Induction of tumor-specific T lymphocyte responses in vivo by prothymosin α

We have recently reported that administration of Pro T to DBA/2 mice before the inoculation of syngeneic L1210 leukemic cells prolonged the survival of these animals by (a) inducing tumoricidal peritoneal macrophages, (b) enhancing natural killer (NK) and inducing lymphokine-activated killer (LAK) activities in splenocytes and (c) inducing the production of interleukin-2 and tumor necrosis factor [Papanastasiou et al. (1992) Cancer Immunol Immunother 35:145; Baxevanis et al. (1994) Cancer Immunol Immunother 38:281]. In this report we demonstrate that Pro T , when administered simultaneously with L1210 tumor cells, is capable of generating in DBA/2 animals tumorspecific CD8⁺ cytotoxic T lymphocytes (CTL). The Pro T -induced CD8⁺ CTL lysed their syngeneic L1210 targets in a major histocompatibility complex (MHC)-restricted fashion since monoclonal antibodies (mAb) against the H-2K^d allelic product could inhibit the cytotoxic response. Mice receiving only Pro T developed non-MHC-restricted cytotoxic activity (NK, and LAK activities) whereas those receiving Pro T and L1210 tumor cells developed both MHC-restricted (CTL) and non-MHC-restricted cytotoxic activities and survived longer. The Pro T -induced CD8⁺ CTL activity was regulated by Pro T -induced L1210-specific syngeneic CD4⁺ cells. This was shown in two different ways: first, CD8⁺-cell-mediated cytotoxic responses against L1210 targets were associated with L1210-specific and MHC-restricted proliferative responses of syngeneic CD4⁺ cells and, second, CD4⁺ cells from mice that had received both Pro T and L1210 tumor cells could enhance in vitro the otherwise weak, MHC-restricted and L1210-specific cytotoxicity of syngeneic CD8⁺ cells from mice that had received only L1210 cells. Our data suggest that Pro T is capable of inducing nonspecific, as well as tumor-specific CTL responses in vivo. This is of importance since Pro T may prove to be useful in clinical protocols aimed at cancer immunotherapy.This work was supported by a CEC grant to Dr. M. Papamichail     

Induction of lipolysis in vitro and loss of body fat in vivo by zinc-α2-glycoprotein

Loss of adipose tissue in cancer cachexia has been associated with tumour production of a lipid-mobilizing factor (LMF) which has been shown to be homologous with the plasma protein zinc-α₂-glycoprotein (ZAG). The aim of this study was to compare the ability of human ZAG with LMF to stimulate lipolysis in vitro and induce loss of body fat in vivo, and to determine the mechanisms involved. ZAG was purified from human plasma using a combination of Q Sepharose and Superdex 75 chromatography, and was shown to stimulate glycerol release from isolated murine epididymal adipocytes in a dose-dependent manner. The effect was enhanced by the cyclic AMP phosphodiesterase inhibitor Ro20-1724, and attenuated by freeze/thawing and the specific β3-adrenoreceptor antagonist SR59230A. In vivo ZAG caused highly significant, time-dependent, decreases in body weight without a reduction in food and water intake. Body composition analysis showed that loss of body weight could be attributed entirely to the loss of body fat. Loss of adipose tissue may have been due to the lipolytic effect of ZAG coupled with an increase in energy expenditure, since there was a dose-dependent increase in expression of uncoupling protein-1 (UCP-1) in brown adipose tissue. These results suggest that ZAG may be effective in the treatment of obesity.     

