Mitogenic Concentrations Research Articles

In vitro thymocyte differentiation in MHC class I-negative Xenopus larvae

CTX is a surface antigen whose expression in larval and adult Xenopus is primarily restricted to MHC class I-negative immature cortical thymocytes. In adult Xenopus, surface expression of CTX marks a population of MHC class I − CD8 + immature thymocytes that appears to be the equivalent of the mammalian CD4CD8 double positive subset. The present study reveals that transient in vitro exposure of immature CTX + thymocytes from MHC class I-negative tadpoles to suboptimal mitogenic concentrations of phorbol ester (PMA) plus ionomycin, induces larval cells to differentiate into more mature T-lymphoblasts that express high level of surface CD5 and CD45. These T-lymphoblasts have downregulated CTX, Rag 1 and TdT genes, whereas TCR-β genes remain actively transcribed. Signaling induced by PMA/ionomycin modulates both class I and class II expression of MHC class I/II-negative larval thymocytes. This study also reveals that larval T-lymphoblasts are composed of two distinct subsets: CD5 highCD8 − and CD5 highCD8 high.

Antisense Inhibition of Decorin Expression in Myoblasts Decreases Cell Responsiveness to Transforming Growth Factor β and Accelerates Skeletal Muscle Differentiation

Decorin is a member of the family of the small leucine-rich proteoglycans. In addition to its function as an extracellular matrix organizer, it has the ability to activate the epidermal growth factor receptor, and it forms complexes with various isoforms of transforming growth factor beta (TGF-beta). Decorin is expressed during skeletal muscle differentiation and is up-regulated in dystrophic muscle. In this study we investigated the role of decorin in TGF-beta-dependent inhibition of myogenesis. To probe the function of decorin during myogenesis, C(2)C(12) myoblasts were stably transfected with a plasmid expressing antisense decorin mRNA. The resulting inhibition of decorin expression led to the expression of myogenin, a master transcription factor for muscle differentiation, under growth conditions and accelerated skeletal muscle differentiation as determined by the expression of creatine kinase. In contrast myogenin expression was inhibited by adenovirally induced decorin expression or by adding exogenous decorin. Reduced synthesis of decorin resulted in a 7-fold decreased sensitivity to TGF-beta-mediated inhibition of myogenin expression. In contrast, adenovirally induced decorin expression in wild type cells resulted in a 5-fold increased sensitivity to TGF-beta-mediated inhibition of myogenin expression. Transfection studies with the TGF-beta-dependent promoter of the plasminogen activator inhibitor-1 coupled with luciferase revealed that the transducing receptors for TGF-beta1 and TGF-beta2 were involved in the different responses of wild type and antisense decorin myoblasts. These results demonstrate that a reduction of decorin expression or of decorin availability results in a decreased responsiveness to TGF-beta. These findings strongly suggest a new role for decorin during skeletal muscle terminal differentiation by activating TGF-beta-dependent signaling pathways.

Dextran-methylprednisolone succinate as a prodrug of methylprednisolone: in vitro immunosuppressive effects on rat blood and spleen lymphocytes

The in vitro immunosuppressive activity of a conjugate of methylprednisolone (MP) with dextran 70 kDa (DEX-MPS) was tested using the lymphocyte proliferation assay after stimulation of lymphocytes with concanavalin A (Con-A). Blood and spleen lymphocytes, isolated from drug-free male Sprague–Dawley rats, were used in the assay. First, the optimum concentration of Con-A for stimulation of lymphocytes was determined. The inhibition of the lymphocyte proliferation was then tested in the presence of 0.25, 0.5, 1.0, 2.5, 5.0, 10, 20, and 50 nM concentrations (MP equivalent) of DEX-MPS or free MP. The maximum stimulation of lymphocytes with Con-A was observed at mitogen concentrations of 2.5 and 10 μg/ml for the spleen and blood lymphocytes, respectively. For free MP, sigmoidal relationships were observed between the effect (% inhibition of lymphocyte proliferation) and the logarithm of MP concentration. Additionally, the maximum inhibitory effect ( I max) and MP concentration producing half of I max (IC 50) were, respectively, 98% and 1.38 nM for the blood and 86% and 3.1 nM for the spleen lymphocytes. For MP conjugated to dextran, the response–log concentration curves were substantially shifted to the right with IC 50 values of 40 and 52 nM for the blood and spleen lymphocytes, respectively. It is concluded that compared with free MP, the steroid attached to dextran has minimal immunosuppressive activity. Therefore, to be effective in vivo, DEX-MPS should release MP in the body.

