Hormonal Regulation of the MHC Class I Gene in Thyroid Cells: Role of the Promoter "Tissue-Specific" Region.

In previous studies we have demonstrated that the expression of the Major Histocompatibility Complex (MHC) class I gene in thyrocytes is controlled by several hormones, growth factors, and drugs. These substances mainly act on two regions of the MHC class I promoter a "tissue-specific" region (-800 to -676 bp) and a "hormone/cytokines-sensitive" region (-500 to -68 bp). In a previous study, we have shown that the role of the "tissue-specific" region in the MHC class I gene expression is dominant compared to that of the "hormone/cytokines-sensitive" region. In the present report we further investigate the dominant role of the "tissue-specific" region evaluating the effect of thyroid stimulating hormone (TSH), methimazole (MMI), phenylmethimazole (C10), glucose and thymosin-α1. By performing experiments of electrophoretic mobility shift assays (EMSAs) we show that TSH, MMI and C10, which inhibit MHC class I expression, act on the "tissue-specific" region increasing the formation of a silencer complex. Glucose and thymosin-α1, which stimulate MHC class I expression, act decreasing the formation of this complex. We further show that the silencer complex is formed by two distinct members of the transcription factors families activator protein-1 (AP-1) and nuclear factor-kB (NF-kB), c-jun and p65, respectively. These observations are important in order to understand the regulation of MHC class I gene expression in thyroid cells and its involvement in the development of thyroid autoimmunity.

Proteasome inhibitors restore the STAT1 pathway and enhance the expression of MHC class I on human colon cancer cells.

BACKGROUND: A new strategy, particularly a novel combination, for immunotherapy in microsatellite stable metastatic colorectal cancer (mCRC) treatment needs to be formulated. Studies on the interferon-γ (IFN-γ)/ Janus kinase (JAK)/ signal transducer and activator of transcription (STAT)1 pathway provide new directions in this regard. METHODS: Our study applies three colon cancer cell lines, including microsatellite stable (MSS) cell lines, which are SW480 and SW620, and microsatellite instability-high (MSI-H) cell line, which is DLD-1. We compared the expressions of immune surface markers on colon cancer cells in response to IFN-γ. We elucidated these mechanisms, which involved the upregulation of immune surface markers. Furthermore, we examined real-world clinical samples using the PerkinElmer Opal multiplex system and NanoString analysis. RESULTS: We established that the baseline expression of major histocompatibility complex (MHC) class I alleles and programmed death-ligand 1 (PD-L1) were generally low in cell line models. The immune surface markers were significantly increased after IFN-γ stimulation on SW480 but were notably unresponsive on the SW620 cell line. We discovered that STAT1 and phosphorylated STAT1 (pSTAT1) were downregulated in the SW620 cell line. We verified that the STAT1/pSTAT1 could be restored through the application of proteasome inhibitors, especially bortezomib. The expression of MHC class I as downstream signals of STAT1 was also up-regulated by proteasome inhibitors. The similar results were reproduced in DLD-1 cell line, which was also initially unresponsive to IFN-γ. In real-world samples of patients with mCRC, we found that higher STAT1 expression in tumor cells was strongly indicative of a highly immunogenic microenvironment, with significantly higher expression levels of MHC class I and PD-L1, not only on tumor cells but also on non-tumor cells. Furthermore, tumor infiltrating lymphocytes (TILs) were increased in the positive-STAT1 group. Through NanoString analysis, we confirmed that the mRNA expressions of IFN-γ, human leukocyte antigen (HLA)-A, HLA-E, and HLA-G were also significantly higher in the positive-STAT1 group than those in the negative-STAT1 group. CONCLUSION: Our study provides a novel rationale for the addition of bortezomib, a proteasome inhibitor, into new immunotherapy combinations.

IL-10 Restores MHC Class I Expression and Interferes With Immunity in Papillary Thyroid Cancer With Hashimoto Thyroiditis.