Differential regulation of immature articular cartilage compressive moduli and Poisson’s ratios by in vitro stimulation with IGF-1 and TGF-β1

Mechanisms of articular cartilage growth and maturation have been elucidated by studying composition-function dynamics during in vivo development and in vitro culture with stimuli such as insulin-like growth factor-1 (IGF-1) and transforming growth factor-beta 1 (TGF-β1). This study tested the hypothesis that IGF-1 and TGF-β1 regulate immature cartilage compressive moduli and Poisson’s ratios in a manner consistent with known effects on tensile properties. Bovine calf articular cartilage from superficial-articular (S) and middle-growth (M) regions were analyzed fresh or following culture in medium with IGF-1 or TGF-β1. Mechanical properties in confined (CC) and unconfined (UCC) compression, cartilage matrix composition, and explant size were assessed. Culture with IGF-1 resulted in softening in CC and UCC, increased Poisson’s ratios, substantially increased tissue volume, and accumulation of glycosaminoglycan (GAG) and collagen (COL). Culture with TGF-β1 promoted maturational changes in the S layer, including stiffening in CC and UCC and increased concentrations of GAG, COL, and pyridinoline crosslinks (PYR), but little growth. Culture of M layer explants with TGF-β1 was nearly homeostatic. Across treatment groups, compressive moduli in CC and UCC were positively related to GAG, COL, and PYR concentrations, while Poisson’s ratios were negatively related to concentrations of these matrix components. Thus, IGF-1 and TGF-β1 differentially regulate the compressive mechanical properties and size of immature articular cartilage in vitro. Prescribing tissue growth, maturation, or homeostasis by controlling the in vitro biochemical environment with such growth factors may have applications in cartilage repair and tissue engineering.     

Prior stimulation of antigen-presenting cells with Lactobacillus regulates excessive antigen-specific cytokine responses in vitro when compared with Bacteroides

The development of allergy is related to differences in the intestinal microbiota. Therefore, it is suggested that the immune responses induced by different genera of bacteria might be regulated through adaptive as well as innate immunity. In this study, we examined whether antigen-specific immune responses were affected by stimulation with the different genera of intestinal bacteria in vitro. Mesenteric lymph node (MLN) cells isolated from germ-free ovalbumin (OVA)-specific T cell receptor transgenic (OVA-Tg) mice were stimulated with OVA and intestinal bacteria. Cecal contents from conventional mice but not germ-free mice could induce OVA-specific cytokine production. Among the murine intestinal bacteria, Bacteroides acidofaciens (BA) enhanced OVA-specific IFN-γ and IL-10 production while Lactobacillus johnsonii (LA) increased OVA-specific IL-10 production only. The expression of cell surface molecules and cytokine production by antigen-presenting cells (APCs) from germ-free Balb/c mice were analyzed. BA increased the expression of MHC II and co-stimulatory molecules on APCs compared with LA. BA increased IL-6 and IL-10 production but induced less IL-12p40 than LA. To examine the effects of prior stimulation of APCs by intestinal bacteria on the induction of antigen-specific immune responses, cytokine production was determined following co-culture with OVA, CD4⁺ T cells from OVA-Tg mice, and APCs which were pre-stimulated with the bacteria or not. APCs pre-stimulated with LA did not enhance OVA-specific cytokine production while BA stimulated OVA-specific IL-10 production. These results suggest that the prior stimulation of intestinal immunocytes by Lactobacillus might regulate excessive antigen-specific cytokine responses via APCs when compared with prior stimulation by Bacteroides.     

A critical regulation of Th2 cell responses by RORα in allergic asthma

Allergic asthma is a chronic inflammatory disease of the lung and the airway, which is characterized by aberrant type 2 immune responses to otherwise unharmful aeroallergens. While the central role of Th2 cells and type 2 cytokines in the pathogenesis of allergic asthma is well documented, the regulation and plasticity of Th2 cells remain incompletely understood. By using an animal model of allergic asthma in IL-4-reporter mice, we found that Th2 cells in the lung expressed higher levels of Rora than those in the lymph nodes, and that treatment with an RORα agonist SR1078 resulted in diminished Th2 cell responses in vivo. To determine the T cell-intrinsic role of RORα in allergic asthma in vivo, we established T cell-specific RORα-deficient (Cd4creRora^f/f) mice. Upon intranasal allergen challenges, Cd4creRora^f/f mice exhibited a significantly increased Th2 cells in the lungs and the airway and showed an enhanced eosinophilic inflammation compared to littermate control mice. Studies with Foxp3^YFP-creRora^f/f mice and CD8⁺ T cell depletion showed that the increased Th2 cell responses in the Cd4creRora^f/f mice were independent of Treg cells and CD8⁺ T cells. Our findings demonstrate a critical regulatory role of RORα in Th2 cells, which suggest that RORα agonists could be effective for the treatment of allergic diseases.

