Retinoic acid and α-galactosylceramide differentially regulate B cell activation in vitro and augment antibody production in vivo

All-trans-retinoic acid (RA) promotes the maturation and differentiation of B cells, which are known as a type of professional antigen-presenting cells. We show here that CD1d, a major histocompatibility complex class I-like molecule that presents lipid antigens, is expressed in the mouse spleen B cells and is increased by RA. Thus, we hypothesized that RA and the CD1d ligand, α-galactosylceramide (αGalCer), could interact to promote the differentiation, maturation, and antibody response of antigen-activated B cells. In isolated B cells, αGalCer alone markedly stimulated, and RA further increased B cell proliferation, synergizing with the B cell antigen receptor ligation via anti-μ antibody (P < 0.05). The significantly increased cell proliferation stimulated by αGalCer was abrogated in the B cells of CD1d-null mice. RA alone and combined with αGalCer also promoted B cell differentiation by the enrichment of sIgG1-, CD138-, and PNA/Fas-positive B cells (P < 0.05), suggesting a plasmacytic cell differentiation. In vivo, wild-type mice treated with RA and/or αGalCer during primary immunization with tetanus toxoid produced a higher serum anti-tetanus IgG response and had more bone marrow anti-tetanus antibody-secreting cells as determined by enzyme-linked immunospot assay (P < 0.05) in the secondary response, a finding indicative of heightened long-term memory; however, the increased antibody secretion after αGalCer treatment was abolished in CD1d-null mice. We provide evidence here that RA, together with αGalCer, can effectively regulate B cell proliferation and differentiation, ultimately promoting a more efficient antibody response to protein antigen. The results suggest that the combination of RA and αGalCer could be a useful adjuvant combination in vaccine strategies.     

Transforming growth factor Β1 influences glycosylation of α1-protease inhibitor in human hepatoma cell lines

We have previously shown that changes in acute-phase protein glycosylation result from alterations occurring within hepatocytes as a result of regulation by cytokines, that the glycosylation patterns of proteins secreted by Hep 3B and Hep G2 cells respond differently to the crude mixtures of cytokines found in conditioned medium from LPS-stimulated monocytes, and that interleukin-6 (IL-6) causes increased concanavalin A (Con A) binding ofl protease inhibitor in Hep 3B cells and decreased Con A binding of this protein in Hep G2 cells. In the present study we found that transforming growth factorl (TGF-), like IL-6, led to secretion of forms ofl-protease inhibitor with increased Con A binding in Hep 3B cells, and that IL-6 and TGF- in combination were additive. In contrast, in Hep G2 cells, TGF- had an effect opposite to that produced by IL-6, leading to secretion of forms ofl-protease inhibitor with increased Con A binding. When employed in combination with IL-6, TGF- abolished the effect of that cytokine. These studies indicate that TGF- influences glycosylation of al-protease inhibitor in two human hepatoma cell lines in a manner that can be differentiated from that of IL-6. The identification of TGF- as a second defined cytokine capable of influencing glycoprotein glycosylation and the demonstration that the effect of one cytokine can be modulated by another cytokine support the view that changes in glycosylation of plasma proteins are mediated by combinations of cytokines.     

