Proliferation Of Murine Splenocytes Research Articles

Mac1 positive cells are required for enhancement of splenocytes proliferation caused by bisphenol a.

We examined the effects of bisphenol A (BPA) on immune cells and it was shown that BPA upregulated the proliferation of murine splenocytes stimulated with Concanavalin A (ConA). The upregulating effects of BPA were removed with depleting Mac1+ cells from the splenocytes. This study provides evidence for the first time that Mac1+ cells were required for enhancement of splenocytes proliferation caused by bisphenol A.

FR252921, a novel immunosuppressive agent isolated from Pseudomonas fluorescens no. 408813. I. Taxonomy, fermentation, isolation, physico-chemical properties and biological activities of FR252921, FR252922 and FR256523.

Novel immunosuppressive agents, FR252921, FR252922 and FR256523 were isolated from the cultured broth of a bacterial strain No. 408813. The strain was identified Pseudomonas fluorescens from morphological and physiological characteristics. FR252921, FR252922 and FR256523, novel compounds containing macrolactone ring, showed immunosuppressive activity against murine splenocyte proliferation stimulated with lipopolysaccharide (LPS) or anti-CD3 mAb in vitro.

Synthetic analogues of the bacterial signal (quorum sensing) molecule N-(3-oxododecanoyl)-L-homoserine lactone as immune modulators.

Comparative immune modulatory activity for a range of synthetic analogues of a Pseudomonas aeruginosa signal molecule, N-(3-oxododecanoyl)-l-homoserine lactone (3O, C(12)-HSL), is described. Twenty-four single or combination systematic alterations of the structural components of 3O, C(12)-HSL were introduced as described. Given the already defined immunological profile of the parent compound, 3O, C(12)-HSL, these compounds were assayed for their ability to inhibit murine and human leucocyte proliferation and TNF-alpha secretion by lipopolysaccharide (LPS) stimulated human leucocytes in order to provide an initial structure-activity profile. From IC(50) values obtained with a murine splenocyte proliferation assay, it is apparent that acylated l-homoserine lactones with an 11-13 C side chain containing either a 3-oxo or a 3-hydroxy group are optimal structures for immune suppressive activity. These derivatives of 3O, C(12)-HSL with monounsaturation and/or a terminal nonpolar substituent on the side chain were also potent immune suppressive agents. However, structures lacking the homoserine lactone ring, structures lacking the l-configuration at the chiral center, and those with polar substituents were essentially devoid of activity. The ability of compounds selected from the optimal activity range to modulate mitogen-driven human peripheral blood mononuclear cell proliferation and LPS-induced TNF-alpha secretion indicates the suitability of these compounds for further investigation in relation to their molecular mechanisms of action in TNF-alpha driven immunological diseases, particularly autoimmune diseases such as psoriasis, rheumatoid arthritis, and type 1 (autoimmune) diabetes.

Renal tubular epithelial expression of the costimulatory molecule B7RP-1 (inducible costimulator ligand).

MHC class II-expressing renal tubular epithelial cells (TEC) are able to present foreign peptide antigens to T cells. The costimulatory signals that are required for effective T cell activation upon antigen presentation by TEC have not been characterized. Various cultured TEC lines were examined for expression of the recently described costimulatory molecule B7RP-1 (B7h), a ligand of the T cell molecule inducible costimulator (ICOS), and expression was compared with that of B7.1, B7.2, and CD40. B7RP-1 and CD40 were abundantly expressed by cultured murine and human TEC, whereas B7.1 and B7.2 could not be detected. Stimulation with lipopolysaccharide or tumor necrosis factor-alpha did not induce B7.1 or B7.2 expression and did not alter B7RP-1 expression. Interestingly, interleukin-2 production by T cell hybridomas after antigen presentation by TEC was enhanced by blocking antibodies to B7RP-1 and ICOS. In contrast, blocking antibodies to B7RP-1 or ICOS exerted inhibitory effects on anti-CD3-activated murine splenocyte proliferation. Immunohistochemical staining of normal human kidneys demonstrated strong constitutive B7RP-1 expression in distal tubules, collecting ducts, and urothelium. In human kidneys with allograft rejection or interstitial nephritis, distinct B7RP-1 staining was also detected in proximal tubules, in areas of mononuclear infiltration. In conclusion, the B7RP-1/ICOS pathway negatively regulates T cell activation upon MHC class II-restricted antigen presentation by TEC. Because B7RP-1 is also expressed by tubules in vivo, it can be speculated that the B7RP-1/ICOS pathway could play an inhibitory role in TEC-mediated immune activation in the kidney.

