Concentrations Of PHA Research Articles

Humic Acid Modified by Being Incorporated Into Phosphate Fertilizer Increases Its Potency in Stimulating Maize Growth and Nutrient Absorption.

Humic acid-enhanced phosphate fertilizer (HAP) is widely applied in Chinese agriculture due to its high efficiency. Although the structural composition and physicochemical properties of humic acid (HA) are significantly altered during HAP production, a clear understanding of the mechanisms underlying the biological effects of HA extracted from HAP fertilizer (PHA) on plant growth is still lacking. In the current study, we extracted PHA from HAP and assessed its effects on the dry biomass, phosphorus (P) and nitrogen (N) uptake, and P absorption rate of maize seedlings when supplied at different concentrations (2.5, 5, 10, and 25 mg C L−1) in the hydroponic culture. The root vigor, root plasma membrane H+-ATPase activity, and root nitrate reductase activity were also determined as the representative indicators of the root capacity for nutrient absorption, and used to clarify the mechanism by which PHA affects the maize growth and nutrient absorption. The results showed that the dry biomass, phosphorus uptake, nitrogen uptake, and average phosphorus absorption rates were significantly higher by 14.7–27.9%, 9.6–35.1%, 17.9–22.4%, and 22.1–31.0%, respectively, in plants treated with 2.5–5 mg C L−1 PHA compared to untreated controls. Application of 10–25 mg C L−1 raw HA resulted in similar stimulatory effects on plant growth and nutrient absorption. However, higher levels of PHA (10–25 mg C L−1) negatively impacted these indicators of plant growth. Furthermore, low PHA or high raw HA concentrations similarly improved root vigor and root plasma membrane H+-ATPase and nitrate reductase (NR) activities. These results indicate that lower concentrations of PHA can stimulate maize seedling growth and nutrient absorption to an extent that is comparable to the effect of higher concentrations of raw HA. Thus, the proportion of HA incorporated into HAP could be lower than the theoretical amount estimated through assays evaluating the biological effects of raw HA.

Modeling phaeopigment concentrations in water from a shallow mesotrophic lagoon.

Concentrations of phaeopigments (Pha) and chlorophyll a (Chl) were determined in surface waters from a temperate lagoon during six sampling campaigns at high and at low tide. In order to develop models for phaeopigment concentration in water, it was necessary to replace Chl with photosynthetic pigment concentration (Pt =Pha+Chl) as one of the explanatory variables. Under first approximation, food availability and water temperature (T) could be considered as independent variables. The concentrations of Pha were then determined following seasonal change of response curves of the consumer community on T. However, multiple regression models with Pt , T and, eventually, salinity as explanatory variables were better able to depict Pha. All equations, developed with Pt , were also solved using Chl as an input variable. Although part of the performance was lost, such back-transformed models can be used at low/medium T and moderate to high concentrations of Chl. The developed equations about middle to long-term variations of Pha could be applied to study the biogeochemistry of contaminants related to Pha and to evaluate the dependence on temperature of phytoplankton utilization by consumers. PRACTITIONER POINTS: Phaeopigment concentration depicted by chlorophyll (Chl), temperature (T), and salinity. Better results obtained at low to medium T and moderate to high Chl concentration. Multiple regression (MR) better for extrapolation than model (S) with variable separation. Thermal response of consumer community in mesotrophic lagoon studied using model S.

Toxic and deterrent effects of phytohemagglutinin on the grain aphid Sitobion avenae

The toxicity and deterrent effects of phytoheamagglutinin (Phaseolus vulgaris agglutinin, PHA) to the grain aphid (Sitobion avenae F.) was investigated in the laboratory by use of liquid diets or gels containing various PHA concentrations. The addition of PHA to the liquid diet increased the aphid pre-reproductive period, mortality, and generation time and decreased aphid fecundity and intrinsic rate of natural increase. Aphids fed on sucrose-agarose gels with higher concentrations of PHA exhibited no phloem-feeding behavior. The number of gel penetrations was reduced and duration of the pathway phase was increased as PHA concentration increased. These results indicate that PHA is detrimental to S. avenae and that the effect of PHA is at least partially explained by suppression of feeding behaviour. PHA could be useful for managing S. avenae.