     

In vitro and in vivo pro-angiogenic effects of thymosin-β4-derived peptides

Thymosin-β4 (Tβ4) is a G-actin sequestering peptide involved in regeneration and remodeling of injured tissues. In this work, we have designed and synthesized three peptide sequences containing the N-terminus (TYB4-n), the central part (TYB4-i) or the C-terminus (TYB4-c) of Tβ4. All fragments are overlapping on the main central binding actin site. After a structural characterization, we have evaluated in vitro and in vivo their pro-angiogenic effects. The results of this study have shown that: (i) each fragment reproduces the native conformation; (ii) Tβ4-derived peptides exert both in vitro and in vivo pro-angiogenic effects; (iii) their in vitro effect seem to be related to the activation of several signaling pathways and is positively modulated by the N-terminus of Tβ4.     

Immunotherapy with MVA-BN?-HER2 induces HER-2-specific Th1 immunity and alters the intratumoral balance of effector and regulatory T cells

MVA-BN?-HER2 is a new candidate immunotherapy designed for the treatment of HER-2-positive breast cancer. Here, we demonstrate that a single treatment with MVA-BN?-HER2 exerts potent anti-tumor efficacy in a murine model of experimental pulmonary metastasis. This anti-tumor efficacy occurred despite a strong tumor-mediated immunosuppressive environment characterized by a high frequency of regulatory T cells (T(reg)) in the lungs of tumor-bearing mice. Immunogenicity studies showed that treatment with MVA-BN?-HER2 induced strongly Th1-dominated HER-2-specific antibody and T-cell responses. MVA-BN?-HER2-induced anti-tumor activity was characterized by an increased infiltration of lungs with highly activated, HER-2-specific, CD8+CD11c+ T cells accompanied by a decrease in the frequency of T(reg) cells in the lung, resulting in a significantly increased ratio of effector T cells to T(reg) cells. In contrast, administration of HER2 protein formulated in Complete Freund's Adjuvant (CFA) induced a strongly Th2-biased immune response to HER-2. However, this did not lead to significant infiltration of the tumor-bearing lungs by CD8+ T cells or the decrease in the frequency of T(reg) cells nor did it result in anti-tumor efficacy. In vivo depletion of CD8+ cells confirmed that CD8 T cells were required for the anti-tumor activity of MVA-BN?-HER2. Furthermore, depletion of CD4+ or CD25+ cells demonstrated that tumor-induced T(reg) cells promoted tumor growth and that CD4 effector cells also contribute to MVA-BN?-HER2-mediated anti-tumor efficacy. Taken together, our data demonstrate that treatment with MVA-BN?-HER2 controls tumor growth through mechanisms including the induction of Th1-biased HER-2-specific immune responses and the control of tumor-mediated immunosuppression.     

Induction of prophage λ by daunorubicin and derivatives correlation with antineoplastic activity

The antineoplastic drug daunorubicin and 15 other anthracyclines were tested for their ability to induce prophage λ in Escherichia coli K12. Prophage λ induction by daunorubicin was obtained in excision-repair deficient uvr⁻ bacteria at doses about 3-fold lower than in excision-repair proficient uvr⁺ cells; this suggests that some of the lesions produced in DNA by daunorubicin are subject to excision repair and may be adducts. Daunorubicin seems to be converted to active species capable of causing prophage inducing lesions in DNA by bacterial enzymes. The antineoplastic and prophage inducing potencies of the anthracyclines were compared in a blind test. These two parameters were correlated for two thirds of the compounds. Such a correlation supports the idea that the antineoplastic activity of the anthracyclines is a consequence of their capacity to damage DNA.     