Involvement of aldose reductase in vascular smooth muscle cell growth and lesion formation after arterial injury.

Abnormal proliferation of vascular smooth muscle cells (VSMCs) is an important feature of atherosclerosis, restenosis, and hypertension. Although multiple mediators of VSMC growth have been identified, few effective pharmacological tools have been developed to limit such growth. Recent evidence indicating an important role for oxidative stress in cell growth led us to investigate the potential role of aldose reductase (AR) in the proliferation of VSMCs. Because AR catalyzes the reduction of mitogenic aldehydes derived from lipid peroxidation, we hypothesized that it might be a potential regulator of redox changes that accompany VSMC growth. Herein we report several lines of evidence suggesting that AR facilitates/mediates VSMC growth. Stimulation of human aortic SMCs in culture with mitogenic concentrations of serum, thrombin, basic fibroblast growth factor, and the lipid peroxidation product 4-hydroxy-trans-2-nonenal (HNE) led to a 2- to 4-fold increase in the steady-state levels of AR mRNA, a 4- to 7-fold increase in AR protein, and a 2- to 3-fold increase in its catalytic activity. Inhibition of the enzyme by sorbinil or tolrestat diminished mitogen-induced DNA synthesis and cell proliferation. In parallel experiments, the extent of reduction of the glutathione conjugate of HNE to glutathionyl-1,4-dihydroxynonene in HNE-exposed VSMCs was decreased by serum starvation or sorbinil. Immunohistochemical staining of cross sections from balloon-injured rat carotid arteries showed increased expression of AR protein associated with the neointima. The media of injured or uninjured arteries demonstrated no significant staining. Compared with untreated animals, rats fed sorbinil (40 mg. kg(-1). d(-1)) displayed a 51% and a 58% reduction in the ratio of neointima to the media at 10 and 21 days, respectively, after balloon injury. Taken together, these findings suggest that AR is upregulated during growth and that this upregulation facilitates growth by enhancing the metabolism of secondary products of reactive oxygen species.

Mitogenicity and release of vascular endothelial growth factor with and without heparin from fibrin glue.

Fibrin glue (FG) has been used for local cytokine delivery on both vascular grafts and angioplasty sites. We measured the diffusive release of vascular endothelial growth factor (VEGF) and heparin from FG and the mitogenic activity of VEGF with and without heparin in FG on canine endothelial cells (ECs) and smooth muscle cells (SMCs). Release of VEGF labeled with iodine 125 and tritiated heparin from FG into the overlying media was serially measured over 96 hours, and the data are reported as the mean percent released +/- SD. Proliferation assays measuring tritiated thymidine incorporation were performed for ECs and SMCs plated in media with 10% serum on FG containing various concentrations of VEGF and heparin. Media was placed on the FG for 24 hours and removed before plating cells to minimize the effect of the released, soluble VEGF and heparin. At 24 hours, 54% +/- 1% and 58% +/- 1% of the radioactive VEGF and heparin were released, respectively, with minimal release thereafter (58% +/- 1% and 66% +/- 1% at 96 hours). The ECs, SMCs, or media only (no cells) was plated on FG containing radioactive VEGF in an immediate or 24-hour delayed fashion for 72 hours to determine the percent release of VEGF into the media with the two different methods of plating. Cell type and the presence or absence of cells did not affect VEGF release, but there was three times more VEGF in the media for the immediate versus delayed plating (P <.001). Without heparin, VEGF at 100 ng/mL or more in the FG was needed to induce EC proliferation. Heparin at 5 U/mL enhanced EC proliferation at the VEGF dose of 100 ng/mL as compared wtih no heparin (P <.001), but not at the VEGF dose of 1000 ng/mL, which likely represents a maximal response. With heparin at 500 U/mL, the ECs died. In contrast, VEGF, in the presence or absence of heparin, did not affect SMC proliferation. We conclude that FG with VEGF at 1000 ng/mL and heparin at 5 U/mL is the optimal concentration for in vivo use because this may encourage EC, but not SMC, proliferation. The VEGF at 1000 ng/mL should leave mitogenic concentrations of VEGF intact after the initial, diffusive loss, and the addition of heparin at 5 U/mL may enhance VEGF mitogenic activity.

