Immunomodulating activity of p-hydroxyphenyllactic and ascorbic acids

p-Hydroxyphenyl lactic acid (PHA) in a concentration of 5 . 10(-5) M produced a significant inhibition of cell proliferation in response to alloantigens in a one-way mixed lymphocyte culture (MLC) in colonic cancer patients and in blast transformation in response to suboptimal doses of Con A. Multiple administration of ascorbic acid in an optimal concentration to the culture increased the proliferative response of lymphocytes to alloantigens and Con A. PHA and ascorbic acid did not exhibit any immunomodulating action during the use of healthy donors' lymphocytes or lymphocytes from colonic cancer patients, transformed with optimal mitogen doses. PHA did not affect the production of cytotoxic T lymphocytes in the MLC of the spleens of allogeneic mice but inhibited lymphocyte proliferation in response to alloantigens in the MLC of the spleens obtained from B6 and vitamin A deficient animals.     

Correction of biochemical and immunological indices in colonic cancer using optimal doses of retinyl acetate and ascorbic acid

Blood plasma retinol level in normal donors and patients with colonic carcinoma was measured by high-pressure liquid chromatography and the concentration of p-hydroxyphenyl lactic and homogentizine acids by Gas Chromatograph Mass Spectrometer MAT-311A using 2H4-p-hydroxyphenylacetic acid as internal reference. The functional activity of lymphocytes was estimated from the proliferative response to alloantigens in a one-way mixed lymphocyte culture and in blast transformation reaction to Con A and pokeweed mitogen. After systematic intake of retinyl acetate and ascorbic acid in optimally high doses, the patients manifested an increase in vitamin C level in plasma and lymphocytes and a lowering of p-hydroxyphenyl lactic acid excretion. Blood plasma retinol remained unchanged. Daily intake of retinyl acetate and ascorbic acid for 8-12 days produced a significant increase of lymphocyte proliferation in response to alloantigens in mixed lymphocyte culture and blast transformation reaction to suboptimal mitogen doses.     

Immunomodulatory role of thyroid hormones: in vitro effect of tetraiodothyronine (T4) on mitogen induced blastogenic response of lymphocytes

An attempt was made to find out the immunomodulatory role of thyroid hormone, tetraiodothyronine (T4), and its effect on in vitro mitogen induced blastogenesis. Human peripheral blood lymphocytes (PBL) were subjected to phytohaemagglutinin (PHA) concanavalin-A (Con. A) and pokeweed mitogen (PWM) in presence or absence of T4. Basal blastogenic response was significantly enhanced in dose related manner by T4. PHA and Con.A induced response was depressed significantly (r = -0.975 and r = -0.945) whereas less than 50 ng T4 in presence of PHA showed mild stimulation. On the other hand, PWM induced response in presence of T4 was enhanced significantly in dose related manner.     

Stimulatory effect of Bestatin,a new specific inhibitor of aminopeptidases,on the blastogenesis of guinea pig lymphocytes

A potent protease-inhibitor of Actinomycetes origin, Bestatin. which is of dipeptide nature and inhibits aminopeptidase B and leucine-aminopeptidase competitively, strongly stimulates blastogenesis of small lymphocytes triggered with polyclonal mitogen. such as phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM) and lipopolysaccharide of Escherichiae coli (LPS), whereas it inhibits DNA synthesis of normal resting lymphocytes. The stimulatory effect is non-selective with respect to the category of small lymphocytes, i.e. T- and B-lymphocytes, but strikingly selective with respect to the stage of blastogenesis: the stimulation is greatest at a relatively early stage, diminishes as mitogen-activation proceeds, and is not appreciable at a later stage of lymphocyte blastogenesis.The pattern of Bestatin stimulation on lymphocyte blastogenesis is specific for the mitogen used: in T-lymphocyte activation with PHA or Con A, the stimulation first increases and then decreases with increase in mitogen concentrations, whereas in B-lymphocyte activation with LPS, with increasing concentrations of the mitogen, the stimulation increases to a plateau at approximately 100 μg/ml of mitogen. The optimum concentration of Bestatin was found to be approximately 50 μg/ml (0.16 mM) for either PHA or Con A activation, and 50 to 75 μg/ml for B-cell activation with LPS. Bestatin must remain in cultures of T- and B-lymphocytes with polyclonal mitogens for at least about 24 and 16 hr, respectively, to exert its stimulatory effect on blastogenesis.Biochemical results, together with those from autoradiographic analyses, indicate that Bestatin increases the number of blastoid-transformed lymphocytes with polyclonal stimulants. It is suggested that aminopeptidases, possibly located at the cell surface, may play a role in the control of lymphocyte activation during immune responses.     