Immunomodulating properties of Argentine plants with ethnomedicinal use.

Five Argentine medicinal plants selected according to folk traditional or ethnomedical use, references and primary pharmacological screening; were chosen to elucidate their immunomodulating properties. Dichloromethane, methanolic and aqueous extracts of the aerial parts of Achyrocline flaccida (A. flaccida), Eupatorium arnottianum (E. arnottianum) and Eupatorioum buniifolium (E. buniifolium), leaves of Lithraea molleoides (L. molleoides) and leaves and stems of Phyllanthus sellowianus (P. sellowianus) were analyzed to disclose their effects on murine normal and tumor cell growth as well as on complement hemolytic activity. Modulation of cell growth was evaluated by tritiated thymidine incorporation while inhibition of complement activity was measured on both classical and alternative complement pathways (CP and AP respectively). The results obtained show that most of the extracts exerted inhibitory effect on tumor as well as on mitogen activated normal spleen cell growth. On tumor cells, IC50 ranged between 1-75 microg/ml for most of the extracts with the exception of dichloromethane of L. molleoides and P. sellowianus which required concentrations higher than 100 microg/ml to produce the effect. On mitogenic activated splenocytes, IC50 ranged between < 1 to 85 microg/ml with the exception of methanolic extract of E. buniifolium or P. sellowianus which were not effective on ConA or LPS stimulated splenocytes respectively. Only E. buniifolium was active on murine normal splenocytes proliferation (IC50 0.5-1.5 microg/ml). Finally, one (7%) of 15 extracts showed inhibition of complement activity on CP and 6 extracts (40%) presented moderate activity on CP. The dichloromethane extract of E. arnottianum was the most active (IC50 5 microg/ml), although remarkable effect was also obtained with dichloromethane and methanolic extracts of P. sellowianus (IC50 11.2 and 17.3 microg/ml respectively). Besides, 2 extracts (13%), dichloromethane extract of E. arnottianum and aqueous extract of P. sellowianus, showed moderate inhibition on AP.

Isolation of cicadin, a novel and potent antifungal peptide from dried juvenile cicadas

A single-chained antifungal protein with a molecular weight of 6.5 kDa and displaying a novel N-terminal sequence was isolated from dried juvenile cicadas which are used in traditional Chinese medicine, by using ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose and then gel filtration on a Superdex peptide column. The peptide, designated cicadin, exerted potent antifungal activity with IC 50 values at nonomolar concentrations against a variety of fungi including Botrytis cinerea, Mycosphaerella arachidicola, Fusarium oxysporum, Rhizoctonia solani and Coprinus comatus. Cicadin suppressed the activity of HIV-1 reverse transcriptase and stimulated the proliferation of murine splenocytes.

A Bowman–Birk-Type Trypsin-Chymotrypsin Inhibitor from Broad Beans

An isolation procedure comprising affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Toyopearl, and fast protein liquid chromatography on Mono S was used to purify a peptide from broad beans which manifested antifungal activity toward Mycosphaerella arachidicola, Fusarium oxysporum, and Botrytis cinerea. The peptide demonstrated a molecular mass of 7.5 kDa. N-terminal sequence analysis disclosed the identity of the antifungal peptide to be a trypsin-chymotrypsin inhibitor. The trypsin-chymotrypsin inhibitor also exerted an inhibitory action on chymotrypsin activity and HIV-1 reverse transcriptase activity. Proliferation of murine splenocytes was stimulated in the presence of the trypsin-chymotrypsin inhibitor. This report constitutes the first observation of antifungal activity of a leguminous peptidic protease inhibitor.

