Search

The ovarian tumor microenvironment (TME) is characterized by the accumulation of immunosuppressive tumor-associated macrophages (TAMs) and granulocytic cells. Very small size particles (VSSP), comprised of the ganglioside NAcGM3 and Neisseria meningitidis derived outer membrane vesicles, is being developed as a nanoparticulated modulator of innate immunity. Prior studies have shown that VSSP enhanced antigen-specific cytotoxic T cell responses and reduced the suppressive phenotype of splenic granulocytic cells in tumor-bearing mice. Here, we hypothesized that intraperitoneal VSSP would modify myeloid cell accumulation and phenotypes in the ovarian TME and abrogate suppressor function of TAMs and tumor-associated granulocytic cells. In the ID8 syngeneic model of epithelial ovarian cancer, VSSP reduced peritoneal TAMs and induced M1-like polarization in TAMs. In addition, VSSP stimulated peritoneal inflammation characterized by increased granulocytes and monocytes, including inflammatory monocytic cells. VSSP treatment resulted in peritoneal TAMs and granulocytic cells being less suppressive of ex vivo stimulated CD8 + T cell responses. VSSP alone and combined with anti-PD-1 modestly but significantly prolonged survival in tumor-bearing mice. In addition, ex vivo treatment with VSSP induced M1-like polarization in TAMs from patients with metastatic ovarian cancer and variably abrogated their suppressor phenotype. VSSP treatment also partially abrogated the induction of suppressor function in healthy donor neutrophils exposed to ascites supernatants from patients with ovarian cancer. Together, these results point to VSSP reprogramming myeloid responses resulting in abrogation of suppressive pathways and raise the potential for administration of VSSP into the TME to enhance anti-tumor immunity., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Cellular exposure to mild stress (39.5°C - 41.5°C) induces thermotolerance, rendering cells resistant to a subsequent heat shock (>42°C) insult. We found that mild hyperthermia at 39.5°C leads to elevations in dicer, a protein well-known for its role in microRNA processing and for its role in cellular stress responses. However, whether elevated dicer protein levels play a role in sustaining a thermotolerant phenotype has, to our knowledge, not been reported. Here we demonstrate that elevated dicer protein is linked to a thermotolerant phenotype in the cervical carcinoma cell line HeLa and in murine embryonic fibroblasts (MEF), and demonstrate that dicer plays a role in mediating PKR and eIF2α phosphorylation. These findings suggest that dicer's role in thermotolerance may be to relay signals to key ER stress pathway components. Moreover, utilizing a MEF cell line defective in microRNA processing, we suggest that dicer's influence on PKR and eIF2α phosphorylation is likely distinct from its microRNA processing role. ATF4 and CHOP are well characterized stress response factors proximal to eIF2α. Evidence is presented that elevated dicer protein in thermotolerant cells differentially modulates ATF4 and CHOP levels to promote a pro-survival phenotype. This work contributes new information on dicer's role in cellular stress responses by defining a pro-survival phenotype in heat stress resistant cells which is sustained, at least in part, by elevated dicer protein levels. Our results suggest an ancillary role for dicer in the cellular stress pathways activated by mild hyperthermia that is likely distinct from its role in microRNA processing., Competing Interests: CONFLICTS OF INTEREST The author(s) declare no potential conflicts of interest.

microRNAs (miRs) are small noncoding RNAs that regulate/fine tune many cellular protein networks by targeting mRNAs for either degradation or translational inhibition. Dicer, a type III endoribonuclease, is a critical component in miR biogenesis and is required for mature microRNA production. Abnormal Dicer expression occurs in numerous cancer types and correlates with poor patient prognosis. Recent reports have demonstrated that epigenetic agents, including histone deacetylase inhibitors (HDACi), may regulate Dicer and miR expression. HDACi are a class of epigenetic agents used to treat cancer, viral infections, and inflammatory disorders. However, little is known regarding the epigenetic regulation of miR biogenesis and function. We therefore investigated whether clinically successful HDACi modulated Dicer expression and found that Panobinostat, a clinically approved HDACi, enhanced Dicer expression via posttranscriptional mechanisms. Studies using proteasome inhibitors suggested that Panobinostat regulated the proteasomal degradation of Dicer. Further studies demonstrated that Panobinostat, despite increasing Dicer protein expression, decreased Dicer activity. This suggests that Dicer protein levels do not necessarily correlate with Dicer activity and mature miR levels. Taken together, we present evidence here that Panobinostat posttranscriptionally regulates Dicer/miR biogenesis and suggest Dicer as a potential therapeutic target in cancer.

MicroRNAs (miRs) are small non-coding RNAs that regulate most cellular protein networks by targeting mRNAs for translational inhibition or degradation. Dicer, a type III endoribonuclease, is a critical component in microRNA biogenesis and is required for mature microRNA production. Abnormal Dicer expression occurs in numerous cancer types and correlates with poor patient prognosis. For example, increased Dicer expression in melanoma is associated with more aggressive tumors (higher tumor mitotic index and depth of invasion) and poor patient prognosis. However, the role that Dicer plays in melanoma development and immune evasion remains unclear. Here, we report on a newly discovered relationship between Dicer expression and tumor immunogenicity. To investigate Dicer's role in regulating melanoma immunogenicity, Dicer knockdown studies were performed. We found that B16F0-Dicer deficient cells exhibited decreased tumor growth compared to control cells and were capable of inducing anti-tumor immunity. The decrease in tumor growth was abrogated in immunodeficient NSG mice and was shown to be dependent upon CD8+ T cells. Dicer knockdown also induced a more responsive immune gene profile in melanoma cells. Further studies demonstrated that CD8+ T cells preferentially killed Dicer knockdown tumor cells compared to control cells. Taken together, we present evidence which links Dicer expression to tumor immunogenicity in melanoma., Competing Interests: The author(s) declare no potential conflicts of interest.