The incidence of papillary thyroid cancer (PTC) with concomitant Hashimoto thyroiditis (HT) is increasing. Interleukin (IL)-10 is a cytokine previously reported to be elevated in this condition. Evidence from multiple human malignancies showed IL-10 participated in tumor immunity and exhibited therapeutic potential. The aim of this study is to investigate whether IL-10 interferes with tumor immunity in PTC with concomitant HT. Expression of IL-10 and major histocompatibility complex (MHC) class Ⅰ were compared with PTC tissues with or without concomitant HT. PTC cell lines K1 and TPC-1 were stimulated with IL-10 and analyzed for MHC class Ⅰ expression afterward. T-cell activation, production of IL-2 and interferon (IFN)-γ and programmed death-1 (PD-1) expression were assessed by coculture of donor peripheral blood lymphocytes (PBLs) with IL-10-pretreated PTC cells. Programmed death-ligand 1 (PD-L1) expression was measured in PTC tissues and IL-10-pretreated cells of K1 and TPC-1. Increased levels of IL-10 and MHC class Ⅰ were observed in PTC with concomitant HT. IL-10 stimulation increased MHC class Ⅰ expression of PTC cells in vitro. Coculture of PBLs with IL-10-pretreated PTC cells enhanced T-cell activation (% cluster of differentiation [CD]25+ of CD3+T cells) and increased IL-2 production along with decreased IFN-γ secretion and PD-1 expression. Reduced PD-L1 expression was seen in PTC + HT tissue samples and IL-10-stimulated PTC cell lines. Elevated IL-10 expression in PTC with concomitant HT restores MHC class Ⅰ expression and interferes with tumor immunity. The potential mechanism of IL-10 in tumor immunity needs further investigation.

Inhalation of a racemic mixture (R,S)-linalool by rats experiencing restraint stress alters neuropeptide and MHC class I gene expression in the hypothalamus.

Some odorants have physiological and psychological effects on organisms. However, little is known about the effects of inhaling them, particularly on the central nervous system. Using DNA microarray analysis, we obtained gene expression profiles of the hypothalamus from restraint stressed rats exposed to racemic (R,S)-linalool. Hierarchical clustering across all probe sets showed that this inhalation of (R,S)-linalool influenced the expression levels of a wide range of genes in the hypothalamus. A comparison of transcription levels revealed that the inhalation of (R,S)-linalool restored the expression of 560 stress-induced probe sets to a normal status. Gene Ontology (GO) analysis showed that these genes were associated with synaptic transmission via neurotransmitters including anxiolytic neuropeptides such as oxytocin and neuropeptide Y. These genes also included several major histocompatibility complex (MHC) class I molecules necessary for neural development and plasticity. Moreover, Upstream Regulator Analysis predicted that the hormone prolactin would be activated by the inhalation of (R,S)-linalool under stress. Our results reveal some of the molecular mechanisms associated with odor inhalation in the hypothalamus in organisms under stress.

Temozolomide increases MHC-I expression via NF-κB signaling in glioma stem cells.

Gliomas are the most common and primary tumors of the central nervous system in adults. Temozolomide (TMZ) is the main drug used to treat glioma; however, prognosis remains poor for most patients. Glioma stem cells (GSCs) are thought to enable glioma initiation and evasion from immune surveillance; their immunogenicity can be determined by expression of major histocompatibility complex (MHC)-I. The present study investigated the effect of TMZ on MHC-I expression in GSCs. Glioma spheres were cultured in serum-free medium containing epidermal growth factor, basic fibroblast growth factor, and B27; MHC-I expression was detected by immunocytochemistry, quantitative real-time PCR, and flow cytometry. Nuclear factor (NF)-κB expression in glioma stem cells was detected by Western blot. TMZ enhanced MHC-I expression in GSCs, and NF-κB was activated. TMZ treatment increased MHC-I expression via modulation of NF-κB signaling in GSCs. In addition to being a chemotherapeutic agent, TMZ may also serve as an immunomodulatory agent in the treatment of glioma patients.

Decitabine Treatment of Glioma-Initiating Cells Enhances Immune Recognition and Killing.