Prediction of intracellular storage polymers using quantitative image analysis in enhanced biological phosphorus removal systems

The present study focuses on predicting the concentration of intracellular storage polymers in enhanced biological phosphorus removal (EBPR) systems. For that purpose, quantitative image analysis techniques were developed for determining the intracellular concentrations of PHA (PHB and PHV) with Nile blue and glycogen with aniline blue staining. Partial least squares (PLS) were used to predict the standard analytical values of these polymers by the proposed methodology. Identification of the aerobic and anaerobic stages proved to be crucial for improving the assessment of PHA, PHB and PHV intracellular concentrations. Current Nile blue based methodology can be seen as a feasible starting point for further enhancement. Glycogen detection based on the developed aniline blue staining methodology combined with the image analysis data proved to be a promising technique, toward the elimination of the need for analytical off-line measurements.

Feed contaminated with Fusarium toxins alter lymphocyte proliferation and apoptosis in primiparous sows during the perinatal period

Two groups of pregnant primiparous sows (day 89±2 of gestation) were 54days (±1day) fed either with an experimental diet (5.08mgkg−1 deoxynivalenol – DON, 0.09mgkg−1 zearalenone and 21.61mgkg−1 fusaric acid) or control diet (0.25mgkg−1 DON). The consummation of feed was significantly higher in the control group. Lymphocyte stimulation in culture from peripheral blood lymphocytes (PBL) was measured by BrdU incorporation test using two different concentrations of mitogen PHA 10 and 20μgml−1, two different concentrations of DON (5 and 1μgml−1) and a combination of both, PHA and DON (PHA 10+DON 5, PHA 10+DON 1 and PHA 10+DON 0.1μgml−1). The lymphocyte proliferation, except for PHA 10μgml−1, was significantly higher in the experimental group. Further on, using the photometric enzyme immunoassay, the apoptosis was studied in non-stimulated 72h lymphocyte culture or stimulated with 1μgml−1 of DON. The significantly higher apoptosis was in non-stimulated lymphocyte cultures from the experimental group (P=0.036). The results suggest that such feed may affect the peripheral lymphocyte population in the direction of their proliferation response and programmed cell death.

Effect of Ivig On Human T Cells: Interference with Activation Rather Than Suppression of Activated Cell Functions.

Abstract 4738 IntroductionIVIg is known to have immunosuppressive effects in a variety of inflammatory and autoimmune diseases, which may be caused by modulations of T cell functions in treated patients. The mechanisms responsible for these modulations have been mostly investigated using in vitro stimulated T cells. These studies revealed that IVIg inhibited the proliferation of activated T cells possibly by interfering with the secretion of cytokines important for T cell proliferation, such as IL-2. In the present study, we sought to determine the precise mechanism by which IVIg inhibited cytokine secretion by stimulated T cells. MethodsHuman PBMC and Jurkat T cells were stimulated with PHA. Human T cells purified from PBMC were stimulated with CD3/CD28 T cell expander beads. Cells were cultured in presence or not of 10 mg/ml of IVIg for 24 hours. IL-2 secretion was measured in the culture supernatants by ELISA. IVIg was depleted of PHA-reactive IgG by passage on a PHA-Sepharose column. The extent of depletion was evaluated by ELISA using PHA as capture antigen. The role of F(ab')2 fragments in the inhibitory effect of IVIg on IL-2 secretion was determined using pepsin-generated fragments. ResultsIVIg inhibited IL-2 secretion by PHA-stimulated T cells, as previously reported. However, the use of increasing concentrations of PHA for T cell stimulation led to a decreased ability of IVIg to inhibit IL-2 production, suggesting that IVIg acted by neutralizing PHA or by competition for receptor occupancy on the cell surface. Pre-incubation of T cells with IVIg followed by washing and addition of PHA did not result in inhibition of IL-2 secretion, indicating that competition for receptor was not involved in this IVIg-mediated inhibition. In contrast, inhibition of IL-2 production by IVIg was completely abrogated using IVIg depleted from PHA-reactive IgG, indicating that IVIg-mediated inhibition of IL-2 secretion was the consequence PHA neutralization. Testing of F(ab')2 fragments of IVIg showed that these fragments bound to PHA and inhibited IL-2 secretion as efficiently as IVIg. Using another activation strategy, we showed that IVIg could also decrease IL-2 secretion by purified human T cells following anti-CD3/CD28 stimulation. Preliminary data using light microscopy indicated that IVIg interfered with the binding of CD3/CD28 beads on T cells, therefore reducing cell activation as evaluated by IL-2 secretion. ConclusionAltogether, our results suggest that the inhibition of T cell responses by IVIg occurs during the cell activation step, by preventing the binding of mitogens or antibody-coated beads on the cell surface. These observations emphasize the importance of ruling out the possible interactions of IVIg with culture medium additives, mitogens or activating agents before deriving strong conclusions on the mechanisms of action of IVIg based on their apparent immunomodulatory effects observed in vitro assays. Disclosures:No relevant conflicts of interest to declare.