Dysregulation of microRNA expression has been shown in multiple sclerosis (MS); however, the mechanisms underlying these changes, their response to therapy and the impact of microRNA changes in MS are not completely understood. Dicer mediates the cleavage of precursor microRNAs to mature microRNAs and is dysregulated in multiple pathologies. Having shown that interferons regulate Dicer in vitro, we hypothesized that MS patient IFNβ1a treatment could potentially alter Dicer expression. Dicer mRNA and protein levels, as well as microRNA expression, were determined in MS patient and healthy control PBL. Acute responses to IFNβ1a were assessed in 50 patients. We found that Dicer protein but not mRNA levels decreases in MS patients while both are selectively induced in patients responding well to IFNβ1a. Potential microRNA biomarkers for relapsing remitting multiple sclerosis (RRMS), secondary progressive multiple sclerosis (SPMS) and IFNβ1a response are described. Contrasts in Dicer and microRNA expression levels between patient populations may offer insight into mechanisms underlying disease courses and responses to IFNβ1a therapy. This work identifies Dicer regulation as both a potential mediator of MS pathology and a therapeutic target., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Dicer is an enzyme of the RNase III endoribonuclease family, which is crucial for RNA interference (RNAi) in eukaryotes. Dicer is a component of the protein machinery (the RNA Induced Silencing Complex [RISC]) which is involved in catalyzing the formation of mature microRNAs from their precursors in the process of microRNA biogenesis. RISC-associated microRNAs bind to specific sequences in the 3' untranslated region of cognate mRNAs largely through complementary base pairing, resulting in either translational inhibition and/or the degradation of a specific mRNA pool. MicroRNAs epigenetically regulate the cellular levels of receptors, transcription factors and signaling proteins that govern the developmental pathways and functions of multiple cellular processes. The pivotal role played by Dicer in microRNA formation has also piqued the interest of molecular immunologists who have sought to understand the biological relevance of microRNAs in the development and function of the immune system. Here, we review the major findings of these studies and provide an overview of the role of Dicer and microRNAs in immune cell development and function. Additionally, we highlight deficiencies in our knowledge and new research areas that may enhance our understanding of the role of Dicer and microRNAs in immunity.

Purpose: To investigate whether mild heat stress at 39.5°C altered Dicer protein and miRNA expression patterns in several cell types., Methods: Multiple human and mouse cell types were cultured during the course of 9 h at temperatures from 37°C to 39.5°C. Dicer mRNA levels and microRNAs were quantified by TaqMan RT-qPCR assays and Dicer protein by western blotting., Results: Dicer protein was substantially elevated on western analysis in response to heat stress at 39.5°C in the absence of significant changes in Dicer mRNA by RT-qPCR., Conclusions: Heat-induced regulation of Dicer expression occurs primarily post- transcriptionally, and the expression levels of Dicer protein are increased and often oscillate in response to fever-range hyperthermia in multiple mouse and human cells. Our studies suggest a potential role for Dicer and microRNAs in the response to mild thermal stress. Additional studies on the mechanisms involved in the stress-induced oscillations of Dicer protein and microRNAs will be of interest.

MicroRNAs have been shown to regulate gene expression both transcriptionally and translationally. Here, we examine evidence that various stresses regulate miRNAs which, in turn, regulate immune gene levels. Multiple studies are reviewed showing altered microRNA levels in normal cells under stress and in various disease states, including cancer. Unexpected was the finding that Dicer expression is altered by treatments with several agents, such as interferons and cortisone, employed in the treatment of immune disorders. Potential signal transduction pathways, including JAK/Stat, PI3K and PKR, that may regulate Dicer and microRNA levels in normal and stressed mammalian cells are discussed.

Trophoblast cells and many cancer cells that harbor foreign antigens may evade immunity by epigenetic silencing of key immune response genes, including MHC class I and II and CD40. Chromatin active agents, such as histone deacetylase inhibitors (HDACi), induce immune response gene expression but often the expression levels are low and the cells lack a robust antigen presentation response. We show here that pre-treatment of trophoblast cells and certain cancer cells with agents that activate stress pathways (Ras oncogene, PMA or H2O2) and induce senescence can substantially enhance the induction of immune response genes (MHC class II, CD40, MICA, MICB) by HDACi and restore a vigorous IFN-gamma response in trophoblast cells and tumor cells. These results could potentially impact the development of novel anti-cancer therapeutic strategies.

The generation of microRNAs is dependent on the RNase III enzyme Dicer, the levels of which vary in different normal cells and in disease states. We demonstrate that Dicer protein expression in JAR trophoblast cells, and several other cell types, was inhibited by multiple stresses including reactive oxygen species, phorbol esters and the Ras oncogene. Additionally, double-stranded RNA and Type I interferons repress Dicer protein in contrast to IFN-gamma which induces Dicer. The effects of stresses and interferons are primarily post-transcriptional. The findings suggest that Dicer is a stress response component and identifies interferons as potentially important regulators of Dicer expression.

In this review we discuss specific examples of regulation of cytokine genes and focus on a new mechanism involving post-transcriptional regulation via miRNAs. The post-transcriptional regulation of cytokine genes via the destabilizing activity of AU-rich elements [AREs] and miRNAs is a pre-requisite for regulating the half-life of many cytokines and achieving the temporal and spatial distributions required for regulation of these genes.