Induction of Abortion by Extra-amniotic Administration of Prostaglandins E2 and F2 α

The use of prostaglandins E₂ and F₂α, administered by extra-amniotic instillation, for the induction of abortion was studied in 94 patients in the first and second trimesters of pregnancy. Abortion was successfully induced in 87% of patients within 36 hours and in 94% within 48 hours. The mean abortion time was 22·4 hours. In 60% of patients abortion was complete.Though the differences were not statistically significant, on average multigravid patients aborted more quickly than primigravidae, while the mean abortion time in PGE₂-treated patients was less than in those receiving PGF₂α.No serious complications occurred. Some side effects were observed. Occasional vomiting was the commonest symptom but the incidence of side effects was lower than with alternative routes of administration. A leucocytosis was often noted but there were no significant instances of infection.The method has proved a safe and effective means of terminating pregnancies in the second trimester.     

Induction of aromaticl-amino acid decarboxylase mRNA by interleukin-1β and prostaglandin E2 in PC12 cells

Aromatic 1-amino acid decarboxylase (AADC) is involved in the synthesis of the putative neurotransmitters dopamine (DA), norepinephrine (NA) and 5-hydroxytryptamine (5-HT). We report here that the gene expression of AADC can be regulated by interleukin (IL) 1- and prostaglandin (PG) E₂ in PC12 cells. The cells were treated with different doses of IL 1- and PGE₂ for 3 days. Slot blot hybridization was performed to detect AADC mRNA and Western immunoblot to detect AADC protein. The cDNA probe for rat AADC was generated by the PCR method. IL 1- and PGE₂ produced a dose- and time-dependent up-regulation in AADC mRNA levels (up to 200% of the control values) which was followed by a stable increase in AADC protein. The data further support the suggestion that AADC is a regulated enzyme and that the regulation occurs at the level of gene expression. Because IL-1 is synthesized, and acts locally, within the brain to influence neuronal and glial functions, it has been proposed to be a mediator with both beneficial and detrimental responses to inflammation and injury. The regulation of AADC by IL-1 may indicate a possible involvement for AADC in neuronal injury and recovery. Since IL-1 promotes PGE₂ formation, its effects may be occurring by increasing level of PGE₂.Abbreviations AADC aromatic 1-amino acid decarboxylase - IL-1 interleukin 1 - PGE₂ prostaglandin E₂ - GITC guanidinium isothiocyanate - DEPC diethyl pyrocarbonate - MOPS 3-(4-morpholino)propanesulfonic acid - SSPE 0.18M NaCl, 0.001M sodium phosphate, and 0.001M EDTA Special issue dedicated to Dr. Bernard W. Agranoff.     

Characterization of a micro-roughened TiO2/ZrO2 coating： mechanical properties and HBMSC responses in vitro

Previous studies have shown phase to bioceramics can that using ZrO2 as a second significantly increase the bonding strength of plasma-sprayed composite material. In the present study, micro-roughened titanium dioxide/ zirconia （TiO2/ZrO2） （30 wt% ZrO2） coating and TiO2 coating were plasma-sprayed onto Ti plates. The microstructural characteristics and mechanical properties of both coatings were investigated. Furthermore, the biological behavior and osteogenic differentiation of human bone marrow mesenchymal stem cells （HBMSCs） on both TiO2/ZrO2 and TiO2 coatings were compared. The results indicated that the shear bond strength and microhardness of TiO2/ZrO2 coating were statistically higher than those of TiO2 coating. Scanning electron microscope observation revealed that more irregularly shaped protuberances and denser pores were formed on the surface of TiO2/ ZrO2 coating compared with those of TiO2 coating. Further comparative analysis of HBMSC proliferation and osteogenic differentiation on both coatings showed that significantly higher cellular alkaline phosphatase activity and expression levels of Runx2 and Osterix at day 10 after osteogenic culture were found on TiO2/ZrO2 coating compared with TiO2 coating, while no statistically significant difference in cell proliferation and extracellular calcium deposition was observed. The present study suggests that TiO2/ZrO2 coating may be favorable for dental implant applications.     