Cytokine adjuvancy of BVDV DNA vaccine enhances both humoral and cellular immune responses in mice

The effect of cytokine adjuvancy on a bovine viral diarrhoea virus (BVDV) DNA vaccine expressing the major glycoprotein E2 was investigated in mice. Murine interleukin-2 (IL-2) and granulocyte-macrophage colony-stimulating factor (GM-CSF) were chosen for their potential ability to enhance the humoral and cellular immune responses involved in protection against BVDV. Both cytokines, co-administered as separate plasmid constructs, had a marked effect on ELISA and neutralising antibody titres, improving the spectrum of neutralisation induced by the E2 DNA vaccine, as demonstrated in heterologous neutralisation assays. The predominance of IgG2a isotypes, in sera from all DNA injected groups, indicated a Th1 biased immune response. Antigen specific proliferation of murine splenocytes from immunised mice was enhanced by cytokine co-administration, with the highest stimulation indexes observed in the group co-injected with the GM-CSF construct. These results obtained in the mouse (Balb/c; H2-k d) animal model demonstrate the value of the two cytokines as adjuvants for the E2 DNA vaccine. The need for an adjuvant in this system was emphasised by the MHC restriction observed when C57BL/6 mice (H2-k b) were immunised with the E2 DNA construct. Antibody levels were dramatically lower in this mouse strain.

Effects of KE-758; an active metabolite of the new anti-rheumatic drug KE-298, d-penicillamine, bucillamine and auranofin on the proliferation of murine lymphocytes, and the production of nitric oxide by murine macrophages

2-Mercaptomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (KE-758), which is the active metabolite of 2-acetylthiomethyl-4-(4-methylphenyl)-4-oxobutanoic acid (KE-298), is a novel sulphydryl anti-rheumatic drug. In this study we analyzed the effect of KE-758 on the proliferation of murine lymphocytes, and on the production of nitric oxide (NO) by RAW264.7 murine macrophage cells. We compared its effect with other sulphydryl drugs such as d-penicillamine, bucillamine and auranofin. The proliferation of lymphocytes was measured by 3H-thymidine incorporation assay. Nitrite was measured using Griess Reagent. In the absence of copper ions, KE-758, d-penicillamine and bucillamine rarely affected the proliferation of concanavarin A (ConA) activated murine splenocytes. However, in the presence of copper, pharmacological concentrations of KE-758 but not d-penicillamine and bucillamine suppressed the proliferation of murine splenocytes through a hydrogen peroxide-dependent mechanism. Auranofin markedly suppressed the proliferation regardless of the presence of copper ions by reducing the cellular viability. Furthermore, only KE-758 markedly suppressed the proliferation of phorbol myristate acetate (PMA) plus ionomycin activated murine whole blood lymphocytes (WBL) even in the absence of exogenous copper ions by a hydrogen peroxide-independent mechanism. Meanwhile, lipopolysaccharide (LPS) or LPS plus interferon-gamma (IFN-γ) induced NO production by RAW264.7 cells were suppressed by KE-758 and auranofin but not by d-penicillamine and bucillamine. In conclusion, KE-758 is a novel immunosuppressive drug, which inhibits both lymphocyte and macrophage functions and its unique anti-rheumatic profile is distinct from that of d-penicillamine, bucillamine and auranofin.

Effects of Fructus Ligustri Lucidi Extracts on Concanavalin A-Stimulated Proliferation of Isolated Murine Splenocytes

Fructus Ligustri Lucidi (FLL), the fruit of Ligustrum lucidum Ait., was fractionated into petroleum ether (FLL-Pe), chloroform (FLL-Ch), butanol (FLL-Bu) and aqueous (FLL-Aq) fractions. The effects of FLL fractions and oleanolic acid (OLA), an active ingredient of FLL, on concanavalin A (Con A)-stimulated proliferation of murine splenocytes were examined under both in vitro and ex vivo conditions. In vitro administration of FLL-Pe could produce an immunostimulatory effect, as evidenced by the significant enhancement of the Con A-stimulated mitogenic response of splenocytes in culture. While FLL-Ch and OLA did not produce any detectable effect on the mitogenic response, FLL-Bu and FLL-Aq inhibited the in vitro ConA-stimulated splenocyte proliferation. In ex vivo experiments, pretreatment with FLLPe, FLL-Ch or OLA was found to enhance the extent of Con A-stimulated splenocyte proliferation to a varying degree as assessed under in vitro conditions, with FLL-Pe being more potent. The ensemble of results indicates that both FLL-Pe and FLL-Ch were able to produce immunostimulatory action under ex vivo assay conditions, but only FLL-Pe produced a similar effect under in vitro conditions. The immunostimulatory action produced by FLL-Pe could not be fully explained by the presence of OLA.