Histone deacetylase inhibitors (HDACi), including trichostatin A (TSA) and valproic acid, can alter the acetylation of histones in chromatin and enhance gene transcription. Previously we demonstrated that HDACi-treated tumor cells are capable of presenting antigen via the MHC class II pathway. In this study, we show that treatment with HDACi enhances the expression of molecules (TAP1, TAP2, LMP2, LMP7, Tapasin and MHC class I) involved in antigen processing and presentation via the MHC class I pathway in melanoma cells. HDACi treatment of B16F10 cells also enhanced cell surface expression of class I and costimulatory molecules CD40 and CD86. Enhanced transcription of these genes is associated with a significant increase in direct presentation of whole protein antigen and MHC class I-restricted peptides by TSA-treated B16F10 cells. Our data indicate that epigenetic modification can convert a tumor cell to an antigen presenting cell capable of activating IFN-gamma secreting T cells via the class I pathway. These findings suggest that the abnormalities, observed in some tumors in the expression of MHC class I antigen processing and presentation molecules, may result from epigenetic repression.

We studied 613 genes which regulate immunity and, utilizing predictive algorithms, identified 285 genes as microRNA (miRNA or miR) targets. Of these, approximately 250 are newly predicted gene-miR interactions. The frequency of predicted miRNA binding sites in immune gene 3'UTRs indicated preferential targeting of immune genes compared to the genome. Major targets include transcription factors, cofactors and chromatin modifiers whereas upstream factors, such as ligands and receptors (cytokines, chemokines and TLRs), were, in general, non-targets. About 10% of the immune genes were 'hubs' with eight or more different miRNAs predicted to target their 3'UTRs. Hubs were focused on certain key immune genes, such as BCL6, SMAD7, BLIMP1, NFAT5, EP300 and others. NF-kappaB and p53 do not themselves have binding sites for miRNAs but rather these pathways are targeted by miRNAs at downstream sites. MHC class II genes lacked miRNA targets but binding sites were identified in the CIITA gene and were shown experimentally to repress IFN-gamma-induced MHC class II activation. Unexpectedly, factors involved in regulating message stability via AU-rich elements (ARE) were heavily targeted. Moreover, multiple components involved in the generation and effector functions of miRNAs (Dicer and Argonautes) were themselves miRNA targets suggesting that a subset of miRNAs may indirectly control their own production as well as other miRNAs.

We have previously reported that Major Histocompatibility Complex (MHC) class II can be induced by histone deacetylase inhibitors (HDACi) in the absence of class II transactivator (CIITA). Here we characterized the histone modifications associated with the CIITA-dependent (IFN-gamma induced) and -independent (HDACi induced) MHC class II expression. We demonstrate that both IFN-gamma and HDACi induced MHC class II expression exhibited enhanced histone H3, H4 acetylation and H3K4me3 at the MHC class II promoter while H3K9me3 was decreased. In contrast, high levels of H3K36me3 were detected at exons 3 and 5 but not at the promoter or the locus control region (LCR). Interestingly, high levels of H3K79me2 were only detected at the promoter and exon 3 of the B cell lines while the level remained low and unchanged despite active MHC class II expression induced by either IFN-gamma or HDACi treatment. Constitutive expression of the CIITA protein by stable transfection of a CIITA deficient B cell line restored the H3K79me2 to a level comparable to its cell of origin. This data demonstrates that, although regulated by different pathways, both IFN-gamma and HDACi treatments resulted in similar patterns of histone modifications and that HDACi induce both histone methylation and acetylation. In addition, the different spatial distribution of the lysine methylation markers along the gene suggests that these modifications play a distinctive role during different phases of the transcription process.

Epigenetic modifications of chromatin, such as histone acetylation, are involved in repression of tumor antigens and multiple immune genes that are thought to facilitate tumor escape. The status of acetylation in a cell is determined by the balance of the activities of histone acetyltransferases and histone deacetylases. Inhibitors of histone deacetylase (HDACi) can enhance the expression of immunologically important molecules in tumor cells and HDACi treated tumor cells are able to induce immune responses in vitro and in vivo. Systemic HDACi are in clinical trails in cancer and also being used in several autoimmune disease models. To date, 18 HDACs have been reported in human cells and more than thirty HDACi identified, although only a few immune targets of these inhibitors have been identified. Here, we discuss the molecular pathways employed by HDACi and their potential role in inducing immune responses against tumors. We review data suggesting that selection of target specific HDACi and combinations with other agents and modalities, including those that activate stress pathways, may further enhance the efficacy of epigenetic therapies.

Background: Numerous immune genes are epigenetically silenced in tumor cells and agents such as histone deacetylase inhibitors (HDACi), which reverse these effects, could potentially be used to develop therapeutic vaccines. The conversion of cancer cells to antigen presenting cells (APCs) by HDACi treatment could potentially provide an additional pathway, together with cross-presentation of tumor antigens by host APCs, to establish tumor immunity., Methods: HDACi-treated B16 melanoma cells were used in a murine vaccine model, lymphocyte subset depletion, ELISpot and Cytotoxicity assays were employed to evaluate immunity. Antigen presentation assays, vaccination with isolated apoptotic preparations and tumorigenesis in MHC-deficient mice and radiation chimeras were performed to elucidate the mechanisms of vaccine-induced immunity., Results: HDACi treatment enhanced the expression of MHC class II, CD40 and B7-1/2 on B16 cells and vaccination with HDACi-treated melanoma cells elicited tumor specific immunity in both prevention and treatment models. Cytotoxic and IFN-gamma-producing cells were identified in splenocytes and CD4+, CD8+ T cells and NK cells were all involved in the induction of immunity. Apoptotic cells derived from HDACi treatments, but not H2O2, significantly enhanced the effectiveness of the vaccine. HDACi-treated B16 cells become APCs in vitro and studies in chimeras defective in cross presentation demonstrate direct presentation in vivo and short-term but not memory responses and long-term immunity., Conclusion: The efficacy of this vaccine derives mainly from cross-presentation which is enhanced by HDACi-induced apoptosis. Additionally, epigenetic activation of immune genes may contribute to direct antigen presentation by tumor cells. Epigenetically altered cancer cells should be further explored as a vaccine strategy.