The competition between PAOs (phosphorus accumulating organisms) and GAOs (glycogen accumulating organisms) in EBPR (enhanced biological phosphorus removal) systems at different temperatures and the effects on system performance

It is well known and firmly established that the rate of chemical and biochemical reactions slow down as temperature decreases. Nevertheless, several studies have reported that the efficiency of enhanced biological phosphorus removal (EBPR) improves as temperature decreases. However, several recent studies have reported that EBPR reaction rates decrease with temperature decrease in accordance with the Arrhenius relationship. This study was designed to more thoroughly investigate this controversy using two UCT plants fed with a synthetic wastewater consisting primarily of acetate as the COD form, and a small amount of supplemental yeast extract. Experiments were performed over temperatures ranging from 5 to 20 degrees C. The results showed that, even though the kinetic rates decrease as temperature decreases, EBPR systems perform better at colder temperatures. The reason for better system performance is apparently related to reduced competition for substrate in the non-oxic zones, which results in an increased population of PAOs and, thus, greater EBPR efficiency. The proliferation of PAOs apparently occurs because they are psychrophilic whereas their competitors are not. The experiments showed that the EBPR sludges accumulated high concentrations of both PHA and glycogen at 20 degrees C, but accumulated more PHA and much less glycogen at 5 degrees C. Although the results could be interpreted as the result of changes in the PAO-GAO competition, Mann-Whitney non-parametric comparisons of transmission electron microscopy examinations revealed no indication of the presence of GAOs population under any temperature conditions. Regardless, mass balances of the glycogen data showed that the involvement of glycogen is less at cold temperature, even though EBPR was greater. Unlike current EBPR models (e.g. Mino model), the results suggest that glycogen metabolism is not a precursor for EBPR biochemistry. The results also indicate that temperature not only may cause selective pressure on the dominant organisms, but also may force them to use a different metabolic pathway as temperature decreases.

Inhibition of cytokine production and cytokine-stimulated T-cell activation by FK506 (tacrolimus)1.

Insofar as it exerted its immunosuppressive effect by inhibiting cytokine expression, we assessed the effect of FK506 (Tacrolimus) on cytokine-stimulated T-cell activation. Human T cells, treated with FK506, or controls were stimulated with the mitogens PHA + PMA, Con A, and the "CD3-bypass" stimulation regimen, PMA + ionomycin. T-cell proliferation was quantitated by measuring the uptake of tritiated thymidine, and mRNA expression was assessed by RT-PCR. FK506, in a concentration-dependent fashion, inhibited T-cell proliferation and steady-state mRNA expression of IL-2 and IL-7; half-maximal suppression was obtained at 10(-7) to 5 x 10(-8) M. We tested whether FK506 antiproliferative effect could be overcome with exogenously reconstituted rIL-2 and/or rIL-7. Neither rIL-2 nor rlL-7, individually in conjunction with suboptimal concentrations of PHA or Con A, or in combination without any costimulus, was capable of abrogating FK506 antiproliferative effect, indicating that FK506 also acted by inhibiting cytokine-stimulated T-cell activation. To confirm this, T cells were treated with FK506 and stimulated by rIL-2 and rIL-7, individually in conjunction with suboptimal concentration of PHA and Con A. In addition, T cells were stimulated with rIL-2 and rIL-7 without any costimuli. FK506 inhibited T-cell activation stimulated by rIL-2 and by rIL-7, individually and in combination. This confirms that, in exerting its antiproliferative effect, FK506 acts at two levels, by inhibiting cytokine availability and by suppressing cytokine effect on target cells, and explains the beneficial effect of FK506 in attenuating ongoing immune responses.

Melatonin enhances IL-2, IL-6, and IFN-gamma production by human circulating CD4+ cells: a possible nuclear receptor-mediated mechanism involving T helper type 1 lymphocytes and monocytes.