Effects of photoperiod and melatonin on lymphocyte activities in male broiler chickens

Understanding the role of the pineal gland in regulating the immune response and the role of photoperiod in influencing pineal gland secretions are becoming increasingly important. The purposes of the present experiments were to investigate the effects of different photoperiod regimens on T- and B-lymphocyte activities in broiler chickens. Next, the influence of different photoperiod regimens on the responsiveness of lymphocytes to melatonin in vitro was examined. The effect of melatonin in vitro on lymphocyte activities was also studied, regardless of the photoperiod received. Finally, the effects of photoperiod on the profiles of different splenocyte cell types were investigated. To study the effect of photoperiod on lymphocyte activities, different photoperiod regimens were used. These were: constant lighting, 23 h light:1 h darkness; intermediate lighting, 12 h light:12 h darkness; and intermittent lighting, 1 h light:3 h darkness. Peripheral blood and splenic lymphocyte activities were tested at 3 and 6 wk of age by performing a mitogen cell-proliferation assay with a polyclonal T-cell mitogen, concanavalin A (Con A), and T-dependent B-cell mitogen, pokeweed mitogen (PWM). To study the effect of photoperiod on the responsiveness of lymphocytes to melatonin in vitro or the effect of melatonin in vitro on lymphocyte activities regardless of photoperiod received, lymphocytes from the chickens that were exposed to the different photoperiod regimens were incubated with mitogen and different concentrations of melatonin. To study the effect of photoperiod on profiles of different cell types, the percentages of splenocyte subpopulations from birds exposed to different photo-periods were determined using flow cytometry with CD4+, CD8+, CD3+, and B-cell markers. The results of these studies indicate that splenic T and B lymphocytes from 6-wk-old chickens grown in intermittent lighting had higher activities than those from chickens grown in constant lighting. Peripheral blood and splenic lymphocytes from chickens raised under constant lighting were more responsive to melatonin in vitro than those from chickens raised under intermittent lighting. This difference in response may be due to lower levels of melatonin in birds receiving constant lighting, making them more sensitive to melatonin in vitro. Melatonin in vitro enhanced the mitogenic response of peripheral blood T lymphocytes from 6-wk-old chickens, splenic T lymphocytes from 3-wk-old chickens, and splenic T and possibly B lymphocytes from 6-wk-old chickens. Finally, intermittent lighting increased the percentages of splenic CD4+, CD8+, and CD3+ cells but not B-cell subpopulations at 6 wk of age, presumably because of increased levels of melatonin in birds receiving intermittent lighting. Our results re-emphasize the importance of melatonin in regulating host immune response; this regulation could be accomplished through exposing broiler chicks to intermittent lighting.

The role of phosphatidic acid in platelet-derived growth factor-induced proliferation of rat hepatic stellate cells.