Immunoregulatory effects of α-GalCer in a murine model of autoimmune myocarditis

This study was designed to investigate the role of α-galactosylceramide (α-GalCer) on experimental autoimmune myocarditis (EAM), and to explore the underlying mechanisms. Balb/c mice were immunized with porcine cardiac myosin to establish the EAM model. All the immunized mice were divided into two groups, the α-GalCer group and the EAM group. α-GalCer or vehicle was given intraperitoneally at the time of immunization. Then α-GalCer or PBS was injected on alternate days for 6 weeks. Myocardial inflammation was evaluated by H & E staining and the expression levels of C/EBPβ and α-SMA were determined by immunohistochemistry. CD4⁺CD25⁺Foxp3⁺ Tregs and iNKT cells were analyzed and sorted by flow cytometry. Western blot analysis was performed to detect MMP-2 and MMP-9 protein expression. Following α-GalCer treatment for 6 weeks, myocardial inflammation improved significantly in the α-GalCer treated group compared to the EAM group. The proportions of CD4⁺CD25⁺Foxp3⁺ regulatory T cells and NK1.1⁺ iNKT cells were statistically increased in the α-GalCer treated group compared to the EAM and normal control groups. In contrast to the EAM group, α-GalCer treatment significantly increased myocardial MMP-2 and MMP-9 expression. Expression of C/EBPβ increased significantly in the EAM group compared to the other two groups. In contrast, the expression of α-SMA did not differ significantly among the three groups. This study demonstrated that α-GalCer alleviates EAM. Thus, α-GalCer represents a potential therapeutic target for autoimmune-inflammation mediated cardiac damage. α-GalCer protects EAM through upregulation of the proportion of iNKT and Tregs and increased expression of myocardial MMP-2 and MMP-9.     

Ex-vivo α-galactosylceramide activation of NKT cells in humans and macaques

NKT cells are key mediators of antiviral and anticancer immunity. Experiments in mice have demonstrated that activation of NKT cells in vivo induces the expression of multiple effector molecules critical to successful immunity. Human clinical trials have shown similar responses, although in vivo activation of NKT cells in humans or primate models are far more limited in number and scope. Measuring ex vivo activation of NKT cells by the CD1d-restricted glycolipid ligand α-Galactosylceramide (α-GalCer) through cytokine expression profiles is a useful marker of NKT cell function, but for reasons that are unclear, this approach does not appear to work as well in humans and non-human primate macaque models in comparison to mice. We performed a series of experiments on human and macaque (Macaca nemestrina) fresh whole blood samples to define optimal conditions to detect NKT cell cytokine (TNF, IFNγ, IL-2) and degranulation marker (CD107a) expression by flow cytometry. We found that conditions previously described for mouse splenocyte NKT cell activation were suboptimal on human or macaque blood NKT cells. In contrast, a 6h incubation with brefeldin A added for the last 4h, in a 96-well plate based assay, and using an α-GalCer concentration of 1 μg/ml were optimal methods to stimulate NKT cells in fresh blood from both humans and macaques. Unexpectedly, we noted that blood NKT cells from macaques infected with SIV were more readily activated by α-GalCer than NKT cells from uninfected macaques, suggesting that SIV infection may have primed the NKT cells. In conclusion, we describe optimized methods for the ex vivo antigen-specific activation of human and macaque blood NKT cells. These assays should be useful in monitoring NKT cells in disease and in immunotherapy studies.     

Combination of pentoxifylline and α-galactosylceramide with radiotherapy promotes necro-apoptosis and leukocyte infiltration and reduces the mitosis rate in murine melanoma

Despite the success for the treatment of melanoma such as targeted molecular therapy, the use of such treatments are expensive For this reason, this study was carried out to explore the anti-cancer properties of available drugs that are able to modify the melanoma prognosis. The study was conducted in two phases: Evaluation of pharmacological effects of pentoxifylline (PTX) administered above (60 mg/kg) which is the therapeutic dose that is aimed at reducing the side-effect of radiotherapy, and of α- galactosylceramide (GalCer) administered at 100 μg/kg, as well as their combination using a murine model (BDF1 mice) of melanoma cell line (B16-F1, ATCC). For the radiotherapy phase, 9 Gy was applied in the tumor area, before (3 days), during (30 min) and after (3 days) the PTX + GalCer treatment. In both study phases, the mitosis rate, leukocyte infiltration and necro-apoptosis were assessed using histological and immunohistochemical approach and tumor volume evaluation as biomarkers. All treatments showed good prognosis results estimated as reduction of mitosis rate (PTX + GalCer after radiotherapy and GalCer), increased leukocyte infiltrate (PTX + GalCer after radiotherapy and GalCer) and necro-apoptosis augmentation (PTX + GalCer after radiotherapy and radiotherapy control). Nevertheless, a lower development of tumor volume was found in GalCer treatment. In this way, it is possible to suggest that the integrated treatment with immuno-stimulators such as GalCer, plus drug used for peripheral vascular disease (PTX) after radiotherapy is probably an alternative for controlling aggressive melanoma in murine model.     