This paper shows that melatonin is able to activate human Th1 lymphocytes by increasing the production of IL-2 and IFN-gamma in vitro. Th2 cells appear not to be affected by melatonin, since IL-4, which is mostly produced by Th2 cells, is not modified by the hormone. Melatonin also enhances IL-6 production by PBMCs. The activation by melatonin of IL-6 production is apparently related to the presence of monocytes, rather than to Th2 cells, in the cell preparation, since PBMCs depleted of monocytes (CD14+ cells) were not activated. Activation of PBMCs by melatonin was dependent on the dose and, measured by cytokine production, was observed only when cells were either not activated or only slightly activated by low concentrations of PHA, or when cell activation was achieved by incubating the cells with previously irradiated cells. Using a different approach to identify what type of cells among the PBMC subsets was activated by melatonin, the expression of CD69, a marker of cell activation, was studied. Melatonin increased the percentage of cells expressing the CD69 Ag in CD4+ but not in CD8+ cells. We have also achieved enhanced production of IL-2 and IL-6 using CGP 52608, a specific ligand of the putative nuclear melatonin receptor RZR/ROR, raising the possibility of direct effects of melatonin on gene regulation in both Th1 cells and monocytes. The results suggest that melatonin may be involved in the regulation of human immune functions by modulating the activity of Th1 cells and monocytes via nuclear receptor-mediated transcriptional control.

Specific induction of interleukin-4-producing cells in response to in vitro allergen stimulation in atopic individuals

Background and Objective CD4+ T cells can be divided into two major subsets. T helper (TH)1 and TH2 cells. Interleukin-4 (IL-4) is produced by TH2 cells and induces switching of immunoglobulin (Ig) M/lgG to IgE. Interferon-γ (IFNγ) produced by TH1 cells counteracts the IgE-promoting effects of IL-4. In this study we wanted to investigate whether the number of IL4-producing cells could be a direct measurement of allergen exposure in vitro, and whether this was correlated to the elevated serum IgE-levels seen in atopic persons. Methods We compared the number of IL-4- and IFNγ-producing cells using an enzyme-linked immunospot assay (ELISPOT) in response to allergens from birch and cat in peripheral mononuclear cells from atopic and healthy individuals. Results In the two sensitized groups there was an increase in the number of lL-4producing cells in response to the specific allergen which was not seen in the healthy group (1/20000 cells and 1/200 000 cells, respectively, P < 0.001 for birch). In criss-cross experiments where birch-sensitized individuals were stimulated with cat allergen, no lL-4-producing cells were seen, indicating a high degree of specificity. In individual subjects, the elevated numbers of IL-4-producing cells were significantly correlated with their allergen-specific serum IgE levels. When allergen was combined with a suboptimal dose of PHA, there was a synergistic increase in the number of allergen-induced IL-4-producing cells (1/4000 cells) in the atopic donors, which was not seen with the number of IFNγ-producing cells. Conclusions Allergen-specific IL-4 producing cells in a peripheral blood mononuclear cell (PBMC) culture can be detected by ELISPOT and the response can synergistically be enhanced by suboptimal concentrations of PHA.

Co-operative effect between insulin-like growth factor-1 and interleukin-2 on DNA synthesis and interleukin-2 receptor-alpha chain expression in human lymphocytes.

Recently we demonstrated that IGF-1 has the same effect on DNA synthesis and G1-phase length during human lymphocyte proliferation as IL-2. In order to determine the link between IGF-1 and IL-2 on lymphocyte proliferation, we tested the ability of these cytokines, alone or in combination, to induce DNA synthesis and increase the number of cells expressing IL-2-alpha chain receptors. Our results showed that the increase in DNA synthesis produced by the addition of IGF-1 to cells cultured with saturable concentrations of IL-2, correlated well with an increase in the number of cells expressing IL-2-alpha chain receptor. The effect on both DNA synthesis and IL-2-alpha chain receptor expression was greater at lower concentrations of PHA. This study suggests that the proliferative effect of IGF-1 might be due to separate stimulation of different cell populations and/or by activation of a tyrosine-kinase signal transduction pathway complementary to the IL-2 activation signal.