Platelet-derived growth factor (PDGF) is the most potent mitogen for hepatic stellate cells (HSCs) in vitro. The aim of this study was to investigate the role of the lipid-derived second messenger phosphatidic acid (PA) in mediating this effect and, in particular, to determine its interaction with the extracellular signal-regulated kinase (ERK) cascade. HSCs were isolated from rat livers. PA production was determined by lipid extraction and thin-layer chromatography (TLC) after prelabeling cells with [(3)H]myristate. ERK activity was measured by an in vitro kinase assay after immunoprecipitation. Mitogenic concentrations of PDGF, but not those of the relatively less potent mitogen, transforming growth factor alpha (TGF-alpha), stimulated the sustained production of PA from HSCs. Exogenous PA stimulated HSC proliferation and a sustained increase in ERK activity, and proliferation was completely blocked by the inhibition of ERK activation with PD98059. The stimulation of ERK by PDGF was of a similar magnitude but more sustained than that caused by TGF-alpha. These results suggest that the potent mitogenic effect of PDGF in HSCs may be caused, in part, by the generation of PA and subsequently by a more sustained activation of ERK than occurs with less potent mitogens that do not induce the production of this lipid second messenger.

Effects of Extracellular Calcium on the Proliferation, Differentiation and Gene Expressions of Bone Morphogenetic Proteins-2 and -4 in Human Gingiva-Derived Fibroblasts.

The current study was undertaken to determine the effects of elevated extracellular calcium on the proliferation, differentiation, and gene expressions of bone morphogenetic proteins-2 and-4 (BMP-2 and-4) and transforming growth factor-β1 (TGF-β1) in human gingiva-derived fibroblasts (HFB-G cells) in vitro. Extracellular calcium was increased by the addition of varying concentrations of CaCl2 (0.1-1.2mM) into DMEM (1.8mM CaCl2). An increase in extracellular calcium significantly increased cell proliferation with an optimal dose of 1.2mM, as assessed by [3H] thymidine incorporation into DNA. Elevated extracellular calcium failed to affect the cellular alkaline phosphatase (ALP) activity in HFB-G cells. The effects of mitogenic concentrations of extracellular calcium on the gene expressions of BMP-2 and-4 and TGF-β1 in HFB-G cells were evaluated by semiquantitative reverse transcription-polymerase chain reaction (RT-PCR) after 0.5-24 hours. Exposure to CaCl2 increased the BMP-2 mRNA levels to 0.4 mM and the BMP-4 mRNA levels to 0.1mM at 0.5and 24 hours, but it did not affect the mRNA levels of TGF-β1. By contrast, decreased extracellular calcium, induced by the addition of EGTA, markedly decreased the mRNA levels of BMP-2 and -4 after incubation for 0.5-24 hours at 1.0mM. The present study offers new evidence that an increase in extracellular calcium can stimulate the mRNA levels of BMP-2 and -4, as well as the cell proliferation, in human fibroblasts in vitro.

A new quantitative RT–PCR assay for thymidylate synthase mRNA in blood leukocytes applied to cancer patients and healthy controls

Thymidylate synthase (TS) is the target enzyme for 5-fluorouracil (5-FU). TS mRNA and protein levels in colorectal tumours are among the most important determinants for tumour response to 5-FU. TS mRNA levels in blood leukocytes may give information on pharmacokinetic and pharmacodynamic actions of 5-FU on TS as it has previously been shown that inhibition of TS levels by 5-FU in bone marrow leukocytes resembles the degree of TS inhibition in colorectal tumours. The aim of this study was to develop a quantitative high-throughput RT–PCR assay for TS mRNA expression in blood leukocytes (CURT–PCR). Furthermore the TS mRNA levels in blood of patients with colorectal cancer and healthy controls was compared. TS mRNA levels in 17 patients with colorectal cancer did not differ from 20 matched controls whereas a group of 14 younger controls had significantly lower TS mRNA expression than patients and matched controls. In order to investigate the sensitivity of the assay towards cellular reactions such as proliferative stimuli, isolated blood leukocytes were stimulated with phytohemagglutinin both in mitogenic and non-mitogenic concentrations and an induction of TS mRNA expression was measured in both cases. TS activity and cellular proliferation also increased but only at mitogenic concentrations, suggesting that TS mRNA expression is an early leukocyte activation marker. This new CURT–PCR assay may allow improved studies of functional kinetics of drugs with impact upon TS. Further studies are required to establish the possible clinical benefit of TS mRNA measurements in blood leukocytes.