Potentiation of antitumor effect of NKT cell ligand, α-galactosylceramide by combination with IL-12 on lung metastasis of malignant melanoma cells1

The combined therapeutic effect of natural killer T (NKT) cell ligand α-galactosylceramide (α-GalCer) and IL-12 against highly metastatic B16-BL6-HM melanoma cells was investigated. In comparison with a single administration of α-GalCer or IL-12, the combined treatment of tumor-bearing mice with α-GalCer plus IL-12 caused a super-induction of serum IFN-γ levels, though α-GalCer-induced IL-4 production was rather inhibited. In parallel with the augmented IFN-γ production, the natural killing activity against YAC-1 cells and syngeneic B16- BL6-HM melanoma was greatly augmented by the combined therapy. The major effector cells responsible for natural killing activity induced by α-GalCer plus IL- 12 were enriched in both NK1.1⁺TCRαβ⁺ NKT cells and NK1.1⁺TCRαβ⁻ NK cells. The preventing effect of α-GalCer or IL-12 alone against lung metastasis of B16-BL6-HM was also enhanced by the combination therapy. The antitumor activity of α-GalCer was totally abolished in NKT-deficient mice. However, IL- 12-induced antitumor activity was not eliminated in NKT-deficient mice though it was inhibited by anti-asialo GM1 Ab treatment. These findings suggested that α-GalCer synergistically act with IL-12 to activate both NKT cells and NK cells, which may play a critical role in the strong prevention of distant tumor metastasis at early stages of tumor-bearing. These data will provide a novel tool for the prevention of tumor metastasis using NKT-specific ligands α-GalCer and IL-12. This revised version was published online in July 2006 with corrections to the Cover Date.     

Direct selection of hepatoma cell variants deficient in α1-antitrypsin gene expression

Expression plasmids containing the human ₁-antitrypsin (₁ AT) promoter fused to either adenine phosphoribosyltransferase (aprt) or xanthine-guanine phosphoribosyltransferase (gpt) coding sequences were sequentially introduced into APRT^– HPRT^– rat hepatoma cells. Stable transfectants expressing both transgenes were isolated and characterized. Nonexpressing variants were subsequently obtained by selecting against expression of one or both transgenes. Variants isolated by selecting against expression of either transgene alone generally displayed deficiency phenotypes incis, as only three of 20 clones tested were affected for expression of ₁AT mRNA. In contrast, double selection yielded predominantlytrans effects: 12 of 14 lines tested showed impaired ability to express their chromosomal ₁AT genes. Furthermore, expression of several other liver genes, including the gene encoding the HNF-1trans-activator, was repressed in many of the variant lines. Thus, double selection using chimeric transgenes is a useful approach for generating variant cell lines deficient in expression of specific mammalian genes.     

Inhibitory effects of dimethyl α-ketoglutarate in hepatic stellate cell activation

Aim: The activation of Hepatic stellate cell (HSC) is a pivotal event in the initiation and progression of hepatic fibrosis and a major source of collagen deposition. A recent study found that autophagy fuels the HSC activation. α-ketoglutarate (AKG), an intermediate in the Kerbs CYCLE, has been shown to regulate the level of autophagy. In this study, we aim to investigate the potential effect of dimethyl α-ketoglutarate (DMKG), a membrane-permeable esters of AKG, on the activation of HSC. Methods: HSC and hepatocyte cell lines were treated with DMKG at gradient concentrations, MTT assay was used to assess the cell viability. Concentrations of DMKG that did not affect the cell survival were added to the culture media of HSC cells. Real-time PCR and western blot analysis was carried out to evaluate the expression of fibrogenic genes in HSC after culture for 24 hours. Results: Low dose of DMKG had little cytotoxicity to both HSCs and hepatocytes, while HSCs were more vulnerable to high dose of DMKG than hepatocytes. More importantly, DMKG inhibited the expression of α-SMA and collagen I significantly in HSCs detected by real-time PCR and western blot analysis at the concentrations that didn’t decrease cell viability. Conclusions: DMKG has a significant role of inhibiting the activation of HSC and may attenuate hepatic fibrosis safely.     