Ginkbilobin, a Novel Antifungal Protein from Ginkgo biloba Seeds with Sequence Similarity to Embryo-Abundant Protein

A novel single-chained antifungal protein with a molecular weight of 13 kDa displaying an N-terminal sequence with marked similarity to embryo-abundant protein from the white spruce was isolated from the seeds of Ginkgo biloba using ion exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion exchange chromatography on SP-Sepharose, and then gel filtration on Superdex 75. The protein, designated ginkbilobin, exerted potent antifungal activity against a variety of fungi, including Botrytis cinerea, Mycosphaerella arachidicola, Fusarium oxysporum, Rhizoctonia solani, and Coprinus comatus. Ginkbilobin exhibited a moderate antibacterial action against Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli. It suppressed the activity of HIV-1 reverse transcriptase and the proliferation of murine splenocytes.

Differential impact of nicotine on cellular proliferation and cytokine production by LPS-stimulated murine splenocytes

The immunoregulatory effects of nicotine have not been fully clarified and the reported data are often conflicting. The present study investigated the role of nicotine as an immunomodulator of murine splenocytes stimulated by lipopolysaccharide (LPS), the endotoxin component of gram-negative bacteria. BALB/c female mice of two different ages, young (2–3 months) and old (18–22 months), were used. The cells were incubated with nicotine at two different time points, 3 h pre-incubation and concurrent incubation relevant to LPS stimulation, before further incubation for 48 or 72 h. Treatment of murine splenocytes with nicotine showed an impact on cellular proliferation as well as on the production of the pro-inflammatory cytokines, tumor necrosis factor-alpha (TNF- α) and interleukin-6 (IL-6). The results indicated that nicotine significantly inhibited cellular proliferation of murine splenocytes in a concentration-related manner (32, 64 and 128 μg/ml). Timing of nicotine exposure prior to LPS stimulation was critical in terms of immunological impact on cytokine production. TNF- α and IL-6 production were significantly enhanced by 1 μg/ml of nicotine when cells were pre-incubated with nicotine for 3 h compared to concurrent incubation relative to LPS stimulation. The alteration in cytokine production varied with the age of the mouse. TNF- α production was significantly inhibited by nicotine in young mice, while IL-6 production was significantly inhibited by nicotine in old mice. Since any immunomodulation that alters the profile of these cytokines may cause an imbalance in the immune system impinging on health status, these findings may be important when dealing with the concept of nicotine as a therapeutic agent.

Effect of dietary vitamin E on apoptosis and proliferation of murine splenocytes

Vitamin E (Vit E) is one of the most potent natural lipophilic antioxidants shown to inhibit proliferation of cancer cells and influence apoptosis in in-vitro systems. In the present investigation, we studied the effect of dietary Vit E on splenocyte proliferation, apoptosis, and necrosis. Two-month old male ICR mice were fed an AIN tm 76 formula diet for three months with 10% (W/W) corn oil, supplemented with Vit E at normal (75 IU/kg) or higher (500 IU/kg) levels, or without Vit E as zero Vit E group. In vivo analysis revealed both serum lipid peroxides (MDA) and splenocyte peroxides (dichlorofluorescein staining) were found to be reduced with increased dietary Vit E levels in a dose-dependent manner; whereas, serum Vit E levels were found to be augmented with increased levels of dietary Vit E. Also, apoptosis and necrosis were measured in splenocytes incubated with and without 1 μM dexamethasone (DEX) in RPMI media for eight and 21 hours. Cells were stained with Annexin V and Propidium Iodide (PI), and both apoptotic and necrotic cells were analyzed simultaneously by a FACScan desktop flow cytometer using the Cell Quest Program. High and normal levels of Vit E increased necrosis by 29 and 18% in the cells placed in media, and 67 and 34% in cells incubated with DEX for eight hours, respectively, in comparison to that in the zero Vit E-fed group. However, in splenocytes from the high Vit E group apoptosis was decreased by 32 and 49.5% in cells incubated for eight hours in media alone or with DEX, respectively. Furthermore, in the high Vit E group necrosis was increased by 25% and apoptosis was decreased by 17% in cells incubated for 21 hr in media alone, compared to that of the zero Vit E group. In addition, proliferation of splenocytes, either stimulated or unstimulated with mitogens, was also found to be decreased with increasing levels of dietary Vit E. In conclusion, our results indicate that dietary Vit E appears to alter both cell proliferation and programmed cell death (apoptosis vs. necrosis), by lowering lipid peroxides.