Malignant gliomas are aggressive brain tumours with very poor prognosis. The majority of glioma cells are differentiated (glioma-differentiated cells: GDCs), whereas the smaller population (glioma-initiating cells, GICs) is undifferentiated and resistant to conventional therapies. Therefore, to better target this pool of heterogeneous cells, a combination of diverse therapeutic approaches is envisaged. Here we investigated whether the immunosensitising properties of the hypomethylating agent decitabine can be extended to GICs. Using the murine GL261 cell line, we demonstrate that decitabine augments the expression of the death receptor FAS both on GDCs and GICs. Interestingly, it had a higher impact on GICs and correlated with an enhanced sensitivity to FASL-mediated cell death. Moreover, the expression of other critical molecules involved in cognate recognition by cytotoxic T lymphocytes, MHCI and ICAM-1, was upregulated by decitabine treatment. Consequently, T-cell mediated killing of both GDCs and GICs was enhanced, as was T cell proliferation after reactivation. Overall, although GICs are described to resist classical therapies, our study shows that hypomethylating agents have the potential to enhance glioma cell recognition and subsequent destruction by immune cells, regardless of their differentiation status. These results support the development of combinatorial treatment modalities including epigenetic modulation together with immunotherapy in order to treat heterogenous malignancies such as glioblastoma.

Enhanced MHC class I and costimulatory molecules on B16F10 cells by Ganoderma lucidum polysaccharides.

PURPOSE: It is obvious that malignant cells evade from immune system in patients with manifest malignancy. Deficient major histocompatibility complex (MHC) class I and costimulatory molecules on malignant cells partially consist of evasion strategy since antigen bond MHC and costimulatory molecules provide two signals necessary for T cell activation. Therefore, enhancement of MHC-I and costimulatory molecules may favor restraint of the evasion. For this purpose, Ganoderma lucidum Polysaccharides (Gl-PS) was used on B16F10 melanoma cells in this study. METHODS: Immunocytochemistry and flowcytometry were used to determine the H-2K(b) and H-2D(b) (two prominent MHC class I molecules in C57BL mouse) as well as B7-1 and B7-2 (two prominent costimulatory molecules) expression on B16F10 cells after incubation with Gl-PS, while messenger ribonucleic acid (mRNA) of these molecules was detected by reverse transcription polymerase chain reaction (RT-PCR). RESULTS: The H-2K(b) and H-2D(b), and B7-1 and B7-2 on B16F10 cells and mRNAs of these molecules were enhanced by Gl-PS, and more efficient antitumor cytotoxicity was induced by the Gl-PS treated cells. CONCLUSIONS: The MHC class I molecules and costimulatory molecules may be enhanced by Gl-PS, and more efficient immune cell mediated cytotoxicity against these B16F10 cells may be induced, which may favor cancer therapy.

Ethanol and hepatitis C virus suppress peptide-MHC class I presentation in hepatocytes by altering proteasome function.

BACKGROUND: Previously, we reported that exposure of hepatitis C virus (HCV) core-expressing ethanol (EtOH)-metabolizing cells to EtOH significantly suppresses proteasome activity which exists as 26S (20S and 19S) and as an unassociated 20S particle. The replacement of the constitutive proteasomal subunits with immunoproteasome (IPR) favors antigen processing. Here, we examined the effects of EtOH consumption by HCV core transgenic mice on proteasome activity in hepatocytic lysates and in partially purified 26S proteasome and the impact of these changes on antigen presentation. METHODS: HCV (-) and HCV (+) core transgenic mice were fed chow diet with or without 20% (v/v) EtOH in water for 4 weeks. Following the feeding regimen, hepatocytes were isolated and examined for chymotrypsin-like proteasome activity, oxidative stress, and the presentation of SIINFEKL-H2Kb complex. Additionally, the constitutive proteasome and IPR were purified for further analysis and identification of proteasome-interacting proteins (PIPs). RESULTS: EtOH significantly decreased proteasome activity in hepatocytes of HCV (+) mice, and this finding correlated with oxidative stress and dysregulated methylation reactions. In isolated 26S proteasome, EtOH suppressed proteasome activity equally in HCV (+) and HCV (-) mice. EtOH feeding caused proteasome instability and lowered the content of both constitutive and IPR subunits in the 20S proteasome. In addition, the level of other PIPs, PA28 and UCHL5, were also suppressed after EtOH exposure. Furthermore, in EtOH-fed mice and, especially, in HCV (+) mice, the presentation of SIINFEKL-H2Kb complex in hepatocytes was also decreased. CONCLUSIONS: Proteasomal dysfunction induced by EtOH feeding and exacerbated by the presence of HCV structural proteins led to suppression of SIINFEKL-H2Kb presentation in hepatocytes.