Some requirements for the response of separated T-cell sub-populations to the mitogens phytohaemagglutinin and concanavalin A.

The proliferative response of various separated populations of mouse spleen and thymus lymphocytes to the mitogen phytohaemagglutinin (PHA) was not a direct function of the level of responsive T cells, but was governed by other regulatory effects. These included a stimulation by adherent macrophages, an inhibition by a separate population of adherent cells and an adherent cell independent restriction of proliferation at high cell concentration. In contrast, the proliferative response to Concanavalin A (Con A) was more closely related to the level of responsive T cells. All density and electrophoretically isolated sub-sets of splenic T cells appeared capable of a proliferative response to PHA and Con A, although under some conditions the PHA responsiveness of certain fractions was suppressed. In the thymus, the minor low theta sub-population appeared capable of response to both mitogens, and accounted for all the activity of the unfractioned thymus cells. No response to either mitogen could be obtained from the major, high theta thymocyte population.     

Influence of various mitogens on the yield of sister-chromatid exchanges, induced by chemicals, in human lymphocytes

The fluorochrome-plus-Giemsa (FPG) method of Perry and Wolff was used to compare the frequencies of sister-chromatid exchanges (SCEs) induced by cyclophosphamide (CP) or mitomycin C (MMC) in human lymphocytes stimulated by phytohaemagglutinin (PHA), concanavalin A (Con A), Wistaria floribunda (WFA), or lentil lectin (LcH-A) extracts. These 4 mitogens, differing in lectin valency and/or sugar specificity, are considered as activating primarily thymus-derived (T) lymphocytes. Regardless of the mitogen used, control cultures displayed a mean yield of about 8 SCEs/cell. A contact, of 1 h, with mitomycin alone or with cyclophosphamide and enzymatic activation, resulted in a significant augmentation of SCEs dependent on the mitogen used. An approximately 2-fold, 4-fold, or 6-fold increase in SCEs was observed for the cultures stimulated by PHA, Con A, and WFA or LcH-A respectively. Furthermore, there were mitogen-dependent differences in mitotic indices and cell-cycle kinetics in human lymphocytes harvested 72 h after stimulation.     

Impairments in pyridoxine-dependent sulphur amino acid metabolism are highly sensitive to the degree of vitamin B6 deficiency and repletion in the pig

