Protective immunity against disparate tumors is mediated by a nonpolymorphic MHC class I molecule

Current peptide-based immunotherapies for treatment of model cancers target tumor Ags bound by the classical MHC class I (class Ia) molecules. The extensive polymorphism of class Ia loci greatly limits the effectiveness of these approaches. We demonstrate in this study that the murine nonpolymorphic, nonclassical MHC class I (class Ib) molecule Q9 (Qa-2) promotes potent immune responses against multiple syngeneic tumors. We have previously shown that ectopic expression of Q9 on the surface of class Ia-negative B78H1 melanoma led to efficient CTL-mediated rejection of this tumor. In this study, we report that surface-expressed Q9 on 3LLA9F1 Lewis lung carcinoma and RMA T cell lymphoma also induces potent antitumor CTL responses. Importantly, CTL harvested from animals surviving the initial challenge with Q9-positive 3LLA9F1, RMA, or B78H1 tumors recognized and killed their cognate tumors as well as the other cancer lines. Furthermore, immunization with Q9-expressing 3LLA9F1 or RMA tumor cells established immunological memory that enhanced protection against subsequent challenge with a weakly immunogenic, Q9-bearing melanoma variant. Collectively, the generation of cross-reactive CTL capable of eliminating multiple disparate Q9-expressing tumors suggests that this nonpolymorphic MHC class I molecule serves as a restriction element for a shared tumor Ag(s) common to lung carcinoma, T cell lymphoma, and melanoma.     

Developmental control of a promoter-specific factor that is also regulated by the E1A gene product

We have detected a cellular factor in F9 teratocarcinoma cells that recognizes an adenovirus E1A inducible promoter. This factor, termed E2F, was previously identified in HeLa cells and was found at increased levels as a function of the E1A gene product. Upon differentiation of F9 cells with retinoic acid and cAMP, the factor declines to near undetectable levels, consistent with the control of this factor by E1A and the presence of a cellular E1A-like activity in F9 cells but not in differentiated F9 cells. Finally, if the E1A gene is introduced into differentiated cells by an adenovirus infection, there is a large increase in the level of the factor. We suggest that the control of E2F during F9 differentiation is indeed due to an E1A-like activity.     

14-3-3 protein family members have a regulatory role in retinoic acid-mediated induction of cytokeratins in F9 cells

We have found that the expression of five 14-3-3 protein isoforms is induced during the retinoic acid (RA)-mediated differentiation of mouse embryonal carcinoma F9 cells. The induced expression of the 14-3-3 proteins is presumed to have a role in enhancing the mitogen-activated protein kinase (MAPK) activity during RA-mediated F9 cell differentiation, because using genetically engineered budding yeast we showed that these isoforms enhanced the signaling in the MAPK cascade mainly through the interaction with Raf-1. Then we assessed the role of increased MAPK activity in F9 cell differentiation by interfering with signaling in the MAPK cascade in F9 cells. The exogenous expression of dominant-negative MEK1 efficiently abrogated RA-mediated induction of the cytokeratins EndoA and EndoC in the F9 cells. These results suggest that the 14-3-3 proteins play a role in the efficient induction of the cytokeratins during F9 cell differentiation through their signal enhancing activity in the MAPK cascade.     

An Antibody against a Novel and Conserved Epitope in the Hemagglutinin 1 Subunit Neutralizes Numerous H5N1 Influenza Viruses