Polychlorinated biphenyl mixtures (Aroclors) inhibit LPS-induced murine splenocyte proliferation in vitro

The immune system is believed to be a sensitive indicator for adverse polychlorinated biphenyl (PCB)-induced health effects. Four commercial PCB mixtures (Aroclors) or six individual PCB congeners were evaluated for their effect on splenocyte viability and lipopolysaccharide (LPS)-induced splenocyte proliferation in vitro in two strains of mice, C57B1/6 (high affinity aromatic hydrocarbon receptor (AhR) complex) and DBA/J (low affinity AhR complex). All four Aroclors, the selected individual noncoplanar congeners, or two tertiary mixtures containing one congener from each class significantly decreased the in vitro LPS-induced proliferation of murine splenocytes in either strain of mice without inducing a significant decrease in viability. In contrast, selected individual coplanar or mono- ortho-coplanar congeners did not inhibit splenocyte proliferation or viability at any concentration. These results suggest that mixtures of PCBs and/or congener class (specifically, noncoplanar congeners) may be more highly immunotoxic than individual planar and mono- ortho-coplanar congeners alone. Thus, this in vitro assay has revealed a more complex pattern of immunotoxicity of Aroclors versus individual congeners than has previously been reported or anticipated based on both in vivo derived immunotoxic data and standard comparisons to 2,3,7,8-tetrachlorodibenzo- p-dioxin (TCDD). These results have important practical significance since mixtures of PCB congeners were used industrially and now contaminate the environment.

Isolation and partial characterization of immunostimulating polysaccharides from Imperata cylindrica.

The water-soluble crude extract prepared from Imperata cylindrica (Beauv.) was investigated for its immunomodulating activity. A set of polysaccharides with high molecular weights has been isolated by fractionation using gel filtration and anion-exchange chromatography. Each step of purification was monitored by bioassays. The presence of six monosaccharides has been established by chemical analysis. Quantitative analysis showed that the ratio of these monosaccharides differed from one polysaccharide to another. The crude extract as well as some of the purified polysaccharides enhance the proliferation of murine splenocytes.

Modulating Effects of Sensory and Autonomic Neuropeptides on Murine Splenocyte Proliferation and Cytokine Secretion Indd by Leishmania Major

The intimate, bidirectional link between neuroendocrine and immune systems is now accepted. A modulating effect of the nervous system on immune and inflammatory responses has been corroborated by identification of neuropeptide receptors on immunocompetent cells and the finding that neuropeptides can regulate leukocyte functions. The present study was undertaken to investigate the possible immunomodulatory role of sensory (SOM, CGRP and SP) and autonomic (VIP and NPY) neuropeptides in a murine model of cutaneous leishmaniasis, using two genetically different inbred mouse strains, BALB/c and C57BL/6, respectively susceptible and resistant to Leishmania (L.) major infection. The parameters studied were extent of splenocyte proliferation, as measured by thymidine uptake, and the ability of these cells to secrete IFN-γ and IL-4 by using a two-site ELISA, upon in vitro challenge with L. major parasites and addition of the neuropeptides. The resistant mouse splenocyte proliferation was enhanced by SOM, CGRP, and VIP at 10−5', 10−6 and 10−9 M concentration, respectively, but was inhibited by NPY at 10−5M. Proliferation of the splenocytes from the susceptible strain was inhibited by SOM (10−11 M) and CGRP(10−5 M). Somatostatin, at various concentrations, stimulated IFN-γ secretion in both mouse strain splenocytes, and IL-4 production in the susceptible mouse. Calcitonin gene-related peptide enhanced IFN-γ secretion in susceptible mouse splenocytes at 10−6 10−7 and 10−9 M, as did VIP at 10−10 M and NPY at 10−7 M. Vasuactive intestinal peptide also stimulated IL-4 production in BALB/c splenocytes at all concentrations used Substance P had no effect on either cell proliferation or cytokine sccrction in eithcr of the two mouse strains.These findings indicatc that the nervous system, represented by sensory and autonomic nerve terminals and their content of neuroinediators, may be involved in the pathophysiology of cutaneous leishmaniasis.