The objectives of the current study included the characterization of the temporal changes in indices of sulphur amino acid metabolism in piglets in response to vitamin B6 deficiency and repletion with graded levels of pyridoxine hydrochloride. In Experiment 1, 12 piglets (average initial weight = 5.3 kg; n = 6 per group) were fed a semi-purified diet containing either 0 (deficiency group) or 3 mg (control group) pyridoxine·HCl/kg diet, using a pair-feeding design, for 6 weeks. Piglets consuming vitamin B6-deficient diets exhibited decreased average daily gains on the 4th week and feed conversion efficiency from the 4th week until the end of the trial (P < 0.05). Plasma pyridoxal-5'-phosphate (PLP), in pigs consuming vitamin B6-deficient diets, was significantly lower than controls throughout the experiment (P < 0.01), reaching a nadir of 14% of the control animals' value by the end of the trial. Indices of sulphur amino acid metabolism, including activities of hepatic cystathionine-β-synthase (CBS), cystathionine-γ-lyase (CGL) and serine hydroxymethyltransferase, as well as hepatic-free cysteine concentrations were markedly decreased after 6 weeks of B6 deficiency (P < 0.05). Total hepatic mRNA expressions for CBS and CGL were not affected. Concurrently, hepatic-free homocysteine concentrations increased by more than eight-fold (P < 0.01) at the end of the trial. An examination of plasma total homocysteine and cysteine concentrations revealed significant (P < 0.05) differences between treatments, with evidence of an abrupt shift in concentrations at 3 weeks post-initiation of dietary treatments (>25-fold increase in homocysteine; halving of cysteine values). At the end of Experiment 1, vitamin B6 deficiency significantly increased plasma methionine and serine levels, but decreased plasma glycine concentrations (P < 0.05). In Experiment 2, 20 pigs of 14 days old (initial BW = 5.0 kg) were subjected to a 4-week vitamin B6 depletion protocol, based on results obtained in Experiment 1. After the depletion period and assessment of baseline status (four pigs), remaining pigs were allocated to one of four dietary vitamin B6 repletion treatments: 0.75, 1.5, 2.25 and 3 mg/kg diet as pyridoxine·HCl (n = 4 per level) for 14 days. Significant dose-dependent increases in plasma PLP and cysteine, and decreases in homocysteine were observed, and these were sensitive to the duration of repletion. In conclusion, data from the current studies support the use of both plasma PLP and homocysteine as sensitive indices of vitamin B6 status in the pig. Additionally, the observed patterns of responses in vitamin B6-sensitive metabolites are supportive of an inclusion level of 2.25 mg/kg diet, as pyridoxine·HCl, in diets for young pigs.     

In vitro studies of the rabbit immune system. IV. Differential mitogen responses of isolated T and B cells.

The mitogenic responses of separated rabbit lymphocyte populations functionally analogous to mouse T and B cells have been tested in vitro. Purified T cells were prepared by passage over nylon wool (NW) and purified B cells prepared by treatment with antithymocyte serum and complement (ATS + C). ATS + C kills 70% of peripheral blood lymphocytes (PBL's) and 50% of the spleen cells while passage over NW yields 40% of the applied PBL's and 5–23% of the applied spleen cells. NW-purified T cells from the spleen or PBL's respond fully to concanavalin A (Con A) but have a reduced response to phytohemaglutinin (PHA) and little or no response to goat anti-rabbit immunoglobulin (anti-Ig). PBL's that survive ATS + C (B cells) are stimulated by anti-Ig but not by Con A or PHA. B cells purified from spleen do not respond to Con A or PHA but will respond to anti-Ig under appropriate conditions. A full spleen B-cell response to anti-Ig required removal of Ig produced by the cultures that blocked anti-Ig stimulation. It is concluded that, for rabbit lymphocytes, Con A and PHA are primarily T-cell mitogens and that anti-Ig is primarily a B-cell mitogen. However, the mitogen response of unfractionated PBL or spleen cell populations indicates an overlap in reactivity. This could be due to cells sharing T and B properties, alteration of cell populations by the fractionation procedures used, or recruitment of one population in the presence of a mitogenic response of the other population.     

Effect of calcium depletion and subsequent repletion on parathyroids,parafollicular (C) cells and bone in the growing pig

Summary The ultrastructure of the chief cells of the parathyroid gland and thyroid parafollicular (C) cells and the morphology of bone in calcium depletion and subsequent repletion were examined in young growing pigs. A low calcium diet resulted in osteopenia, increased removal of the cartilaginous core, osteoclasia and osteocytic osteolysis. Subsequent repletion quickly returned bone to normal. In pigs fed the low calcium diet, there was a marked depletion of secretory granules but a striking increase in the number of microtubules in chief cells. Increasing the calcium content of the diet to normal quickly returned the ultrastructural appearance of chief cells to apparent normal. In the initial response to calcium repletion, chief cells exhibited large number of lysosomes and occasionally prominent paracrystalloid bodies. Electron microscopic examination of parafollicular (C) cells of the thyroid gland failed to reveal differences in ultrastructure between test and control pigs. These findings support the view that bone resorption following calcium deficiency may be the result of a secondary hyperparathyroidism rather than of calcium deficiency per se.Supported by U.S.P.H.S. Grant A.M. 12957 from the Division of Arthritis and Metabolic Diseases     

Proliferation of murine thymic lymphocytes in vitro is mediated by the concanavalin A-induced release of a lymphokine (costimulator).