IgA anaphylactic transfusion reactions

IgA anaphylactic transfusion reactions are rare events, estimated to occur in 1 in 20,000 to 47,000 transfusions. The signs and symptoms of these reactions do not differentiate them from other causes of anaphylaxis. The diagnosis of an anaphylactic transfusion reaction is established by showing an IgA-antibody in the patient's serum. Most laboratories that test for IgA antibodies rely on the PHA method, which uses red blood cells that are coated with serologically defined IgA multiple myeloma proteins. We tested sera referred from Red Cross regional blood centers and hospitals from patients with suspected IgA anaphylactic reactions and found an IgA antibody in 76.3% of IgA-deficient patients. However, only 17.5% of all samples referred contained an IgA antibody, indicating that most persons with suspected IgA anaphylactic reactions had experienced acute generalized reactions that were from causes other than anti-IgA transfusion. Using PHIA to measure serum concentrations of IgA and PHA to detect IgA antibodies, we found the frequency of IgA deficiency (< 0.05 mg/dL) and class-specific anti-IgA in random blood donors to be approximately 1 in 1,200. Titers of anti-IgA did not distinguish these seemingly healthy blood donors from patients with a history of an anaphylactic transfusion reaction. Because the frequency of 1 in 1,200 greatly exceeds the observed frequency of anaphylactic reactions in transfused persons, we conclude that using PHA for anti-IgA does not reliably predict risk for an anaphylactic transfusion reaction. Additional research is needed to define a more specific marker to identify those persons who are truly at risk for these serious, but rare, complications of blood transfusion.

Anti-CD3-stimulated Ca2+ signal in individual human peripheral T cells. Activation correlates with a sustained increase in intracellular Ca2+1.

The changes in [Ca2+]i after mitogen stimulation of individual human peripheral T cells were examined by single cell image analysis to determine the relationship between the Ca2+ signal and functional outcome. Marked heterogeneity in the magnitude of increase in [Ca2+]i, in the lag time of the responses, and in the percentage of T cells that responded to mAb to CD3 and to PHA was observed. However, mitogenic stimuli that induced IL-2 production or DNA synthesis consistently generated increases in [Ca2+]i in individual T cells that were sustained for 1 to 2 h. Soluble mAb to CD3 induced an increase in [Ca2+]i that remained elevated at 60 min and led to IL-2 production and proliferation upon costimulation by phorbol ester. In contrast, cross-linking anti-CD3 with a secondary antibody foreshortened the increase in [Ca2+]i, and IL-2 production and DNA synthesis were inhibited. Immobilized anti-CD3, which can stimulate T cell proliferation and IL-2 production in the absence of phorbol ester, produced a constant sustained elevation in [Ca2+]i that lasted more than 2 h. Similarly, functional responses could be generated by concentrations of PHA that resulted in only a slow increase in [Ca2+]i that continued to rise for 1 to 2 h. Examination of the mitogen-induced sustained increases in [Ca2+]i suggested that an elevation in [Ca2+]i as small as 50 to 100 nM above control mean [Ca2+]i was associated with evidence of T cell activation. Spontaneous oscillatory changes in [Ca2+]i were observed in a small percentage of peripheral T cells although they were noted to occur frequently in Jurkat cells. Mitogenic stimulation did not consistently increase oscillations in peripheral T cells, and neither their frequency nor their magnitude correlated with IL-2 production or DNA synthesis. These observations suggest that oscillatory changes in [Ca2+]i are not a primary determinant of T cell activation. Rather, the data indicate that functional activation of T cells by PHA and anti-CD3 is correlated with the induction of a small, but sustained increase in [Ca2+]i.

Substance P induces a rise in intracellular calcium concentration in human T lymphocytes in vitro: evidence of a receptor-independent mechanism

The neuropeptide substance P (SP) has been shown to play an important role as a mediator of neurogenic inflammation. Moreover, in vitro SP is capable of modulating the activity of lymphocytes, monocytes and polymorphonuclear cells. We have examined one of the early events that occur after addition of SP to human peripheral blood mononuclear cells (PBMC). Addition of 10 −6−10 −4 M SP to human peripheral blood mononuclear cells results in a dose-dependent rise in intracellular calcium concentration as determined by FACS analysis. We show that the effect of SP cannot be attenuated by the SP receptor antagonist [D-Pro 4,D-Trp 7,9]-SP(4–11), indicating that the response is not mediated via a SP receptor. Amphiphilic peptides like SP appear to have the capacity to insert themselves into the cell membrane and interact directly with intracellular proteins. This hypothesis is supported by the fact that the amphiphilic analogue of SP, [D-Pro 2,D-Phe 7,D-Trp 9]-SP, is capable of inducing a calcium response in our system, although it is known as an SP receptor antagonist. Functionally, we show that SP increases the proliferative response of T cells induced by suboptimal concentrations of the mitogen PHA. These data provide evidence of a potential role of SP in the regulation of lymphocyte activation.