Human fetal retinal pigment epithelial cells induce apoptosis in allogenic T-cells in a Fas Ligand and PGE2 independent pathway

Purpose. To evaluate the inhibitory effect of human fetal retinal pigment epithelium (HFRPE) on the activation of human T-cells. Methods. Pure cultures of HFRPE cells were incubated with purified human T-cells in three different activation assays: 1) allogenic peripheral blood mononuclear cells; 2) OKT3 coated beads in the presence of accessory cells; and 3) stimulation with phorbol ester and phytohemagglutinin. Results. HFRPE cells suppressed the activation of T-cells in all three assays. The mechanism of HFRPE mediated T-cell suppression was apoptosis. The role of Fas ligand(FasL)/Fas-mediated T-cell suppression was excluded, since FasL protein or mRNA could not be detected on HFRPE cells with flow cytometry and by reverse transcriptase polymerase chain reaction, respectively. Additionally, the inhibitory effect of HFRPE cells could not be blocked by anti-Fas ligand or antagonistic anti-Fas antibodies. Moreover, HFRPE cells suppressed the proliferation of anti-CD3 mAb mediated T-cell proliferation of murine splenocytes isolated from lpr mice. The inhibitory effect of HFRPE cells was not PGE 2 mediated, since indomethacin could not restore the T-cell activation. Although the HFRPE mediated T-cell apoptosis was cell-cell contact independent, it was not induced by secretion of TNF-a, TGF-ß, or IL-10. The ratio between HFRPE and T-cells had a major impact on the HFRPE's inhibitory effect. Conclusions. HFRPE cells suppressed the activation of human T-cells by induction of T-cell apoptosis through a process that involves the secretion of soluble factors. The HFRPE mediated T-cell suppression was dependent on the ratio between HFRPE and T-cells. This undefined pathway of T-cell apoptosis may play a role in the maintenance of immune privilege in the subretinal space and may reduce the severity of the immune response after HFRPE transplantation.

CD28 COSTIMULATION OF PROLIFERATION IN MURINE SPLENIC T CELLS IS SENSITIVE TO INHIBITION BY SP100030, A NOVEL T CELL-SPECIFIC DUAL INHIBITOR OF NF-??B AND AP-1