Glatiramer acetate treatment increases stability of spinal synapses and down regulates MHC I during the course of EAE.

The recent discovery that the major histocompatibility complex of class I (MHC I) expression has a role in the synaptic elimination process, represented an insight into understanding the cross talk between neurons. In the present study, the possibility that glatiramer acetate (GA) treatment influences the MHC class I expression and the synaptic plasticity process in the spinal cord during the course of EAE was investigated. C57BL/6J mice were induced to EAE and submitted to treatment either with a placebo solution or with GA (0.05 mg/animal, subcutaneously, on a daily basis). All the animals were sacrificed at the peak disease (14 days after induction) or at the point of recovery of the clinical signs (21 days after induction). The spinal cords were removed and submitted to immunohistochemical examination, Western blotting and transmission electron microscopy analysis. The results showed that GA treatment was able to decrease synaptic loss during the course of EAE, which correlates with the downregulation of the MHC I complex. The present results reinforce the neuroprotective role of GA treatment, by reducing synaptic loss during the course of the disease. Such action may be associated with the recently described role of MHC I regulation during the synaptic plasticity process.

Liposomal pravastatin inhibits tumor growth by targeting cancer-related inflammation.

The chronic inflammatory environment of tumors is a target for novel antitumor therapeutic strategies. Besides cholesterol lowering effects, statins have been studied for their anti-inflammatory and immunomodulatory properties. These pleiotropic effects result mainly from the altered post-translational modification of GTP-binding proteins which regulate many intracellular pathways involved in cell growth and survival. Although pre-clinical studies suggest that statins may be effective anticancer agents required doses that are 100 to 500 fold higher than those needed to lower cholesterol levels. Furthermore, in view of their wide-ranging effects on cellular metabolism, target site-specific delivery is preferred. In this study, we investigated tumor-specific delivery of pravastatin using small long-circulating liposomes. In vitro studies on the effects of (liposomal) pravastatin on viability and proliferation of tumor cells, endothelial cells and macrophages revealed that the latter were the most sensitive cell type towards (liposomal) pravastatin treatment. In vivo, liposome-encapsulated pravastatin (5mg/kg) inhibited murine B16F10-melanoma growth over 70% as compared to free pravastatin, which was ineffective. As expected, treatments did not influence serum cholesterol levels within the time frame of the study. At 48 h post-injection, 3 µg of pravastatin could still be recovered from the tumors of liposomal pravastatin treated mice, whereas pravastatin could not be detected in tumors of the free drug treated mice (i.e. < 20 ng). In contrast to the free drug, liposomal pravastatin treatment effectively inhibited the production of several pro-inflammatory/pro-angiogenic mediators involved in inflammation and angiogenesis, out of a range of a panel of 24 proteins studied. Furthermore, liposomal pravastatin treatment increased MHC class I protein expression in the tumor tissue whereas free drug showed no effect. Taken together, targeted delivery of statins can improve their tumor growth inhibiting activity by increasing local drug concentration and direct modulation of macrophage function. The antitumor activity seems to result primarily from a local inhibition of tumor inflammation and stimulation of antitumor immune response.

(5R)-5-hydroxytriptolide inhibits the immune response of human peripheral blood mononuclear cells.

AIM: (5R)-5-hydroxytriptolide (LLDT-8) displayed immunosuppressive activities both in vitro and in autoimmune disease models. Here, we aim to further clarify the effect of LLDT-8 on the immune responses of human peripheral blood mononuclear cells (PBMC). METHOD: Cell proliferation of human PBMC from healthy donors was evaluated by [3H]-thymidine uptake. NK cell cytotoxicity was assayed using K562 cells in a [3H] lysis assay. Cytokine production was determined by enzyme-linked immunosorbent assay. The expression of cell surface molecules was detected with flow cytometry. The mRNA expression and the protein phosphorylation levels were detected by RT-PCR and Western immunoblot assay. RESULTS: LLDT-8 at 25 and 50 nM significantly inhibited the PHA- and recall antigens-induced T cell proliferation, and suppressed mixed lymphocyte reaction. LLDT-8 reduced cytokines production (IFN-gamma, IL-2, TNF-alpha) in PHA- and Sac-activated PBMC. LLDT-8 did not alter the increased expression of MHC class I/II and B7.1, but reduced B7.2 by approximately 30%. No effect of LLDT-8 was observed for the expression of T cell activation markers (CD69, CD154). However, LLDT-8 significantly reduced IFN-gamma-expressing T cell percentages and IFN-gamma mRNA transcription in PHA-activated T cells. It also inhibited the phosphorylation levels of JNK and p38. LLDT-8 did not affect NK cytotoxic activity against K562 cells. CONCLUSION: LLDT-8 was a promising immunosuppressant for human immune-related diseases.