IL-4 and IL-2 promote human T-cell proliferation through symmetrical but independent pathways

The role of IL-4 and IL-2 on normal human T-cell activation and proliferation was studied. Both IL-2 and IL-4 were unable to induce proliferation of resting T cells. Therefore, we investigated their effect and the regulation of the T-cell proliferative response in competent T cells. T cells were rendered competent following incubation with PDB/ionomycin for 30 min or suboptimal concentrations of PHA for 60 min. Cells were then washed and recultured with PDB, IL-2, or IL-4 in the second or progression phase of the culture. Cells cultured in medium alone in this phase did not proliferate. IL-2 and IL-4 independently promoted competent T cells to proliferate to a similar degree as the response to PDB and the combination of IL-2 and IL-4 was not additive. The induction of competence and subsequent responsiveness to IL-2 and IL-4 could be maintained for about 24 hr after which time they become gradually less responsive to the interleukin in the progression phase. Addition of anti-IL-2R mAb or anti-IL-2 mAb resulted in selective inhibition of IL-2-mediated proliferation only. Similarly, addition of anti-IL-4 mAb resulted only in inhibition of IL-4-mediated proliferation. Addition of IL-2 during the progression phase led to an enhancement of IL-2R (TAC) expression while IL-4 did not affect IL-2R expression. The production of IL-2 and IL-4 by competent T cells could not be enhanced by the non-corresponding lymphokine. These results on the protein level were confirmed at the mRNA level as well and demonstrated that only PDB and IL-2 could induce IL-2 mRNA and PDB and IL-4 enhanced IL-4 mRNA. The immunosuppressive drug, cyclosporin A, failed to inhibit progression triggered by PDB, IL-2 or IL-4 in competent T cells. These findings suggest that IL-2 and IL-4 trigger T-cell proliferation through symmetrical, but independent pathways.

Effects of changes in membrane potential on the cyclosporin-induced inhibition of T-cell proliferation

Cyclosporin A (CsA) exerts its major immunosuppressive effect by inhibition of T-lymphocyte proliferation. The precise mechanism and target of its action has not yet been completely identified. CsA is also known to induce a rapid membrane depolarization in T lymphocytes. We have tested the role of CsA-dependent depolarization in the inhibition of T-cell proliferation by the drug. In these studies, induced membrane depolarization (in the presence of gramicidin or by replacing the Na+ content of the medium with K+) or hyperpolarization (in the presence of valinomycin) had no influence on the induction of T-cell competence by phorbol dibutyrate/ionomycin or by submitogenic concentrations of PHA, a target for CsA immunosuppression. However, regardless of the state of membrane potential during the induction of T-cell competence, the inhibition by CsA was the same as seen in normally polarized cells. We conclude that the depolarization induced by CsA is not a critical element in its inhibitory effect on T-cell proliferation.

Proliferative capacity of human peripheral blood lymphocytes sorted on the basis of glutathione content

Glutathione (GSH) is important in defense against oxygen free radical damage, in detoxification of xenobiotics, and in mitogenesis. The reducing conditions provided by low molecular weight thiols such as 2-mercaptoethanol (ME) have been shown to promote the growth of lymphocytes in culture. We wished to determine the effects of 2-ME on GSH content, and to determine to what extent GSH status affected lymphocyte proliferation. GSH content was quantitated in human peripheral blood lymphocytes (PBL) using a flow cytometric assay with monochlorobimane. This analysis was performed on PBL as well as on the CD4+ T-cell subset, as identified with fluorescent anti-CD4 monoclonal antibodies (mAb). Cells were viably sorted on the basis of their GSH content, and incubated for 3 days with mitogenic concentrations of PHA (for PBL) or anti-CD3 mAb (for CD4+ cells) in the presence of bromodeoxyuridine (BrdU). BrdU/Hoechst cell cycle analysis was then performed on these cells. High GSH sorted cells had a higher percentage of cells capable of entering the cell cycle than low GSH sorted cells. This data indicates that some of the heterogeneity in proliferative capacity within PBL in culture is directly or indirectly related to GSH content. Incubation of cells in 2-ME prevented the loss of GSH that occurs when cells are cultured. 2-ME improved the proliferative capacity of unsorted cells, and of cells sorted for high and low GSH. Acridine orange staining of anti-CD3 mAb stimulated cells sorted for high and low GSH indicated that an early event in cell activation was affected by GSH content.