Mitogen-induced proliferation of lymphocytes may in theory result directly from the interaction of mitogen with the cells, or indirectly as a result of the mitogen-stimulated release of lymphokines. In the case of murine thymic lymphocytes exposed to concanavalin A (Con A) in tissue culture, we have determined that mitogenesis depends upon a lymphokine. Interaction of the thymic lymphocytes with lectin is necessary, but not sufficient, for mitogenesis. A lymphokine, or costimulator for mitogenesis, is released by normal spleen or thymus cells during the first 16 hr of their exposure to Con A, and in the presence of a phytomitogen it stimulates thymic mitogenesis. Under conditions of low costimulator levels, no mitogenesis follows the interaction of Con A with cells. The response of adult CBA/J mouse thymocytes to phytohemagglutinin (PHA) is very low, compared to their response to Con A. When costimulator is added to PHA, the cells respond as well as they do to Con A. Costimulator does not act through Con A-binding sites on thymus cells. Its production is dependent on both cells carrying omega surface antigen (T lymphocytes) and adherent cells of the macrophage-monocyte series. The adherent population, but not the T cells, may be heavily irradiated without affecting production of costimulator. Costimulator is not a mitogen on its own.     

Changes in lymphocyte populations in protein—Calorie-deficient mice

Changes in lymphocyte subpopulations induced by postweaning malnutrition were studied in C57BL/6 mice kept on a protein-restricted diet (D), by weekly assessment of the “homing” properties and the response to mitogens of thymus and spleen lymphocytes during the first 2 months of diet. Cell loss in the lymphoid organs during the early phase of protein restriction was mainly due to depletion of nonrecirculating cells. This resulted in relative enrichment of medullary cells in the thymus and T₂ cells in the periphery as shown by the rise in the percentage migration of D lymphocytes to the lymph nodes as well as in their response to optimal doses of PHA and Con A and PHA:Con A response ratio. Reversion of the distribution pattern of D lymphocytes, with depressed homing to the lymph nodes and decrease in the response to mitogens, was observed concomitantly with a second phase of partial recovery in the whole-body weight and cell content of the thymus and spleen. The [³H]thymidine uptake by D spleen cells stimulated with supraoptimal doses of mitogen was significantly increased during the whole length of the experiment. The suppression of DNA synthesis induced by high doses of mitogen reappeared after short-term nutritional rehabilitation.     

Evidence that guanine-nucleotide binding regulatory proteins couple cell-surface receptors to the breakdown of inositol-containing lipids during T-lymphocyte mitogenesis

We have studied the role of guanine-nucleotide binding regulatory proteins (G proteins) in the stimulation of inositol lipid breakdown during mitogenic activation of normal human T lymphocytes. The effect of the mitogen phytohemagglutinin (PHA) was compared with the action of two G-protein activators, fluoroaluminate (AlF4-) and guanosine-5'-O-thiotriphosphate (GTP gamma S). PHA and AlF4- stimulated the breakdown of inositol lipids via both the phospholipase A and C pathways when added to intact lymphocytes. PHA, AlF4- and GTP gamma S also triggered both these pathways when added to permeable lymphocytes. The magnitude of the response obtained with AlF4- and GTP gamma S was about four-fold less than with PHA. This difference was attributable to increases in cAMP elicited by AlF4- and GTP gamma S which inhibited the phospholipase pathways. AlF4-, GTP gamma S, and PHA all stimulated the phosphorylation of a 42 kDa protein on tyrosine residues. We propose a model for the early steps following mitogen binding, including sequential activation of a G protein, phospholipase C, protein kinase C and a tyrosine protein kinase. A parallel pathway involving G protein mediated activation of phospholipase A is also implicated.     