Expression and regulation of αB-crystallin in the kidney in vivo and in vitro

αB-crystallin, a major component of the mammalian eye lens, is a small heat shock protein and molecular chaperone that is also abundant in the mammalian kidney. The present study aimed to characterize more closely the intrarenal expression and regulation of αB-crystallin in vivo and in vitro. In normal rat kidney, the expression of αB-crystallin mRNA and protein were both close to the detection limit in cortex, but increased steeply from the outer to the inner medulla where αB-crystallin constitutes approximately 2% of total tissue protein. Immunohistochemistry disclosed papillary collecting duct cells and thin limbs as the major sites for intrapapillary αB-crystallin immunoreactivity. In rats subjected to sucrose diuresis for 3 days, αB-crystallin mRNA expression was reduced by 27 and 46% in outer and inner medulla, respectively. In agreement with the results obtained in vivo, in Madine–Darby canine kidney cells, αB-crystallin mRNA and protein were induced significantly by elevating the medium osmolality to 500 mosm/kg H₂O by the addition of NaCl and raffinose, and also by urea. The NaCl-induced increase in αB-crystallin expression was concentration-dependently blunted by SP600125, a specific JNK inhibitor. Overexpression of αB-crystallin in 293 cells resulted in increased tolerance to acute osmotic stress. These results indicate that αB-crystallin may be regulated by papillary interstitial tonicity in a JNK-dependent process. Moreover, the high abundance of αB-crystallin in the renal medulla may be important for cell survival in an environment characterized by extreme interstitial solute concentrations as present during antidiuresis.     

Cytoprotective effects of high dose of α-galactosylceramide against activation-induced CD4+ T and CD8+ T cell death as an adjuvant

Objective: To investigate the cytoprotective effects of high dose of α-galactosylceramide (α-GC) on the activation-induced CD4+ T and CD8+ T cell death. Methods: Experimental autoimmune encephalomyelitis (EAE) was induced using adoptive transfer of MOGCD4+ cells treated using α-GC into recipient C57BL/6 mice while the MOGCD4+ cells treated using 0.5% polysorbate were set as vehicle group, based on which to investigate the effects of α-GC on activation induced CD4+ T cell death. Additionally, an EG7 tumor-bearing mice model is established using adoptive transfer of CD8+ T cells, based on which to investigate the effect of α-GC on the apoptosis of CD8+ T cells. Results: A higher induction rate was noticed after adoptive transfer of MOGCD4+ cells treated using α-GC together with the severity of EAE compared with the conventional methods. Longer survival duration was noted in the green fluorescent protein (GFP) labeled MOGT in the α-GC group compared with the vehicle group (P < 0.05). Severe inflammatory cell infiltration and myelinoclasis was noted in the white matter of nervous system in the α-GC group. In the EG7 tumor model, more adoptive CD8+ T cells were survived in α-GC group compared with that of vehicle group. The growth of tumor mass was significantly inhibited in α-GC group. Conclusions: high dose of α-GC could be used as an adjuvant for inhibiting activation-induced CD4+ T and CD8+ T cell death. Our study could provide helpful information for the development of adoptive cell therapy with reduced programmed cell death.     

Effect of α1 blockade on structure and function of the renal endocrine system

Research Institute of Cardiology, Ministry of Health of the Uzbek SSR. (Presented by Academician of the Academy of Medical Sciences of the USSR D. S. Sarkisov.) Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 113, No. 2, pp. 211–214, February, 1992.     