Effect of estradiol and exercise on lymphocyte proliferation responses in female mice

Many North American women are encouraged to take estrogen replacement therapy and to begin (or continue) physical activity during the postmenopausal years. This study investigated the effect of estradiol exposure and physical exercise on lymphocyte proliferation responses to T and B cell mitogens in female mice. Sixty C57BL/6 mice were randomized to hormone and exercise treatment conditions: hormone treatment consisted of estradiol in vivo (71.4 μg estradiol per day for 21 days) or placebo pellet following bilateral ovariectomy, or surgical sham (mice were not ovariectomized). Exercise consisted of a single forced treadmill run (26 m/min, 6° slope, 90 min) or a sedentary control condition. Outcomes were thymic and splenic lymphocyte proliferation responses to the mitogens concanavalin A (ConA, 1.0 and 5.0 μg/mL concentrations) and lipopolysaccharide (LPS, 0.5, 1.0, and 2.0 μg/mL concentrations). In the thymus, there was a significant reduction of proliferation to ConA in the Ovx + E2 animals relative to the other conditions at both concentrations of mitogen. At 1.0 μg/mL concentration, there was a significant interaction of hormone and exercise treatments. Sham (control) mice given exercise had a higher proliferation response relative to sedentary counterparts, whereas E2 mice did not differ in proliferation responses, irrespective of exercise condition. In the spleen, exposure to high concentrations of estradiol was associated with reduced proliferation responses to both mitogens; there were, however, no main or interaction effects of exercise. These results suggest that high levels of estradiol exposure following ovariectomy in mice significantly reduces lymphocyte blastogenesis responses, and that thymic immunomodulation after acute exercise is masked by the hormonal effect.

Quantitative and Qualitative Signals Determine T-Cell Cycle Entry and Progression

Cell growth and proliferation as well as cell cycle arrest and apoptosis all play integral roles in the cellular immune response. The signals that lead to cytokine production by antigen- or mitogen-stimulated T cells have been studied in detail. However, it is not fully understood how these signals promote cell cycle entry and progression to DNA synthesis in T lymphocytes, especially in primary cells. We used a model distinguishing between competence and progression phases to examine quantitative and qualitative differences in signal transduction that resulted in cell cycle entry and G1 phase arrest or led to DNA synthesis in human T cells. Resting peripheral blood T cells were rendered competent by stimulation with submitogenic concentrations of phytohemagglutinin (PHA) or they were stimulated to proliferate using mitogenic concentrations of PHA. The competent state (that is, the capacity to proliferate in response to exogenous IL-2) was characterized by calcium mobilization, a protein kinase C-dependent internalization of CD3, increased mitogen-activated protein kinase (MAPK) activity, transient translocation of AP-1 transcription factors to the nucleus, expression of immediate early genes, activation of G1-phase cyclin-dependent kinases, and increased CD25 (IL-2Rα) expression. However, all of these events were of lesser magnitude in T cells rendered competent than in T cells stimulated to proliferate. Furthermore, the mitogenic stimulus induced a different pattern of MAPK activation and sustained translocation of AP-1 to the nucleus with concomitant IL-2 production. The data indicate that quantitative and qualitative differences in early signaling events distinguish the acquisition of the competent state or the induction of cytokine production with a commitment to T-cell proliferation.

Vascular endothelial growth factor VEGF-like heparin-binding protein from the venom of Vipera aspis aspis (Aspic viper).