Prostaglandin E(2) modulation of gene expression in an Atlantic salmon (Salmo salar) macrophage-like cell line (SHK-1).

Following lipopolysaccharide (LPS)-stimulation of Atlantic salmon (Salmo salar) macrophage-like SHK-1 cells, prostaglandin E(2) (PGE(2)) exhibited dose-dependent inhibition of the antigen presenting molecules major histocompatability class I and II and the pro-inflammatory cytokine interleukin-1 beta gene expression. Prostaglandin E(2) was found to be stimulatory towards cyclooxygenase-2 (COX-2) expression at higher concentrations (1 x 10(-6) and 1 x 10(-8)M) and inhibitory at lower concentrations (1 x 10(-10) and 1 x 10(-12)M) after 4h exposure. After 24h exposure, however, LPS-induced COX-2 expression decreased and was completely inhibited by all PGE(2) concentrations (1 x 10(-6)-1 x 10(-10)M). Incubation of SHK-1 cells with LPS alone had no effect on tumour necrosis factor alpha (TNFalpha)-like gene or transforming growth factor beta-like gene expression after 4h, however, LPS and PGE(2) showed a synergistic effect on TNFalpha-like gene expression after 24h. This study provides evidence for the existence of a PGE(2)-mediated negative feedback mechanism in the control of PGs through down-regulation of COX-2, as well as for inflammatory responses by the down-regulation of both COX-2 and IL-1 beta. The differential regulation of immune-related genes under these conditions further demonstrates the usefulness of the SHK-1 cell line for studying aspects of salmonid immunology.

Apoptotic and necrotic cells induced by different agents vary in their expression of MHC and costimulatory genes.

We have recently reported, in a murine tumor model, that apoptotic cells induced by different agents may vary in their ability to elicit host immunity. The basis for this observation is unclear but may involve varying efficiencies of cross-presentation and/or direct activation of immunity by different apoptotic preparations. As a first step in addressing this issue, we compared expression patterns of selected immune genes (MHC class I, class II, CD40, B7-1, B7-2) on viable and apoptotic populations induced by four different agents. The histone deacetylase inhibitor trichostatin A (TSA) induced MHC class II expression on viable and apoptotic cell populations, while LPAM, H2O2 and gamma-irradiation did not activate class II. Each agent employed elicited a different expression pattern of costimulatory molecules (CD40, B7-1, B7-2) on both apoptotic and 7-AAD+ 'necrotic' populations. In striking contrast to the TSA induction of MHC class II, class I cell surface protein was diminished on the apoptotic populations. These effects were not a result of changes in the cell cycle produced by the various treatments. The data demonstrate that distinctive gene expression patterns on viable and apoptotic cells are elicited by different apoptosis inducing agents. We discuss how expression patterns on dead or dying tumor cells could potentially affect the tumor's ability to elicit immunity.

Modulation of major histocompatibility complex Class I molecules and major histocompatibility complex-bound immunogenic peptides induced by interferon-alpha and interferon-gamma treatment of human glioblastoma multiforme.