101 Introduction: SP100030 (SP) is the only known T cell-specific dual inhibitor of both NF-κB and AP-1, and its inhibition of these transcription factors suppresses cytokine production in T cell lines. Our goal has been to investigate the in vivo efficacy of SP and to define its mechanism of action on normal T cells. We previously showed that SP suppressed host vs graft murine popliteal lympho node hyperplasia, rejection of murine heart allografts, and inhibited murine splenocytes proliferation in vitro by suppressing IL-2 production. Now we show that SP inhibits not only CD3-or Ca2+/PMA-triggered T cell proliferation but also CD28-mediated costimulation of cell proliferationin vitro, and eventually blocks cell cycle progression of T cells at the G0-G1 stage. Methods: (1) Cell culture: C3H splenic T cells were isolated by positive selection using anti-thy1.2 microbeads. Cells were stimulated with plate-adherent anti-CD3 mAb ± soluble anti-CD28 mAb or different combinations of PMA, ionomycin (INM) and anti-CD28 mAb. The cells were incubated with or without drugs (SP or cyclosporine, CsA) at 37°C in 5% humidified CO2. (2) Cell proliferation assay: After 30 hrs of culture, cells were pulsed for 12 hrs with 3H-TdR and incorporated3 H-TdR was measured at 42 hr. (3) PCNA and cell cycle analysis: Intracellular expression of PCNA and DNA content in the cells were simultaneously measured using cytometry at 42 hr. Results: Both SP and CsA suppressed CD3-triggered T cell proliferation by >90%. Addition of CD28 costimulation to CD3 triggering augmented T cell proliferation by 24 fold at the highest concentration of CsA, but only 6 fold at the highest concentration of SP. Although T cell proliferation induced with PMA+INM was inhibited by >90% by both SP and CsA, stimulation both by PMA+CD28 and by PMA+INM+CD28 was inhibited by>90% by SP, but not at all by CsA. Inhibition of 3H-TdR incorporation by SP or CsA correlated inversely with PCNA expression and the number of cells in S+G2/M phase; both drugs increased the number of apoptotic cells and cells in G0 phase. Conclusions: (1) CD28 costimulation for CD3-triggered T cell proliferation is sensitive to inhibition by SP, but resistant to inhibition by CsA. (2) CD28 costimulation is sensitive to inhibition by SP, but insensitive to inhibition by CsA when T cells were stimulated with PMA alone or PMA plus INM. (3) SP as well as CsA blocks T cell cycle progression at the G0-G1 stage. Therefore SP inhibits not only CD3- and Ca2+/PMA-triggered T cell proliferation but also CD28-mediated costimulation resistant to CsA, eventually blocking cell cycle progression of T cells at the G0-G1 stage.

An immunoaffinity-purified Trypanosoma cruzi antigen suppresses cellular proliferation through a TGF-beta-mediated mechanism.

Two subfractions with opposite immunological properties were obtained from the flagellar antigens (FF) of Trypanosoma cruzi epimastigotes by immunoaffinity chromatography. The ligand-bound material (Ag 123) contained four polypeptide bands of 97, 55, 38 and 14 kDa. The nonretained flow-through (FT), induced a potent proliferation of murine naive splenocytes. In contrast, Ag 123 inhibited the proliferative capacity of the FT as well as the proliferation mediated by the mitogen Concanavalin A (Con A). The suppressive effect of Ag 123 on the Con A-mediated proliferation was neutralized by an anti-TGF-beta monoclonal antibody. Both Ag 123 and FF stimulated high serum levels of TGF-beta in injected mice. Ag 123 also induced in vitro secretion of TGF-beta by murine splenocytes. These results demonstrate that Ag 123 is a potent stimulator of TGF-beta both in vivo and in vitro. Oligopeptides derived from the 38 kDa protein present in Ag 123 showed homology with human and rat alpha-fetoproteins (AFP). Ag 123 seems to have a key role in the immunosuppression that develops during early stages in the infection with T. cruzi.

In vitro effects of smokeless tobacco extract on tumor necrosis factor- α (TNF- α) and interleukin-1 β (I l-1 β) production, and on lymphocyte proliferation

The use of smokeless tobacco (moist snuff) products is associated with mucosal lesions, gingival recession, attachment loss, and oral cancer. Despite numerous reports on the general toxic effects of smokeless tobacco extract, little information is available regarding the specific effects of smokeless tobacco on immune response. Inflammatory cytokines released as a result of smokeless tobacco-induced irritation may play a role in the development of oral mucosal lesions at habitual tobacco placement sites in smokeless tobacco users. Consequently, the purpose of this study was to determine whether an aqueous extract of smokeless tobacco (STE) affects the secretion of tumor necrosis factor- α (TNF- α), interleukin -1 β (I L-1 β), and the proliferation of lymphocytes. A macrophage cell line (J774-A1) was used to measure the effects of STE on tumor necrosis factor- α (TNF- α) and interleukin -1 β (I L-1 β) secretion. Mouse splenocytes were used to assess the effects of STE on lymphocyte proliferation. We found that STE at low concentrations enhanced the production of both TNF- α and I L-1 β. Furthermore, STE at similar concentrations enhanced mitogen-induced murine splenocyte proliferation. Overall, these data suggest that smokeless tobacco upregulated two key proinflammatory cytokines and also induces lymphocyte proliferation.