The heparin-binding dimeric hypotensive factor (HF) was purified from Vipera aspis aspis (Aspic viper) venom [Komori, Y. and Sugihara, H. (1990) Toxicon 28, 359-369]. In this study, the amino acid sequence, and structure and function of HF, were elucidated. By electrospray ionization mass spectrometry (ESI-MS), the molecular weight of HF was determined to be 25 072.1. The complete amino acid sequence of HF was determined by Edman sequencing of the S-pyridylethylated HF and its peptides derived from enzymatic digestion. The theoretical molecular mass calculated from the primary structure agrees well with the molecular weight determined by ESI-MS. HF consists of two homogeneous monomers bound covalently. The monomer with an N-terminal blocked by pyroglutamic acid contains 110 amino acid residues, including eight cysteine residues, two of which are considered to be involved in intermolecular disulfide bonds. Sequential homology search revealed that the primary structure of HF is similar to that of vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) with a sequential homology of 45 and 22%, respectively. When injected intradermally into a rat, an increase in capillary permeability was observed with HF or VEGF. On the other hand, only HF exerted a strong hypotensive effect after intravenous injection of samples into a rat. Purified HF has a mitogenic effect on endothelial cells. Through the use of bovine aortic endothelial cells (BAEC), the half-maximal mitogenic concentration of HF was determined to be 5-5. 5 nM (125-138 ng/mL). Similarly, VEGF had a mitogenic concentration at 0.5-1 nM. When incubated with HF and cycloheximide or HF and heparin, the cell growth was inhibited, suggesting that the mechanism of action of HF is similar to that of VEGF.

Transplantation of CD34 human cells into mice with severe combined immunodeficiency results in functional T cells 4 weeks after transplantation

Objective: Our purpose was to determine whether transplantation of fetal human CD34 + cells into mice with severe combined immunodeficiency results in functional T cells. Study Design: The cells used in this study were isolated from fetal human liver tissue obtained after elective termination of normal 18- to 24-week pregnancies. Women with medical conditions that could confound the outcome were excluded. Cells were labeled with fluorochrome-conjugated antibodies that recognized CD34 or other cell surface antigens. The cells were then sorted with the use of a fluorescein-activated cell sorter. The human sorted cells were injected intraperitoneally in mice with severe combined immunodeficiency. Four groups of mice were studied: group 1, injected with 10 5 CD34 + cells (n = 17); group 2, injected with 10 5 CD34 – cells (n = 14); group 3, injected with 10 6 unsorted cells (n = 19); and group 4, sham-injected with phosphate-buffered saline solution as controls (n = 14). At 1, 2, and 4 weeks after transplantation, the peripheral blood monocytes of the study mice were analyzed for functional T cells. Aliquots of cells (10 5) were incubated for 48 hours with 0, 5, 10, and 20 μg of phytohemagglutinin. Thereafter the cells were treated with 1 μCi of tritiated thymidine. Subsequently the incorporation of tritiated thymidine was determined by liquid scintillation counting. Results: Cells from mice transplanted with either unsorted cells, sorted CD34 + cells, or CD34 – cells showed a response to phytohemagglutinin that varied with time and with the mitogen concentration. Even though unsorted fetal human liver cells had a maximal response at 2 weeks, this posttransplantation response was not statistically significant. CD34 + cell response to phytohemagglutinin was significant at 4 weeks after transplantation. CD34 – cells also had a peripheral blood cell response at 4 weeks after transplantation; however, this response was not statistically significant. In addition, all mice transplanted with fetal human liver cells had some functional T cells at 4 weeks; however, this response was statistically significant only for CD34 + cells. Conclusion: Transplantation of either sorted CD34 (positive or negative) cells or unsorted fetal human liver cell preparations into mice with severe combined immunodeficiency results in functional T cells. However, only the mice with transplanted CD34 + cells demonstrated a statistically significant response. (Am J Obstet Gynecol 1999;181:80-6.)

CD38 Stimulation Lowers the Activation Threshold and Enhances the Alloreactivity of Cord Blood T Cells by Activating the Phosphatidylinositol 3-Kinase Pathway and Inducing CD73 Expression