A highly bone marrow metastatic murine breast cancer model established through in vivo selection exhibits enhanced anchorage-independent growth and cell migration mediated by ICAM-1

To understand the mechanisms underlying bone marrow metastasis precisely, we established the highly metastatic 4T1E/M3 murine breast cancer cell line. 4T1 murine breast cancer cells were transfected with the neomycin resistance gene, selected in G418, intravenously injected into mice, and harvested from bone marrow. By repeating this protocol three times, we established the 4T1E/M3 cells. The clonality of 4T1E/M3 cells was markedly high confirmed by genomic southern analysis using neo-gene probe. When tissues harvested from mice after intravenous injection of 4T1E/M3 cells were examined histologically, markedly enhanced bone marrow metastasis was observed; 77% of spines from 4T1E/M3-injected mouse showed metastasis as compared to 14% metastasis seen with the parent cells. In vitro, 4T1E/M3 cells attached more strongly to the plastic plate and to bone marrow-derived endothelial cells. DNA micro arrays, real time RT-PCR and FACS analyses revealed that the expression of ICAM-1 and beta2 integrin was upregulated in 4T1E/M3 cells at both the mRNA and cell surface protein levels. 4T1E/M3 cells also showed greater anchorage-independent proliferation in soft agar, and migrated markedly faster than the parent cells in wound healing assays. Anti-ICAM-1 antibodies strongly inhibited both the colony formation and the migration activity of 4T1E/M3 suggesting the importance of the role of ICAM-1. Our newly established highly metastatic 4T1E/M3 cells may provide a potentially powerful tool to study the molecular mechanisms of bone marrow metastasis and to identify new molecular targets for therapeutic interventions.     

Concentration-dependent inversion of antioxidant and prooxidant effects of β-carotene in tissuesin vivo

Low doses (below 20 mg/kg) of oral β-carotene inhibit, while high doses (above 100 mg/kg) activate ascorbate-dependent LPO in rat liver and myocardial cells. Administration of β-carotene in a dose of 20 mg/kg decreased by 34% infarction zone in coronary occlusion, while the dose of 100 mg/kg was ineffective. Anin vivo concentration-dependent inversion of β-carotene from antioxidant to prooxidant in tissues is hypothesized. Pharmacological efficacy of β-carotene is determined by its antioxidant effect, while high doses provoke the prooxidant effect and can lead to negative consequences. Translated fromByulleten' Eksperimental'noi Biologii i Meditsiny, Vol. 128, No. 9, pp. 314–316, September, 1999     

The effects of β1-adrenergic blockade on cardiovascular oxygen flow in normoxic and hypoxic humans at exercise

At exercise steady state, the lower the arterial oxygen saturation (SaO₂), the lower the O₂ return A linear relationship between these variables was demonstrated. Our conjecture is that this relationship describes a condition of predominant sympathetic activation, from which it is hypothesized that selective 1-adrenergic blockade (BB) would reduce O₂ delivery and To test this hypothesis, we studied the effects of BB on and in exercising humans in normoxia and hypoxia. O₂ consumption cardiac output heart rate, SaO₂ and haemoglobin concentration were measured on six subjects (age 25.5±2.4 years, mass 78.1±9.0 kg) in normoxia and hypoxia (inspired O₂ fraction of 0.11) at rest and steady-state exercises of 50, 100, and 150 W without (C) and with BB with metoprolol. Arterial O₂ concentration (CaO₂), and were then computed. Heart rate, higher in hypoxia than in normoxia, decreased with BB. At each was higher in hypoxia than in normoxia. With BB, it decreased during intense exercise in normoxia, at rest, and during light exercise in hypoxia. SaO₂ and CaO₂ were unaffected by BB. The changes under BB were parallel to those in was unaffected by exercise in normoxia. In hypoxia the slope of the relationship between and was lower than 1, indicating a reduction of with increasing workload. was a linear function of SaO₂ both in C and in BB. The line for BB was flatter than and below that for C. The resting in normoxia, lower than the corresponding exercise values, lied on the BB line. These results agree with the tested hypothesis. The two observed relationships between and SaO₂ apply to conditions of predominant sympathetic or vagal activation, respectively. Moving from one line to the other implies resetting of the cardiovascular regulation.     