The spread of the recently emerged, highly pathogenic H5N1 avian influenza virus has raised concern. Preclinical studies suggest that passive immunotherapy could be a new form of treatment for H5N1 virus infection. Here, a neutralizing monoclonal antibody (MAb) against the hemagglutinin (HA) of the influenza A/chicken/Hatay/2004 H5N1 virus, MAb 9F4, was generated and characterized. MAb 9F4 binds both the denatured and native forms of HA. It was shown to recognize the HA proteins of three heterologous strains of H5N1 viruses belonging to clades 1, 2.1, and 2.2, respectively. By use of lentiviral pseudotyped particles carrying HA on the surface, MAb 9F4 was shown to effectively neutralize the homologous strain, Hatay04, and another clade 1 strain, VN04, at a neutralization titer of 8 ng/ml. Furthermore, MAb 9F4 also neutralized two clade 2 viruses at a neutralizing titer of 40 ng/ml. The broad cross-neutralizing activity of MAb 9F4 was confirmed by its ability to neutralize live H5N1 viruses of clade 2.2.2. Epitope-mapping analysis revealed that MAb 9F4 binds a previously uncharacterized epitope below the globular head of the HA1 subunit. Consistently, this epitope is well conserved among the different clades of H5N1 viruses. MAb 9F4 does not block the interaction between HA and its receptor but prevents the pH-mediated conformational change of HA. MAb 9F4 was also found to be protective, both prophylactically and therapeutically, against a lethal viral challenge of mice. Taken together, our results showed that MAb 9F4 is a neutralizing MAb that binds a novel and well-conserved epitope in the HA1 subunit of H5N1 viruses.The highly pathogenic avian influenza A subtype H5N1 virus was first isolated from geese in Guangdong province, China, in 1996 (44). Since 2003, the H5N1 strains have caused major morbidity and mortality in poultry populations across Asia, Europe, and Africa (3, 25). In 1997, the virus was transmitted from chickens to humans in Hong Kong, causing 18 reported cases of illness, including 6 deaths (6, 7, 37). As of September 2009, there were 442 confirmed human infections in 15 countries, with an alarming fatality rate of 59% (42). Although occurrences of human H5N1 infection are sporadic and rare, its rapid dissemination, the ongoing evolution of the avian H5N1 virus, and the absence of anti-H5N1 herd immunity in humans raise concerns regarding a possible H5N1 influenza pandemic (2, 4, 13). Since human infections are associated with severe disease and high mortality, the consequences of a pandemic could be catastrophic.Current strategies against influenza include vaccination and antiviral drug treatment (1). Due to the existence of multiple antigenic clades and subclades of the H5N1 virus, the difficulty of predicting the major strain that may cause the next pandemic is the main obstacle to current vaccine development. Moreover, resistance to M2 ion channel inhibitors (rimantidine and amantidine) has been reported in H5N1 isolates (1, 5), and the neuraminidase inhibitors (oseltamivir and zanamivir) require higher doses and prolonged treatment (45), and resistance has been reported in children (21). Passive immunotherapy is now increasingly used to treat numerous human infectious diseases (28, 33). Convalescent-phase blood and serum products were used to improve clinical outcomes for severely ill influenza patients during the 1918 influenza pandemic (27). Promising results with mouse models using a neutralizing monoclonal antibody (MAb) for H5N1 influenza treatment (17, 26) and a report of the recovery of an H5N1 virus-infected patient after treatment with convalescent-phase plasma (47) indicate that MAbs could be a potential treatment against H5N1 viruses.The hemagglutinin (HA) protein is one of the two major surface glycoproteins on the envelope of influenza A virus, with 16 distinct types identified in the avian species. The HA protein is responsible for receptor binding to host cells and for viral entry and is therefore the primary target of neutralizing antibodies (Abs) (35). It is a homotrimer, with each subunit made up of two disulfide-linked polypeptides, HA1 and HA2. Structurally, each subunit consists of a membrane-proximal helix-rich stem structure and a membrane-distal receptor binding globular domain (35).In this study, we describe a MAb, named MAb 9F4, raised against the recombinant baculovirus-expressed HA protein of A/chicken/Hatay/2004 H5N1 virus. Its neutralizing property was investigated, and epitope mapping was performed. The MAb 9F4 binding site was found to lie outside previously characterized antigenic sites in the HA protein. This epitope is well conserved among the different clades of H5N1 viruses, consistent with the cross-neutralizing activity of MAb 9F4. The mode of inhibition was also investigated, and MAb 9F4 was found to mediate postattachment neutralization in a dose-dependent manner. Finally, the protective ability of MAb 9F4 was also evaluated in a mouse model, and it was shown to protect against lethal H5N1 challenge both prophylactically and therapeutically. Taken together, the data could provide new information for the design of an H5N1 vaccine, and MAb 9F4 may be a possible candidate for use in passive immunotherapy.     