Essential role of intracellular glutathione in controlling ascorbic acid transporter expression and function in rat hepatocytes and hepatoma cells

Although there is in vivo evidence suggesting a role for glutathione in the metabolism and tissue distribution of vitamin C, no connection with the vitamin C transport systems has been reported. We show here that disruption of glutathione metabolism with buthionine-(S,R)-sulfoximine (BSO) produced a sustained blockade of ascorbic acid transport in rat hepatocytes and rat hepatoma cells. Rat hepatocytes expressed the Na(+)-coupled ascorbic acid transporter-1 (SVCT1), while hepatoma cells expressed the transporters SVCT1 and SVCT2. BSO-treated rat hepatoma cells showed a two order of magnitude decrease in SVCT1 and SVCT2 mRNA levels, undetectable SVCT1 and SVCT2 protein expression, and lacked the capacity to transport ascorbic acid, effects that were fully reversible on glutathione repletion. Interestingly, although SVCT1 mRNA levels remained unchanged in rat hepatocytes made glutathione deficient by in vivo BSO treatment, SVCT1 protein was absent from the plasma membrane and the cells lacked the capacity to transport ascorbic acid. The specificity of the BSO treatment was indicated by the finding that transport of oxidized vitamin C (dehydroascorbic acid) and glucose transporter expression were unaffected by BSO treatment. Moreover, glutathione depletion failed to affect ascorbic acid transport, and SVCT1 and SVCT2 expression in human hepatoma cells. Therefore, our data indicate an essential role for glutathione in controlling vitamin C metabolism in rat hepatocytes and rat hepatoma cells, two cell types capable of synthesizing ascorbic acid, by regulating the expression and subcellular localization of the transporters involved in the acquisition of ascorbic acid from extracellular sources, an effect not observed in human cells incapable of synthesizing ascorbic acid.     

Flow cytometry of blastogenesis and the concomitant viability assay of lymphocytes stained with 4', 6 diamidino-2-phenylindole

The blastogenic response of lymphocytes induced by concanavalin A (Con A), phytohemagglutinin (PHA), pokeweed mitogen (PWM) and by paraperiodic acid (H5IO6) was assayed by flow cytometry of 4',6 diamidino-2-phenylindole (DAPI) stained nuclei as well as by thymidine (3H-TdR) incorporation rates. The DNA content in DAPI-stained nuclei of viable and dead human, rat and mouse lymphocytes, was readily distinguishable from each other by flow cytometric methods (FCM).     

Suppression of Con A mitogen-induced proliferation of normal spleen cells by macrophages from chickens with hereditary muscular dystrophy

Spleen cells from chickens with hereditary muscular dystrophy (MD) give low blastogenic responses to the T cell mitogen concanavalin A (Con A) while exhibiting normal mitogen stimulated blastogenic responses to the T cell mitogen phytohemagglutinin (PHA). The addition of MD spleen cells to normal spleen cells caused a marked suppression of the Con A response of the normal cells while not affecting the PHA response of the normal cells. The suppressive activity by the MD spleen cells requires viable cells and is contact mediated. The suppressive activity is attributed to the presence in MD spleens of a population of suppressor cells with characteristics typical of macrophages. The suppressor cell activity was not removable by complement-mediated lysis using anti-T or anti-B sera, but it was reversible by treatment with carrageenan or carbonyl iron magnet, by passage through a Sephadex G-10 column, and by adherence to plastic petri dishes or glass beads. MD spleen cells depleted of the suppressor cell population remained unable to respond to Con A.     

Dose-dependent hyporreactivity to phytohemagglutinin in systemic lupus erythematosus

The response of peripheral blood lymphocytes to stimulation with optimal and suboptimal doses of PHA was measured in patients with active SLE before initiation of therapy. The [³H]thymidine uptake of SLE patient's lymphocytes was significantly lower than that of their matched controls when cells were stimulated with suboptimal PHA doses in the presence of autologous plasma. A moderate improvement in the PHA response was observed by culturing washed patient's lymphocytes in medium supplemented with pooled normal human plasma, but only in one case the response reverted to normal values. A significant inhibitory effect of SLE plasma on the response of normal donor's lymphocytes to stimulation with low PHA doses, which was independent from the presence of lymphocytotoxic antibodies and persisted after complement inactivation was observed in further experiments.The results indicate that depression of lymphocyte transformation could be demonstrated in patients with active SLE using suboptimal doses of PHA and suggest that this depression may be caused by both a defect in the responding lymphocyte populalation and the presence of inhibitory factor(s) in SLE plasma.     