Role of α5β1 and αvβ3 integrins in relation to adhesion and spreading dynamics of prostate cancer cells interacting with fibronectin under in vitro conditions

Interaction of cell integrins with the ECM (extracellular matrix) proteins is commonly assumed to be associated with cell dissemination and tumour metastases. Since these processes depend on the mechanism of cell-protein interaction, we have attempted to show the contribution of α5β1 and αvβ3 integrins of the prostate cancer PC-3 cells in in vitro interaction with FN (fibronectin) adsorbed on defined polystyrene surfaces. Cell adhesion, spreading and cytoskeleton organization were studied using antibodies against integrins or a GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro) peptide. The results show that blocking the α5β1 integrin causes: (i) a decrease in the number of the adherent cells in the early phase of adhesion and (ii) a decrease in the dynamics of cell spreading and cell shape changes, and weaker reorganization of cytoskeletal proteins than in the control cells. Conversely, the blocking of the αvβ3 integrin: (i) causes no observable effect on the number of the adhered cells; however, (ii) causes an increase in the dynamics of cell spreading and cell shape changes, and stronger reorganization of cytoskeletal proteins than in the control cells. Interestingly, the blocking of integrins with a GRGDSP peptide strongly decreases the number of the adhered cells, and a complete inhibition of cell spreading. Our results strongly suggest that the α5β1 integrin plays the main role in the adhesion and spreading of PC-3 cells interacting with FN, whereas the αvβ3 integrin seems to regulate other receptors in the spreading process. Moreover, integrin-FN interaction through the RGD sequence evidently curbed the cell adhesion and spreading.     

Adaptive immune responses during Shigella dysenteriae type 1 infection: an in vitro stimulation with 57 kDa major antigenic OMP in the presence of anti-CD3 antibody

An effort was made to understand the role of the 57 kDa major antigenic fraction of Shigella outer membrane protein (OMP) in the presence of T-cell antigen receptor in activation of adaptive immune responses of the cell mediated immune (CMI) restored patients. The expression of HLA-DR/CD4 out of CD3⁺ T-cells was significantly dominant over the HLA-DR/CD8 and comparable to unstimulated cells of infected or healthy controls. CD4⁺ T-cell activation together with HLA-DR is associated with the expression of CD25⁺ (IL2Rα) for IL-2 growth factors with decreased IL-4 levels, required for maintaining the homeostasis of CD4⁺ T cell. Furthermore, the positive expression of the CD45 antigen is possibly required for acquiring the memory for CD4⁺ cells signals and facilitates the interaction with CD54 antigen. As a result, antigen-specific secondary signal is generated for B-cell activation to produce IgG2a and IgG2b. This suggests that antibody mediated-adaptive immune responses are generated due to anti-CD3 induced helper T-cell activity. The above mentioned findings reflect that the antigen alone might not exacerbate the selective T-cell responses. But these antigens in the presence of anti-CD3 antibody might help to elicit adaptive immune response via T-cell receptor (TCR) activation.     

An orally active Th1/Th2 balance modulator, M50367, suppresses Th2 differentiation of naïve Th cell in vitro

We previously reported an orally active anti-allergic agent, M50367, modulated Th1/Th2 balance to down-regulate Th2 response in a murine model of atopic asthma. In this study, we examined the effect of M50354, the active metabolite of M50367, on the differentiation of na?ve Th cells into Th1/Th2 cells. M50354 at 3 microM decreased the generation of Th2 cells by 0.2-fold and increased that of Th1 cells by 1.6-fold from na?ve Th cells primed with antigenic peptide and antigen-presenting cells. Its effect was also seen when na?ve Th cells were primed with anti-T cell receptor and anti-CD28 agonistic antibodies instead of antigen and antigen-presenting cells. M50354 decreased early endogenous IL-4 production in the nai;ve Th cell priming culture without affecting interferon-gamma production and proliferation. In contrast, M50354 had no effect on interferon-gamma and IL-4 production from mature Th1 and Th2 cells. These results suggest that M50354 directly acts on na?ve Th cells to suppress their differentiation into Th2 cells.