We have recently described in cord blood T cells (CBTC) a novel pathway linking CD38 and CD73, two signal transducers with ecto-enzyme activity. The aim of this study was 2-fold: first, to characterize the mechanisms by which CD38 regulates CD73 expression; and second, to determine whether surface-induced CD73 modulates CBTC responses. A marked increase in CD73 expression was observed in CD38+ cells after incubation with the appropriate CD38 mAbs. The induction of CD73 was blocked by wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3-K). CD38 stimulation induced tyrosine phosphorylation of the p85 regulatory subunit of PI3-K and its association with other tyrosine-phosphorylated proteins. Surface-induced CD73 was as efficient in delivering activatory signals as the CD73 constitutively expressed on adult T cells. Highly CBTC, totally unresponsive to mitogenic concentrations of plastic-immobilized CD3 mAb, proliferated vigorously when exposed to the combination of plastic-immobilized CD3 and CD73 mAbs. The reactivity to allogeneic irradiated PBMC was also significantly enhanced by CD38 stimulation and was dependent on CD73 expression. Thus, CD38 stimulation lowers the activation threshold of CBTC by the CD3/TCR complex and enhances their reactivity to allogeneic cells via activation of the PI3-kinase pathway and CD73 expression.

In vitro differentiation of a CD4/CD8 double-positive equivalent thymocyte subset in adult Xenopus.

The thymus is the major site of selection and differentiation of T cells in mammals and birds. To begin to study the evolution of thymocyte differentiation, we have developed, in the frog Xenopus, an in vitro system that takes advantage of cortical thymocyte antigen (CTX), a recently discovered T cell antigen whose expression is restricted to Xenopus cortical thymocytes. Upon transient stimulation with suboptimal mitogenic concentrations of the phorbol ester phorbol myristate acetate (PMA) plus ionomycin, Xenopus thymocytes are induced to differentiate into cycling T lymphoblasts that actively synthesize and express high levels of surface MHC class I and class II molecules. This appearance of T lymphoblasts correlates with a rapid down-regulation of both surface CTX protein and CTX mRNA. A thymocyte subset with an immature phenotype (CTX+, CD8+, class II- or class II low and class I-) was characterized by depleting class II+ cells or by panning with anti-CTX mAb. This immature CTX+ thymocyte subset displays a limited proliferative capacity compared to total, class II+ or to CTX- thymocytes, and can be induced, by PMA/ionomycin, to differentiate into more mature T lymphoblasts expressing surface class II and class I molecules. These results provide the first in vitro evidence in an ectothermic vertebrate of a conserved intrathymic pathway of thymocyte differentiation. In addition, our data reveal that CTX can serve as a differentiation surface marker of a population of immature thymocytes that appears to be the equivalent of the mammalian CD4/CD8 double-positive subset.

Stimulation by low concentrations of fluoride of the proliferation and alkaline phosphatase activity of human dental pulp cells in vitro

Fluoride has been used for decades, either systemically or topically, to prevent dental caries. The purpose of this study was to clarify the effects of low concentrations of fluoride on proliferation, differentiation and extracellular-matrix synthesis in normal human dental pulp cells (DP-1 and DP-2) in vitro. The effects were compared with those on a human osteoblastic osteosarcoma cell line, TE-85. Fluoride at micromolar concentrations significantly and dose-dependently stimulated [ 3H]thymidine incorporation into DNA in DP-1, DP-2 and TE-85 cells, with optimal effects around 50 μM, by 127±7%, 124±0.6% and 152±13.4%, respectively. To assess the potential influence of fluoride on cell differentiation, the effects of mitogenic concentrations on alkaline phosphatase activity were measured. Fluoride significantly increased the enzyme’s activity in DP-1 and TE-85 by 177±12% and 144±12.3%. To evaluate the effect on extracellular-matrix synthesis, the synthesis of type I collagen was indirectly determined by an assay of procollagen type I c-peptide production. Fluoride significantly increased that production by 150±8.7% in TE-85, but not in either DP-1 or DP-2. These observations suggest that fluoride, if used at low concentrations, could be a useful therapeutic agent where increased regeneration of dentine is desired, such as after pulp amputation, by stimulating the proliferation and differentiation of the dental pulp cells.

5-HT moduline metabolism in mammalian brain: Identification of two enzymatic activities

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.