According to the concept of immune surveillance, the appearance of a tumor indicates that it has earlier evaded host defenses and subsequently must have escaped immunity to evolve into a full-blown cancer. Tumor escape mechanisms have focused mainly on mutations of immune and apoptotic pathway genes. However, data obtained over the past few years suggest that epigenetic silencing in cancer may be as frequent a cause of gene inactivation as are mutations. Here, we discuss the evidence that tumor immune evasion is mediated by non-mutational epigenetic events involving chromatin and that epigenetics collaborates with mutations in determining tumor progression. Since epigenetic changes are potentially reversible, the relative contribution of mutations and epigenetics, to the gene defects in any given tumor, may be a factor in determining the efficacy of treatments. We review new developments in basic chromatin mechanisms and in this context describe the rationale for the current use of epigenetic agents in cancer therapy and for a novel epigenetically generated tumor vaccine model. We emphasize that epigenetic cancer treatments are currently a 'blunt-sword' and suggest future directions for designing chromatin-based programs of potential value in the diagnosis and treatment of cancer.

We evaluated the regulation of the major histocompatibility complex class II (MHC II) transactivator (CIITA) gene expression in two microglial cell lines, EOC2 and EOC20. We demonstrate that interferon-gamma (IFN-gamma) activates type III- and IV-CIITA mRNA and high levels of MHC II in EOC20. However, in EOC2 cells only low levels of type IV-CIITA mRNA and MHC II are detectable following IFN-gamma treatment. Transforming growth factor-beta1 (TGF-beta1) inhibits both type III- and IV-CIITA expression in EOC20 cells while, in EOC2 cells TGF-beta1 enhances IFN-gamma induced pIV-CIITA expression. Trichostatin A (TSA), a histone deacetylase (HDAC) inhibitor, abrogates the TGF-beta1 mediated repression of the IFN-gamma induced CIITA in EOC20. Evidence is presented that the TG-interacting factor (TGIF), a co-repressor known to recruit HDACs, plays a role in determining the effects of TGF-beta1 on microglial cells.

Interferon signaling pathways are critical to both innate and adaptive immunity. We have demonstrated here that the inhibition of heat shock protein 90 (Hsp90) functions by small interfering RNAs or chemical inhibitors blocking interferon-induced gene expression. Hsp90 was required for signal transducers and activators of transcription 1 phosphorylation, and in its absence, Janus kinase (JAK) 1/2 were degraded by the proteosome. JAK1 interacts with Hsp90 and the CDC37 co-chaperone, and both interactions are destabilized by Hsp90 inhibitors. The biological consequences were suggested by experiments showing that T cell activation by interferon-gamma-primed macrophages and the antiviral response of interferons required Hsp90. We conclude that JAK1/2 are client proteins of Hsp90 and that Hsp90 and CDC37 play a critical role in types I and II interferon pathways.

The regulation of MHC class II expression by the class II transactivator (CIITA) is complex and differs in various cell types depending on the relative activity of three CIITA promoters. Here we show that, in plasma cell tumors, the deacetylase inhibitor trichostatin A (TSA) elicits PIII-CIITA but does not activate the IFN-gamma-inducible PIV-CIITA promoter. In trophoblast cells, all CIITA promoter types are constitutively silent and not induced by IFN-gamma or TSA treatment. TSA induction of PI-CIITA was restricted to macrophage and dendritic cell lines. In the Colon 26 tumor IFN-gamma induced endogenous PIV-CIITA but not PIII-CIITA while TSA activated class II in the apparent absence of CIITA. Reporter assays in Colon 26 showed that TSA induced PIII-CIITA but not PIV-CIITA. Transfection of a dominant negative CIITA plasmid in Colon 26 inhibited induction of class II by IFN-gamma but not TSA. Thus, the potential for both CIITA-dependent and -independent pathways of MHC induction exists within a single cell. Further evidence of CIITA-independent class II expression elicited by TSA was obtained using knockout mice with defects in CIITA, STAT-1alpha and IRF-1 expression. TSA treatment can also activate class II expression in mutant cell lines with deficiencies in signaling molecules, transcription factors and the BRG-1 cofactor that are required for IFN-gamma-induced CIITA expression. Importantly, after epigenetic activation by the deacetylase inhibitor, MHC class II is transported and displayed on the cell surface of a plasma cell tumor and it is converted to an efficient antigen presenting cell for protein and class II-peptide presentation.

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.

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.

Exosomes are membrane-bound vesicles derived from multivesicular bodies that are externalized by cells through fusion with the plasma membrane. Exosomes have been implicated in cell-to-cell signaling, and those derived from immunologic cells may be involved in both direct and cross-presentation of antigens to T cells. The research presented here evaluated their efficacy as a prophylactic cancer vaccine in a mouse plasmacytoma model. Plasmacytoma cells were shown to release exosomes in vitro, and vaccination with a single dose (5 microg) of exosome protein protected 80% of mice against challenge with wild-type tumors. Protection could be linked to the immune system since vaccinated mice generated specific cytotoxic T lymphocytes, the effects were not seen in SCID mice, and immunity was tumor-specific. Several proteins involved in immunity, including two potential tumor antigens (P1A and intracisternal A particle protein) as well as Hsp70, were demonstrated to be present in exosomes. The authors conclude that exosomes can induce tumor-specific immunity and prevent tumor development and are a potential strategy for future therapeutic tumor vaccination.