Roles of transforming growth factor-β in graft-versus-host and graft-versus-tumor effects

Transforming growth factor (TGF)-β is a pleiotropic cytokine with widespread and profound effects on immune cells. Consequently, it has generated considerable interest in relation to the immunologic outcomes after allogeneic hematopoietic cell transplantation. The TGF-β pathway has been shown to be an important modulator of alloimmunity, with direct consequences on graft-versus-host disease pathophysiology and graft-versus-tumor response. The TGF-β-related effects can be both beneficial and detrimental to the host, underscoring the complexity of TGF-β biology. This article reviews the evidence linking TGF-β to alloimmune responses in allogeneic hematopoietic cell transplantation and highlights foreseeable strategies that?would maximize the beneficial effects of TGF-β pathway modulation on both graft-versus-host disease pathophysiology and the graft-versus-tumor effect.     

Functional and metabolic dichotomy of murine γδ T cell subsets in cancer immunity

γδ T cells can display a plethora of immune functions, but recent studies have highlighted their importance, in multiple disease models, as sources of the pro-inflammatory cytokines, IL-17A (IL-17), and IFN-γ. These are produced by distinct murine effector γδ T cell subsets that diverge during thymic γδ T cell development. Among the multiple roles these subsets play in peripheral tissues, a striking dichotomy has emerged at tumor sites: whereas IFN-γ⁺ γδ T cells inhibit tumor cell growth, IL-17⁺ γδ T cells promote tumor progression and metastasis formation. In this review, we discuss the main lines of evidence, mostly from preclinical studies in mouse models, for this functional dichotomy in cancer immunity. We further highlight very recent advances in our understanding how metabolic sources and pathways can impact on the balance between IFN-γ⁺ and IL-17⁺ γδ T cells in the tumor microenvironment, which opens a new exciting avenue to explore toward the application of γδ T cells in cancer immunotherapy.     

Role of interferon-γ on the in vivo expression of functional interleukin-2 receptors by murine macrophages

Interferon - activates both in vitro and in vivo macrophage functions. Injection of rat recombinant interferon- (rR-IFN-) induced the expression of interleukin-2 receptors (1L-2R) by peritoneal macrophages from normal BALB/c and MRL-+/+ mice. Moreover, rR-IFN- stimulated in a dose-dependent manner the oxidative burst of cells as revealed by luminol-dependent chemiluminescene (LDCL). Resident peritoneal macrophages from MRL-lpr/lpr (mice that develop a systemic lupus-like syndrome) showed a higher PMA-triggered LDCL response. This enhanced activity was accompanied by an increase in IL-2R expression (30% vs. < 1%). The activated; macrophages from rR-IFN--treated normal mice as well as MRL-lpr/lpr mice did not respond to the addition of recombinant interleukin-2 (rHu-IL-2) by an inc -ease in LDCL. However, rHu-IL-2 triggering became efficient when cells enriched in IL-2R-bearing macrophages were preincubated overnight with rHu-IL-2R. This response may point out a functional role for IL-2R and provide a role for IL-2 in certain macrophage functions.     

The effects of artificial E-cadherin matrix-induced embryonic stem cell scattering on paxillin and RhoA activation via α-catenin

Mechanical forces have been shown to affect stem cell behavior in a large array of ways. However, our understanding of how these mechanical cues may regulate the behavior of embryonic stem cells (ESCs) remains in its infancy. Here, we aim to clarify the effect of cell scattering on the regulation of Rho family GTPases Rac1 and RhoA as well as paxillin. Allowing ESCs to spread and scatter on a synthetically designed E-cadherin substratum causes phosphorylation of paxillin on consensus phosphorylation sites leading to activation of Rac1 and inactivation of RhoA. By culturing cells in presence of RhoA activator or growing cells to a highly confluent state reverses the effect of cell scattering phenotype. Knockdown of E-cadherin-adapter protein α-catenin revealed that it negatively affects paxillin phosphorylation and up-regulates RhoA activity in compact cellular aggregates. Collectively these results indicate that cell scattering might cause a conformational change of α-catenin limiting its capacity to inhibit paxillin phosphorylation that causes an increase in Rac1 activation and RhoA deactivation. Understanding how synthetically designed extracellular matrix affect ESC signaling through mechanical cues brings a new aspect for stem cell engineers to develop technologies for controlling cell function.