Potential role of PHA in producing human monoclonal thyroid autoantibodies of different subclasses

A human monoclonal autoantibody to thyroglobulin (Tg) of subclass IgG2 was developed by fusing a mouse myeloma with Tg antibody secreting Epstein-Barr virus (EBV)-infected B lymphocytes from a Hashimoto patient. Subsequent studies showed that EBV-infected B lymphocytes from this patient synthesized IgG2 Tg antibody while unfractionated blood lymphocytes cultured with pokeweed mitogen secreted IgG1, IgG2, and IgG4 Tg antibodies in amounts proportional to those present in the patient's serum. To investigate this discrepancy further, we cultured EBV-infected lymphocytes from blood, lymph nodes, and thyroid tissue in medium alone and with increasing concentrations of PHA. In individuals with thyroid autoantibodies predominantly of subclass IgG1, PHA enhanced the levels of total Tg antibody synthesis without affecting the IgG subclass distribution. However, in patients with serum autoantibodies of subclasses IgG1, 2, and 4, the increased levels of total Tg antibody synthesis were associated with increased amounts of thyroid autoantibodies of all of these subclasses; in some instances IgG1 and IgG4 autoantibodies were only synthesized in cultures containing PHA. These observations suggest that addition of the T-cell mitogen PHA to cultures of EBV-infected lymphocytes may ensure activation of B-cell precursors committed to synthesizing the IgG subclasses characteristic of serum antibody in the lymphocyte donor. Since Tg antibodies of subclasses IgG2 and IgG4 recognize different epitopes on Tg, the ability to produce human monoclonal antibodies of different IgG subclasses may simultaneously ensure the development of antibodies to different epitopes on the same antigen.

Increased cyclic adenosine monophosphate levels block progression but not initiation of human T cell proliferation.

The involvement of cyclic nucleotides in the regulation of cell growth is well established as are the findings that sustained high levels of intracellular cyclic adenosine monophosphate (cAMP) inhibit ligand-induced lymphocyte proliferation. However, there is considerable controversy concerning the precise role of cAMP in this regard or the target of its action. A number of studies with cAMP analogs or forskolin have implicated the early events after ligand binding as being particularly susceptible whereas others have failed to do so or offered alternate targets. We have examined the effects of cell permeant analogs of cAMP and forskolin on human T cell proliferation. After brief exposure to submitogenic concentrations of PHA or phorbol ester plus ionomycin, we have examined the effects of increased cAMP levels during initial activation (or induction of competence) and progression to DNA synthesis. We find that these drugs that inhibit T cell proliferation have little effect on induction of competence or early activation events including transmembrane Ca2+ uptake, release of Ca2+ from internal stores or induction of the genes encoding c-fos or early growth response gene. In contrast, it is the progression phase of the proliferative response which appears to be the major target for cAMP-protein kinase A-mediated inhibition of human T cell proliferation.

Accessory cell functions in mononuclear cell cultures from uremic patients

Mononuclear cells (PBMC) were isolated from dialyzed patients and healthy control subjects. Lymphocyte responses to stimulation with optimal and suboptimal concentrations of lectin (PHA), or stimulation with T cell receptor antibody (Leu 4) were found decreased in the patient cultures. The separate and the combined effects of exogenous interleukin-1 (IL-1) and interleukin-2 (IL-2) were examined in PHA and Leu 4 stimulated cell cultures. Addition of IL-1 did not normalize the decreased proliferation response of the patient cultures. In contrast, addition of IL-2 alone clearly enhanced and almost normalized the response of patient cultures stimulated with suboptimal concentrations of PHA. The combined addition of IL-1 and IL-2 gave no evidence of an additive effect of IL-1 and IL-2. Cell cultures from uremic and normal HLA-identical relative were examined. Substitution of uremic adherent monocytes with normal adherent monocytes as accessory cells did not improve the uremic T cell responses to stimulation with PHA. Furthermore, uremic adherent cells did not suppress the normal T cell responses. These results suggest that uremic accessory cells support T cell activation and, in particular, do not suppress T cell responses. The effect of IL-2 in the present study as well as previous findings of decreased IL-2 production in patients cultures may indicate that uremia primarily influences the proliferation of T cells.