Previously we have reported serum amyloid P component (SAP) induced cell death in cerebro-cortical cultures of rat brain. In this paper we studied the types of target cells and the molecular mechanism of SAP-induced cell death. Immuno-electron and light microscopy revealed that SAP penetrates the plasma membrane and translocates selectively into the nuclei of neurons. Neuronal cells with SAP immunoreactivity exhibit the morphological hallmarks of apoptosis in vitro. The apoptotic mechanism of cell death is also supported by the increased Bax/Bcl-2 ratio. In addition to neurotoxic effects, we detected elevated beta-amyloid (Abeta) immunoreactivity following SAP treatment. This study supports the thesis that SAP plays an important role in the pathomechanism of neurodegenerative diseases, including Alzheimer's disease by inducing neuronal apoptosis.

MHC class II (Ia) Ag expression is inversely correlated with tumorigenicity and directly correlated with immunogenicity in clones of the mouse L1210 lymphoma (1 ). Understanding the mechanisms by which class II Ag expression is regulated in L1210 lymphoma may facilitate the development of immunotherapeutic approaches for the treatment of some types of lymphoma and leukemia. This study demonstrates that the variation in MHC class II Ag expression among clones of L1210 lymphoma is due to differences in the expression of the class II transactivator (CIITA). Analysis of stable hybrids suggests that CIITA expression is repressed by a dominant mechanism in class II-negative L1210 clones. DNA-alkylating agents such as ethyl methanesulfonate and the chemotherapeutic drug melphalan activate CIITA and class II expression in class II negative L1210 cells, and this effect appears to be restricted to transformed cell lines derived from the early stages of B cell ontogeny. Transient transfection assays demonstrated that the CIITA type III promoter is active in class II(-) L1210 cells, despite the fact that the endogenous gene is not expressed, which suggests that these cells have all of the transacting factors necessary for CIITA transcription. An inverse correlation between methylation of the CIITA transcriptional regulatory region and CIITA expression was observed among L1210 clones. Furthermore, 5-azacytidine treatment activated CIITA expression in class II-negative L1210 cells. Collectively, our results suggest that 1) CIITA gene expression is repressed in class II(-) L1210 cells by methylation of the CIITA upstream regulatory region, and 2) treatment with DNA-alkylating agents overcomes methylation-based silencing of the CIITA gene in L1210 cells.

Background: Impaired allostimulatory function of dendritic cells in patients with AIDS has been reported previously. Granulocyte-macrophage colony-stimulating factor (GM-CSF) can restore the T-cell stimulatory function in transforming growth factor-beta 1 (TGF-beta 1)-inhibited murine accessory cells. We now report the effect of intravenous recombinant human GM-CSF (rhGM-CSF) on accessory cells of HIV-infected patients., Method: The in vivo effect of GM-CSF on allostimulatory function of accessory cells was evaluated. Seventeen individuals with AIDS received a single infusion of rhGM-CSF (125 mg/m(2) over 120 minutes). Samples of peripheral blood lymphocytes (PBL) were taken at 1, 5, and 24 hours after infusion, and the allostimulatory capacity was measured., Results: A single bolus infusion of rhGM-CSF resulted in significantly increased accessory cell function in 13/17 (88%) patients at one or more assayed time points after infusion., Conclusion: These results suggest that the administration of rhGM-CSF can potentially restore allostimulatory function to accessory cells in HIV-infected patients, and this presents a novel way of immune reconstitution. Clinical significance of this approach of immune reconstitution in AIDS patients warrants further investigations.

Epigenetic mechanisms are involved in regulating chromatin structure and gene expression through repression. In this study, we show that histone deacetylase inhibitors (DAIs) that alter the acetylation of histones in chromatin enhance the expression of several genes on tumor cells including: MHC class I, II, and the costimulatory molecule CD40. Enhanced transcription results in a significant increase in protein expression on the tumor cell surface, and expression can be elicited on some tumors that are unresponsive to IFN-gamma. The magnitude of induction of these genes cannot be explained by the effect of DAIs on the cell cycle or enhanced apoptosis. Induction of class II genes by DAIs was accompanied by activation of a repressed class II transactivator gene in a plasma cell tumor but, in several other tumor cell lines, class II was induced in the apparent absence of class II transactivator transcripts. These findings also suggest that the abnormalities observed in some tumors in the expression of genes critical to tumor immunity may result from epigenetic alterations in chromatin and gene regulation in addition to well-established mutational mechanisms.

In cells, genes are contained within chromatin - a highly structured array of DNA wrapped around core histone proteins. Packaged genes are subject to a variety of regulatory modifications including, CpG methylation, histone acetylation and phosphorylation. These epigenetic mechanisms of gene regulation involve higher ordered protein complexes possessing enzymatic activities such as ATP hydrolysis and acetylation that are targeted to specific genes by transcription factors, coactivatorsand coreptessors. In this article, we endeavor to providean overview of current research on mechanisms of transcriptional regulation by chromatin remodeling of MHC and other genes that are of interest in reproductive immunology.

In the present report, the effects of IFN-gamma and transforming growth factor beta1 (TGF-beta1) on major histocompatibility complex class II (MHC-II) gene expression in isolated mouse brain microglial cells, in the MH-S macrophage cell line and in the primary mouse macrophage cultures were examined. IFN-gamma is a potent inducer of MHC-II gene and this induction was further elevated in microglia by TGF-beta1, while TGF-beta1 inhibited IFN-gamma, induction in macrophages. The enhancing effect of TGF-beta1 was also detected in microglia at the protein level. Transient transfection of microglia with 5' deletional mutants of the MHC-II IAalpha promoter linked to the chloramphenicol acetyltransferase reporter gene demonstrated that TGF-beta1 acts at the transcriptional level to enhance the MHC-II expression induced by IFN-gamma.