Fluoxetine Action on Murine T-Lymphocyte Proliferation: Participation of PKC Activation and Calcium Mobilisation

The present study was undertaken to analyse the effect of fluoxetine upon murine T-lymphocyte proliferation. We found that fluoxetine exerted a dual effect, which depended on the degree of lymphocyte activation: at mitogenic concentration (2 μg/mL) of concavalin A (Con A), we observed an inhibitory effect on cellular proliferation, whereas, on submitogenic Con A concentration (1 μg/mL), fluoxetine stimulated the cellular response. Given these facts, we studied PKC activation and calcium mobilisation in both stimulatory and inhibitory effects of fluoxetine on T-cell proliferation. We observed that fluoxetine increased PKC translocation obtained with 1 μg/mL Con A concentration, whereas PKC was degraded when 2 μg/mL was used. This mechanism is thought to be mediated by calcium mobilisation. According to our results, fluoxetine seemed to modulate calcium influx, which, in turn, would influence PKC translocation, modulating the immune response.

Neonatal dendritic cells

The capacity of lymphoid dendritic cells from human cord blood or adult peripheral blood to support a mixed leukocyte reaction in cord blood and adult T cells has been compared. Cord blood dendritic cells have a limited ability to induce either adult or cord blood T cells to proliferate in response to typical concentration of phytohemagglutinin or concanavalin A. Adult blood dendritic cells, on the other hand, induce equivalent mitogen responses in cord blood and adult blood T cells. This relative deficiency can be overcome by increasing the concentration of mitogen or the numbers of dendritic cells in the culture. Neonatal primary immune responses may, in part, reflect the reduced function of dendritic cells.

Iron Deficiency Reduces the Hydrolysis of Cell Membrane Phosphatidyl Inositol-4,5-Bisphosphate during Splenic Lymphocyte Activation in C57BL/6 Mice

Iron deficiency impairs lymphocyte proliferation in humans and laboratory animals by unknown mechanisms. In this study, we investigated whether this alteration can be attributed in part to impaired hydrolysis of cell membrane phosphatidyl inositol-4,5-bisphosphate (PIP2), a required early event of T-lymphocyte activation. The study involved 46 iron-deficient (ID), 26 control (C) and 23 pair-fed (PF) mice, and ID mice that were repleted for 3 (n = 16), 7 (n = 17) or 14 d (n = 18). Mice were killed after 40–63 d (mean, 48 d) of consuming the test diet (0.09 mmol/kg iron) or the control diet (0.9 mmol/kg). The mean (±SEM) hemoglobin concentrations were 57 ± 16.7, 176 ± 2.6 and 181 ± 9.7 g/L for ID, C and PF groups, respectively. After splenic lymphocytes were labeled in vitro with 3H-myoinositol for 3 h, PIP2 hydrolysis was estimated by measuring the radioactivity recovered as a mixture of inositol mono-, di- and triphosphate (IP) from concanavalin A (0, 1, 2.5, 5 and 10 mg/L) activated cells. Although cells from ID mice and those from mice repleted for 3 d incorporated slightly more radioactivity in cellular phospholipids than did cells from C or PF mice, less (P < 0.005) was recovered as IP than in controls, suggesting impaired conversion of the precursor to PIP2. At almost all incubation periods (10–120 min) and mitogen concentrations, the rate of PIP2 hydrolysis expressed as the ratio of radioactivity obtained in Con A–treated to untreated cells was significantly (P < 0.05) reduced in cells from ID mice compared with those obtained from C and PF mice. For cells that were activated for 60 min or less, iron repletion for 14 d significantly (P < 0.05) improved the rate of PIP2 hydrolysis. PIP2 hydrolysis positively and significantly (P < 0.05) correlated (r = 0.27–0.56) with indicators of iron status. Mitogenic response was also significantly (P < 0.05) reduced in ID but not PF mice, and it was corrected by iron repletion for 3, 7 or 14 d. Lymphocyte proliferation positively (r = 0.27–0.37, P < 0.01) correlated with indices of iron status and IP ratios. The data suggest that reduced PIP2 hydrolysis contributes to impaired blastogenesis in iron deficiency.