though the activation raised the IFN-beta 2 mRNA level at 5 h. The disappearance of IFN messengers was prevented when cycloheximide was added 5 h after PHA activation, when the transcription of both messengers had already been turned on, suggesting the presence of the repressor mechanism proposed for IFN-beta 1 and IFN-beta 2 mRNAs in fibroblasts. In the absence of PHA activation, cycloheximide did not induce IFN-beta 2 mRNA transcription as it did in fibroblasts and moreover prevented the accumulation of the messenger observed in the control cells. In contrast to IFN-beta 2 mRNA, cycloheximide treatment of lymphocytes produced a slight accumulation of IFN-gamma mRNA. This accumulation was already detectable 6 h posttreatment and its level remained unchanged for up to 24 h. Addition of actinomycin D, 5 h after PHA activation, did not impair the shut off and accelerated the decay of IFN messengers.

Cells, Cultured↗