OBJECT: Little is known about the quantitative modulation of major histocompatibility complex (MHC) Class I expression on human gliomas that is effected by interferons; even less is known about the immunogenic peptides that are accommodated in the peptide-binding motifs of MHC Class I alleles in these brain tumors. In this article the authors investigated the ability of interferon (IFN)alpha and IFNgamma to upregulate MHC Class I expression and to modulate acid-eluted Class I-bound peptides on human glioblastoma multiforme (GBM) cells in vitro. METHODS: Early-passage primary human GBM cell cultures and U87MG GBM cells were incubated with varying doses of INFalpha or IFNgamma ranging between 0 and 2000 U/ml. Upregulation of MHC Class I expression was assayed by immunocytochemical analysis, flow cytometry, and Western blot analysis. Modulation of acid-eluted MHC Class I-bound peptides from the IFN-treated GBM cells was examined with the aid of mass spectroscopy. The in vitro expression of the MHC Class I molecule was upregulated by both IFNalpha and IFNgamma in a dose-dependent manner. Interferon-gamma exhibited a more potent effect on MHC Class I upregulation, peaking at 10 U/ml; whereas the effect of IFNalpha was less marked, reaching a plateau at 500 U/ml. In addition, a native peptide eluted from MHC Class I molecules of human GBM cells was identified and found to be consistently upregulated by IFN treatment. CONCLUSIONS: Interferon-alpha and IFN-gamma can significantly upregulate the MHC Class I molecules that are expressed on the cell surface of human GBM cells as well as the potentially immunogenic peptides bound to the MHC. These results may help explain the molecular basis for increased immunogenicity with IFN treatment of human GBMs and might provide added insight into the design of future antitumor vaccines for human brain tumors.

An epigenetically altered tumor cell vaccine.

Functional inactivation of genes critical to immunity may occur by mutation and/or by repression, the latter being potentially reversible with agents that modify chromatin. This study was constructed to determine whether reversal of gene silencing, by altering the acetylation status of chromatin, might lead to an effective tumor vaccine. We show that the expression of selected genes important to tumor immunity, including MHC class II, CD40, and B7-1/2 are altered by treating tumor cells in vitro with a histone deacetylase inhibitor, trichostatin A (TSA). Tumor cells treated in vitro with TSA showed delayed onset and rate of tumor growth in 70% of the J558 plasmacytoma and 100% of the B16 melanoma injected animals. Long-term tumor specific immunity was elicited to rechallenge with wild-type cells in approximately 30% in both tumor models. Splenic T cells from immune mice lysed untreated tumor cells, and SCID mice did not manifest immunity, suggesting that T cells may be involved in immunity. We hypothesize that repression of immune genes is involved in the evasion of immunity by tumors and suggest that epigenetically altered cancer cells should be further explored as a strategy for the induction of tumor immunity.

Augmenting major histocompatibility complex class I expression by murine tumors in vivo enhances antitumor immunity induced by an active immunotherapy strategy.

OBJECTIVE: Tumors down-regulate major histocompatibility complex class I expression, escaping recognition by the cellular immune response. We hypothesized that augmentation of tumor cell class I expression by interferon-gamma would enhance the cellular antitumor immune response and cure rate of an active immunotherapy strategy. METHODS: B16.F10 tumor cells were exposed to interferon-gamma in culture, and class I expression was quantified using flow cytometry. Syngeneic mice bearing established tumors were injected with interferon-gamma (5000 U, intraperitoneal), and class I expression was assessed using immunohistochemistry. Tumor-specific cytotoxic T lymphocytes were induced in mice by an intratumoral injection of AdCD40L (5 x 10(10) particles), an adenovirus gene transfer vector-based immunotherapy strategy previously demonstrated to augment cellular antitumor immunity. A conjugate-formation assay and the enzyme-linked immunospot assay were used to evaluate the binding and activation of cytotoxic T lymphocytes, respectively. Interferon-gamma was administered to tumor-bearing mice concomitantly with intratumoral AdCD40L. End points measured included the frequencies of cytotoxic T lymphocytes using the enzyme-linked immunospot assay, tumor size, and mouse survival. The role of class I expression was further evaluated by monoclonal antibody blockade in both in vitro and in vivo experiments. RESULTS: B16.F10 cells exposed to interferon-gamma expressed significantly more class I, both in vitro and in vivo, and were able to bind to and activate cytotoxic T lymphocytes more efficiently than untreated cells. Cytotoxic T-lymphocyte frequencies, tumor regression, and the cure rate induced by AdCD40L were augmented by the addition of a single dose of interferon-gamma in tumor-bearing mice. These in vitro and in vivo effects of interferon-gamma were attenuated by class I monoclonal antibody blockade. CONCLUSIONS: Up-regulation of class I expression using interferon-gamma enhances the cellular antitumor immune response and cure rate of AdCD40L, an active immunotherapy strategy. This approach may be useful for human tumors that lack class I expression.