Astrocytes and microglial cells were examined for expression of two immunologically important molecules, major histocompatibility complex class II (MHC-II) and nitric oxide (NO) following treatment with IFN-gamma and beta-amyloid (betaA) peptides, betaA(1-42) and betaA(25-35). IFN-gamma is a potent inducer of both MHC-II gene expression and NO production. The induction of MHC-II was inhibited by both betaA peptides in astrocytes but they had little or no effect in microglia. betaA peptides had no effect on NO release in astrocytes but on microglia betaA(1-42) synergistically induced NO release with IFN-gamma. Transient transfection of astrocytes with 5' deletional mutants of MHC-II IAalpha promoter linked to the chloramphenicol acetyl transferase reporter gene (IAalpha-CAT), demonstrated that betaA acts at the transcriptional level to downregulate IFN-gamma induced MHC-II gene expression in astrocytes. In previous studies, the induction of MHC-II on glial cells were suggested to be involved in the pathogenesis of neurodegenerative diseases and MHC-II(+) microglial cells were observed at much higher frequency than astrocytes. This study provides information on the regulation of the MHC-II gene expression in astrocytes and in microglial cells by betaA and this pathway may be critically involved in the immune/inflammatory regulation within the central nervous system., (Copyright 1999 Elsevier Science B.V.)

Although the mechanism(s) underlying the failure of the maternal immune system to reject the semiallogeneic fetus have not been clearly defined, the absence of MHC class II antigen expression by fetal trophoblast cells very likely plays a critical role in the maintenance of normal pregnancy. However, the regulation of class II antigen expression in trophoblast cells is poorly understood. Class II transactivator (CIITA) is a transacting factor that is required for both constitutive and IFN-gamma-inducible class II gene transcription. In this report we demonstrate that the inability of trophoblast cells to express class II antigens is due to a lack of CIITA gene expression. Trophoblast cell lines derived from human, mouse, and rat do not express CIITA, and expression is not inducible by IFN-gamma. The absence of CIITA gene expression in trophoblasts treated with IFN-gamma does not result from a defect in the IFN-gamma receptor or the JAK/STAT pathway, because the classical IFN-gamma inducible gene encoding the guanylate-binding protein is expressed. Transfection of CIITA expression vectors into trophoblast cells results in activation of class II promoters, endogenous class II mRNA expression, and subsequent expression of class II antigens on the cell surface. In contrast, class I mRNA is not expressed in human trophoblast cells transfected with CIITA expression vectors. Thus, trophoblast cells contain all of the DNA binding factors necessary for class II transcription, and ectopic expression of CIITA is sufficient to activate class II, but not class I expression. The failure of trophoblast cells to express CIITA, and therefore class II antigens, provides a potential mechanism by which the fetus is protected from the maternal immune system during pregnancy.

Background: The T-cell stimulatory function of accessory cells isolated from peripheral blood lymphocytes of AIDS patients has been reported to be suppressed. These patients also have elevated levels of the immunosuppressive factor transforming growth factor (TGF)-beta1 in their serum and plasma., Objective: To explore the role of TGF-beta1 in the loss of accessory cell function of peripheral blood lymphocytes from AIDS patients., Methods: Fluorescent labeled anti-TGF-beta1 and confocal microscopy were used to detect the presence of TGF-beta1 on the cell membrane of dendritic cells. To assess the role of TGF-beta1 in the inhibition of accessory cell function in AIDS, antibodies against TGF-beta1 or the TGF-beta1 type III receptor, beta-glycan, were added to a mixed lymphocyte reaction., Results: TGF-beta1 was detected on the cell membrane of dendritic cells isolated from AIDS patients. The addition of blocking antibodies against either TGF-beta1 or beta-glycan restored the T-cell stimulatory function to accessory cells from these patients., Conclusions: T-cell stimulatory function was not irreversibly lost in AIDS patients. Our data suggested that beta-glycan-TGF-beta1 immunosuppressive complexes may contribute to the suppression of accessory cell function in these patients.

The maintenance of the fetus during pregnancy has been attributed to the absence of major histocompatibility complex (MHC) class II antigens on fetal trophoblastic cells that make contact with the maternal immune system. However, the mechanism(s) by which class II genes are regulated in trophoblast cells is unclear. We have identified a negative regulatory element (IA alpha NRE) in the promoter of the mouse class II gene IA alpha that represses IA alpha transcription in trophoblast cells. IA alpha NRE, located from-839 to -828, binds transacting factors from rat, mouse and human trophoblast cells, but not from 18 other cell lines tested. These results indicate that IA alpha NRE binding proteins (IA alpha NRE BPs) are conserved in species with hemochordial placentas, and suggest that IA alpha NRE binding activity is restricted primarily to trophoblast cells. Interestingly, the IA alpha NRE BPs bind to the IA alpha NRE antisense strand in a sequence-specific manner. IA alpha NRE represses transcription from the IA alpha promoter in a position-dependent manner, and has a minor down-regulatory effect on the activity of the SV40 promoter/enhancer. Our results demonstrate that MHC class II gene transcription is repressed in fetal trophoblast cells by sequence-specific, single-stranded DNA binding proteins, and suggest a possible mechanism by which the conceptus is protected from immune rejection during pregnancy.