Lymphocyte membrane receptors in cultures treated with mitogens

Lymphocyte membrane receptors for sheep erythrocytes (E) and human erythrocytes sensitized with antibody and complement (HEAC) were used as markers for human T and B cells, respectively. Combining the method of rosette formation with E and HEAC with radioautography, we have studied the effect of in vitro stimulation with phytohemagglutinin (PHA), concanavalin A (Con A), pokeweed mitogen (PWM), staphylococcal filtrate (SF) and mercuric chloride (HgCl₂) on the proportion of small lymphocytes and blasts presenting these receptors. After mitogenic stimulation, small lymphocytes as well as blasts were found forming rosettes with E or HEAC, in variable proportions. PHA, Con A, SF and HgCl₂ showed a similar effect in vitro since most of the blasts obtained after stimulation had receptors for E and a smaller proportion for HEAC.The stimulation with PWM led to a blast population made up of a higher percentage of HEAC than E rosette-forming cells.     

Interaction of vasoactive intestinal peptide with mouse lymphocytes: specific binding and the modulation of mitogen responses

Binding of radioiodinated vasoactive intestinal peptide (VIP) to mouse lymphocytes has been investigated. Specific cell binding of 125I-VIP was demonstrated with lymphocytes from mesenteric lymph nodes, subcutaneous lymph nodes, spleen, and Peyer's patches. The binding of VIP by these cells was accounted for by VIP binding sites upon T cells rather than non-T cells. In the presence of VIP, the in vitro response of lymphocytes to the T cell mitogens concanavalin A (Con A) and phytohemagglutinin (PHA) was inhibited in a dose-dependent fashion, whereas that to the B cell mitogen lipopolysaccharide (LPS) was not. There was a close correlation between the potency of VIP and some structurally related peptides for inhibition of 125I-VIP binding and the effect of those peptides on T cell mitogen responses. These observations demonstrate that mouse T lymphocytes have specific VIP receptors and that VIP can modulate the response of T cells to mitogenic stimulation. VIP may be an important immunoregulatory molecule, and may be implicated in the regulation of T cell function in mucosal tissues innervated by VIP-containing neurons.     

Mitogen Synergism in low-responding CBA/CaJ mice.

Although neither phytohemagglutinin (PHA) nor concanavalin A (Con A) stimulated blood cultures in vitro from low-responding CBA/CaJ mice effectively, a mixture of PHA and Con A over a range of concentrations stimulated a response from CBA/CaJ mouse blood that was greater than the sum of the responses produced by using PHA or Con A individually. This synergistic effect was expressed as the percentage by which the responses to the PHA and Con A mixture exceeded the sum of the responses to PHA alone and Con A alone. When the mitogen concentrations that gave maximum responses individually were used, the synergistic effect averaged 319% in cultures of blood from low-responding CBA/CaJ mice. Apparently simultaneous exposure to PHA and Con A stimulates DNA synthesis in white blood cells of CBA/CaJ mice that fail to respond to either mitogen alone.     

The role of macrophages in thymocyte mitogenesis.

The thymic lymphocytes of CBA/J mice respond to the mitogen Concanavalin A (Con A) only in the presence of adherent cells of the monocyte-macrophage series. Depletion of adherent cells abrogates the response, and macrophage-rich population of cells restore it. The need for macrophages and mitogen is completely provided by irradiated splenic macrophages which have been exposed to Con A and washed free of the soluble mitogen. The mitogenmacrophage effect in this case is apparently not due to soluble factors. Even more striking than the effect of macrophages on fresh cultures of thymic lymphocytes is their ability to restimulate quiescent cells 72 hr after their first stimulation with Con A. The quiescent cells respond immediately and quantitatively to Con A in the presence of fresh macrophages. This stimulation, like that of fresh thymocytes, is also controlled by a lymphokine ("costimulator") produced by mixing macrophages, mitogen, ant T lymphocytes. Our data suggest a model in which two signals are required for mitogenesis. First, the interaction of macrophage, T cell, and mitogen elicits a soluble costimulator, which is itself not mitogenic. Secondly, in the presence of costimulator, the mitogen (either soluble, or, more efficiently, bound to macrophages) induces a proliferative response in the T cell.