Increased expression of CEA and MHC class I in colorectal cancer cell lines exposed to chemotherapy drugs.

PURPOSE: Cancer-specific immunotherapy holds great promise as an emerging treatment for advanced colorectal cancer and may be combined with standard chemotherapy to provide a synergistic inhibitory action against tumor cells. To examine the interrelationship between the immune system and chemotherapy, we studied the induction of both CEA, a tumor-associated antigen, and MHC class I, a major component of the antigen presenting system, in response to a number of chemotherapeutic agents. METHODS: The effect of a selection of standard chemotherapeutics on MHC class I and CEA expression in human colorectal cancer cell lines was determined by flow cytometry and semi-quantitative RT-PCR. In addition, studies using mice bearing tumors derived from an injected murine colon cancer cell line were performed to determine if alteration in MHC class I expression occurs in vivo following continuous infusion of chemotherapeutic agents into the peritoneal cavity, as well as to facilitate correlations between expression of this factor and therapeutic effectiveness. RESULTS: All anti-cancer drugs examined, when given at IC50 values, induced expression of MHC class I protein in the human colon cancer cell line, COLO201. However, expression of CEA mRNA was only induced upon exposure to 5-FU, in contrast to obscure induction following CDDP and SN-38 treatment. Combined treatment with 5-FU and CDDP gave additional effect on CEA expression in COLO201 cells. Regarding the in vivo studies in mice, the size of the murine colon cancer cell-derived tumors was reduced only in response to treatment with CDDP, which also mediated the highest induction of MHC class I expression. CONCLUSION: These results suggest that chemotherapeutic agents trigger the immune system and cancer-specific immunotherapy may be effective when used in combination with systemic chemotherapy.

Injection of FGF6 accelerates regeneration of the soleus muscle in adult mice.

FGF6, a member of the fibroblast growth factor (FGF) family, accumulated almost exclusively in the myogenic lineage, supporting the finding that FGF6 could specifically regulate myogenesis. Using FGF6 (-/-) mutant mice, important functions in muscle regeneration have been proposed for FGF6 but remain largely controversial. Here, we examined the effect of a single injection of recombinant FGF6 (rhFGF6) on the regeneration of mouse soleus subjected to cardiotoxin injection, specifically looking for molecular and morphological phenotypes. The injection of rhFGF6 has two effects. First, there is an up-regulation of cyclin D1 mRNA, accounting for the regulating role of a high FGF6 concentration on proliferation, and second, differentiation markers such as CdkIs and MHC I and Tn I increase and cellular differentiation is accelerated. We also show a down-regulation of endogenous FGF6, acceleration of FGFR1 receptor expression and deceleration of the FGFR4 receptor expression, possibly accounting for biphasic effects of exogenous FGF6 on muscle regeneration.

Granulocyte-macrophage colony stimulating factor is an anti-apoptotic cytokine for thymic dendritic cells and a significant modulator of their accessory function.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a growth-promoting factor for myeloid-derived dendritic cells (DC) but not for lymphoid DC. The data about its effect on thymic DC (TDC), which are both of lymphoid and myeloid origin, are very scarce. Using an in vitro model, we demonstrated in this work that GM-CSF significantly increased the survival of rat TDC in culture by inhibiting their apoptosis and the effect correlated with up-regulation of Bcl-2 expression. GM-CSF also stimulated differentiation and maturation of TDC as judged by higher expression of MHC class I and II molecules, CD54, CD80 and CD86. These changes correlated with stronger stimulatory activity of GM-CSF-pulsed TDC in syngeneic thymocyte proliferation assay and MLR. The stimulatory potential of TDC was further increased when thymocytes were cultivated with an anti-alphabeta TCR (R73) monoclonal antibody (mAb). The influence of unstimulated TDC on proliferation of thymocytes was inhibited by anti-CD86 but not anti-CD80 mAb, whereas in cultures with GM-CSF-treated TDC both mAbs exerted an additive blocking effect. After separation of TDC on CD11b(+) and CD11b(-) we demonstrated that GM-CSF inhibited apoptosis and potentiated accessory activity of both TDC subsets independently of the myeloid marker expression. Cummulatively, our results suggest that GM-CSF is one of the regulatory cytokine involved in survival, maturation, differentiation and accessory function of TDC.