Previous work from this laboratory has shown that transforming growth factor beta 2 (TGF-beta 2) mRNA is abundant in the pregnant uterus. In the present study, we examined the synthesis and secretion of TGF-beta 1,2 and 3 in the rat uterus and mammary gland and show differential secretion and expression of TGF-beta 2 in a tissue specific manner. Elevated levels of TGF-beta 2 were detected in late pregnant maternal plasmas (> 100 pM), and in the milk (> 500 pM) during early lactation. High concentrations of TGF-beta 2 (> 200 pM) were also detected in uterine fluids collected from ovariectomized adult rats after high dose estrogen treatment. TGF-beta 2 mRNA levels were elevated in lobuloalveolar epithelial cells isolated from pregnant mammary gland. Three major transcripts of 3.5, 4.0, and 4.7 kb were seen, of which the 4.7 kb, dominates. Mammary glands of estrogen treated ovariectomized rats showed a similar pattern of TGF-beta 2 transcripts. In contrast, four major TGF-beta 2 mRNA transcripts of 5.7, 4.7, 4.0, and 3.5 kb, with the dominant species of 4.0 and 5.7 kb, were observed in uteri from the estrogen treated animals up to 48 h after the last estrogen injection. This suggests that TGF-beta 2 is regulated in a tissue specific manner. We conclude that the secretion of TGF-beta 2 is tightly regulated by hormones and that estrogen and prolactin are critical factors in the tissue-specific regulation of the local production of TGF-beta 2 in the mammary gland and female reproductive tract.

The local immune response to the presence of tumour is affected by the pattern of cytokine production within the tumour bed. The purpose of this study was to define the pattern of cytokine production in human soft tissue sarcomas, attempting to identify potential immune suppression. Total RNA was extracted from six human soft tissue sarcomas from which cDNA was generated. PCR was carried out in the presence of primer pairs for G3PDH, TGF-beta1, TGF-beta2, TNF-alpha, INF-gamma, IL-2 and IL-10. TGF-beta1 and IL-10 mRNA was detected in all tumours, however, mRNA for IL-2 and INF-gamma was detected in only two out of six sarcomas. Paraffin sections were incubated with alpha-hu-TGF-beta1 or alpha-hu-IL-10 antibodies to localize protein production. TGF-beta1 and IL-10 protein expression was only associated with the tumour cells. These findings suggest potential local immune suppression within the tumour bed.

Here we describe a possible model of the cleavage mechanism in the hammerhead ribozyme. In this model, the 2' hydroxyl of C17 is moved into an appropriate orientation for an in-line attack on the G1.1 phosphate through a change in its sugar pucker from C3' endo to C2' endo. This conformational change in the active site is caused by a change in the uridine turn placing the N2 and N3 atoms of G5 of the conserved core in hydrogen bonding geometry with the N3 and N2 atoms on the conserved G16.2 residue. The observed conformational change in the uridine turn suggests an explanation for the conservation of G5. In the crystal structure of H.M. Pley et al., Nature 372, 68-74 (1994), G5 is situated 5.3A away from G16.2. However, the uridine turn is sufficiently flexible to allow this conformational change with relatively modest changes in the backbone torsion angles (average change of 14.2 degrees). Two magnesium ions were modeled into the active site with positions analogous to those described in the functionally similar Klenow fragment 3'-5' exonuclease (L.S. Beese and T.A. Steitz, EMBO J. 10, 25-33 (1991)), the Group I intron (T.A. Steitz and J.A. Steitz, P.N.A.S. U.S.A. 90, 6498-6502 (1993); R.F. Setlik et al., J. Biomol. Str. Dyn. 10, 945-972 (1993)) and other phosphotransferases. Comparison of this model with one in which the uridine turn conformation was not changed showed that although the changes in the C17 sugar pucker could be modeled, insufficient space existed for the magnesium ions in the active site.

We report that exposure of cells to a single electric pulse (250-1250 V/cm) results in the rapid and persistent activation of the DNA binding activities of a number of transcription factors, including AP-1, SP1, AP-2, and NF-kappa B, and the transient expression of select members of the fos and jun gene families. Induction of gene expression occurs primarily at the level of transcription, although c-jun expression also appears to be regulated posttranscriptionally. Interestingly, maximal induction of gene expression is detected at electrical field strengths that do not result in pore formation in the plasma membrane and that do not significantly affect cell viability. Exposure of cells to electric pulses does not result in the activation of HSF1 DNA binding activity, or the induction of hsp70 or p53 protein synthesis, indicating that the induction of fos and jun gene expression is not coincident with protein or DNA damage. The results of these studies suggest that electrical pulses may represent a novel mechanism for inducing the activities of multiple transcription factors and the expression of select members of the fos and jun gene families.

The initiation of the immune response is regulated, in part, by the effect of cytokines on the level of expression of major histocompatibility complex (MHC) class II antigens on antigen-presenting cells (APC). The expression of class II antigens on B cell and macrophage APC is induced by IFN-gamma and IL-4, and GM-CSF induces class II expression on macrophages (M phi). Our results show that transforming growth factor-beta (TGF-beta) inhibits IL-4- and GM-CSF-induced Ia gene expression on bone marrow macrophages but enhances IFN-gamma-induced gene expression. Nuclear run-on experiments demonstrated that the inhibitory effects of TGF-beta on GM-CSF- and IL-4-induced Ia antigen expression were primarily posttranscriptional and augmentation of IFN-gamma by TGF-beta was largely transcriptionally regulated.

Two hammerhead chimeric RNA/DNA ribozymes (HRz) were synthesized in pure form. Both were 30 nucleotides long, and the sequences were such that they could be targeted to cleave the HIV-1 gag RNA. Named HRz-W and HRz-M, the former had its invariable core region conserved, the latter had a uridine in the invariable region replaced by a guanine. Their secodary structures were determined by 2D NOESY 1H 500 MHz NMR spectroscopy in 90% water and 10% D2(0), following the imino protons. The data show that both HRz-M and HRz-W form identical secondary structures with stem regions consisting of continuous stacks of AT and GT pairs. An energy minimized computer model of this stem region is provided. The results suggest that the loss of catalytic activity that is known to result when an invariant core residue is replaced is not related to the secondary structure of the ribozymes in the absence of substrate.

The transforming growth factor beta family of peptides have diverse actions on the reproductive tracts of primates and rodents. In this study we report the expression of high levels of mRNA of one member of this superfamily, TGF-beta 2, in the pregnant mouse uterus. Using Northern blot analysis and in situ hybridization techniques, we have examined the pattern of expression of TGF-beta 1, TGF-beta 2 and colony-stimulating factor (CSF-1) in mouse maternal and fetal tissue at specific days of gestation. We report here that TGF-beta 2 is synthesized primarily in maternal decidual and uterine epithelial tissues. We observed a shift in the major site of synthesis from decidua to uterus between days 8.5 and 10.5 of gestation. These data demonstrate that the expression of TGF-beta 2 is differentially regulated in the decidua and uterine epithelial cells at various times during gestation. Small amounts of TGF-beta 2 mRNAs were detected in the fetus, and none was detected in placenta, yolk sac, or amniotic membrane. The uterus is likely the major site of synthesis of the TGF-beta 2 found in mouse amniotic fluid. TGF-beta 1 mRNAs are expressed in the uterus at markedly lower levels when compared to TGF-beta 2 mRNAs in both the decidua and uterus. Our results suggest that there is a unique regulation of TGF-beta 2 during pregnancy which may depend on pregnancy hormone(s) and differentiates it from the other mammalian isoforms of the TGF-beta s. TGF-beta 2 may play an important, albeit unknown, role at the maternal/fetal interface.

Interleukin-4 (IL-4) is a multipotent cytokine which stimulates proliferation of B and T lymphocytes, induces B lymphocyte expression of major histocompatibility complex (MHC) class II molecules and Fc epsilon R II (CD23) molecules, and promotes immunoglobulin class switching to IgE and IgG1. The mechanisms by which IL-4 induces these changes are unclear. To study the basis for heterogeneity in induction of class II MHC proteins observed in splenic B cells, three mouse B cell lines were treated with IL-4, and the response of MHC class II A alpha mRNA was analyzed. Each of the three cell lines responded with a distinctive profile. In one line, 70Z/3, A alpha mRNA was induced greater than 10 fold by 65 hr of IL-4 stimulation. Additional studies showed that A alpha mRNA was stabilized by IL-4 treatment of 70Z/3 cells, and that changes in gene transcription accounted for little of the increase in mRNA levels. A second line, WEHI.231, was shown to increase A alpha mRNA levels 4 fold after 48 hr of IL-4 treatment. In contrast to 70Z/3, when A alpha mRNA stability in the IL-4 treated WEHI.231 cells was compared to untreated cells, no difference was observed, IL-4 treatment induced A alpha transcription. The third cell line, M12.4.1, expressed high basal levels of A alpha, and these levels increased only slightly following IL-4 stimulation. The small increase correlated with a comparable transcriptional response. These data shown that the nature of the A alpha gene response to IL-4 differs among B cell lines. This heterogeneity of response is consistent with responses in total splenic B cells, and with the existence of functionally distinct subpopulations of B cells.

This report describes a murine amniotic fluid (MAF) immunosuppressive factor that has properties similar to transforming growth factor beta (TGF-beta). The MAF factor exhibits TGF-beta-like activity in stimulating soft agar colony formation by AKR-2B cells and inhibiting thymidine uptake by Mv1Lu cells. We demonstrate that both the immunosuppressive and TGF-beta-like activities of the MAF factor are completely neutralized by anti-TGF-beta 2-specific antibodies and not by anti-TGF-beta 1-specific antisera. The immunosuppressive factor in MAF is novel in that it appears to be identical or very closely related to TGF-beta 2 and is active in its native state. This active and anti-TGF-beta 2-neutralizable factor chromatographs at approximately 70 kD on Sephadex at neutral pH and appears to be able to complex with alpha-fetoprotein in native amniotic fluid. Chromatography of native MAF under acidic conditions demonstrates a lower molecular mass protein that chromatographs on BioGel in the same position as the mature 25-kD TGF-beta. This protein has the biological properties of TGF-beta and is immunosuppressive. Both of these activities are neutralizable with anti-TGF-beta 2 but not with anti-TGF-beta 1 or other antisera. By Northern analysis, we find high levels of TGF-beta 2 mRNA (with little or no TGF-beta 1) in the pregnant uterus that peak around day 15 of gestation and then fall rapidly by day 19 as birth approaches. The TGF-beta 2-like factor could possibly play a role in maternal immunity, in the retention of the fetal allograft, as well as in regulating fetal and neonatal immunological competence.

A method of isolation of alpha-1-antitrypsin (alpha-1-AT) in good yield from normal human plasma is described. A key step was affinity chromatography employing an antiserum which had been depleted of alpha-1-AT antibodies. The final preparations were homogeneous by immunological and physicochemical criteria. The specific activity of the purified alpha-1-AT was 0.363 mg of active bovine trypsin inhibited per 1.0 mg of inhibitor. Polyacrylamide gel patterns at both alkaline and acid pH of highly pure preparations frequently, but not invariably, showed multiple hands. Molecular weight studies by sedimentation equilibrium ultracentrifugation in aqueous buffer and in 6 M guanidine as well as sodium dodecyl sulfate polyacrylamide gel electrophoresis suggest that alpha-1-AT is a single polypeptide chain having a molecular weight of 49,500. Other physical and chemical properties of the inhibitor are described. A limited N-terminal sequence (Glu-Asp-Pro-Gln-Gly-Asx-Ala-Ala) was obtained. It was found that alpha-1-AT easily forms polymers and higher aggregates when exposed to denaturing agents such as 8 M urea and 6 M guanidine. The results suggest that aggregation is determined by both covalent and noncovalent forces.