Investigating the Role of Endophytic Fungi in <Emphasis Type="Italic">Gentiana scabra</Emphasis> bge. by Cross-Growth Period Inoculation

Gentiana scabra Bge. (gentian) is a Chinese medicinal plant. Endophytic fungi from the roots of gentian were isolated and cross-growth period inoculation was performed to study the roles of three Trichoderma spp. strains (F1, F2, and F9) in their original host plant. In treatments inoculated with F1, F2, and F9, gentiopicroside content increased 33.6, 23.7 and 13% than that in the control. Strains F1, F2, and F9 could also improve polysaccharide content by more than 6.6, 18.7 and 30% compared to the control. The incidence of spot blight in gentian inoculated with F1, F2, and F9 decreased by 31.2, 26.7 and 8.5%. Inconsistent changes in the activity of the three enzymes (superoxide dismutase, catalase and peroxidase) were observed when the plants were attacked by pathogens or inoculated with fungi. High enzymatic activity did not reflect mild disease. Cross-growth period inoculation, which takes into account the original living environment (gentian plant as “substrate” and different microorganisms as symbionts) of endophytic fungi, provides a new idea for studying effects of endophytes on their original hosts. This is the first research about the role of endophytic fungi in Gentiana scabra bge. in vivo.     

Structure of the malaria antigen AMA1 in complex with a growth-inhibitory antibody

Identifying functionally critical regions of the malaria antigen AMA1 (apical membrane antigen 1) is necessary to understand the significance of the polymorphisms within this antigen for vaccine development. The crystal structure of AMA1 in complex with the Fab fragment of inhibitory monoclonal antibody 1F9 reveals that 1F9 binds to the AMA1 solvent-exposed hydrophobic trough, confirming its importance. 1F9 uses the heavy and light chain complementarity-determining regions (CDRs) to wrap around the polymorphic loops adjacent to the trough, but uses a ridge of framework residues to bind to the hydrophobic trough. The resulting 1F9-AMA1-combined buried surface of 2,470 A(2) is considerably larger than previously reported Fab-antigen interfaces. Mutations of polymorphic AMA1 residues within the 1F9 epitope disrupt 1F9 binding and dramatically reduce the binding of affinity-purified human antibodies. Moreover, 1F9 binding to AMA1 is competed by naturally acquired human antibodies, confirming that the 1F9 epitope is a frequent target of immunological attack.     

Nicotinate O-Glucosylation Is an Evolutionarily Metabolic Trait Important for Seed Germination under Stress Conditions in Arabidopsis thaliana

The glycosylation of nicotinate (NA), a key intermediate of the NAD salvage pathway, occurs widely in land plants. However, the physiological function of NA glycosylation is not well understood in planta, and no gene encoding NA glycosyltransferase has been reported to date. NA glycosylation in Arabidopsis thaliana occurs at either the N- or the O-position of the NA molecule, and O-glucosylation appears to be unique to the Brassicaceae. Using gene-enzyme correlations focused on Family 1 glycosyltransferases (GTs; EC 2.4), we identified and characterized three Arabidopsis GTs, which are likely involved in NA glycosylation. These include one NAOGT (UGT74F2; previously identified as a salicylic acid glycosyltransferases) and two NANGTs (UGT76C4 and UGT76C5). Arabidopsis mutants of UGT74F2 accumulate higher levels of free NA, but not salicylic acid, than that of the wild type, and this inversely correlated with seed germination rates under various abiotic stresses. The germination defect of the ugt74f2-1 mutant could be fully complemented by overexpression of UGT74F2. These observations, together with comprehensive chemical analysis, suggest that NA glycosylation may function to protect plant cells from the toxicity of NA overaccumulation during seed germination. Combined with phylogenetic analysis, our results suggest that NAOGTs arose recently in the Brassicaceae family and may provide a fitness benefit. The multifunctionality of UGT74F2 in Arabidopsis is also investigated and discussed.     

Structure determination of a novel uronic acid residue isolated from the exopolysaccharide produced by a bacterium originating from deep sea hydrothermal vents

The exopolysaccharide produced by the bacterium Alteromonas sp. strain 1644 originating from deep sea hydrothermal vents was shown to contain a novel glucuronic acid derivatives:

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acid. The structure of this compound was established on the basis of mass spectrometric data, methylation analysis, preparation of derivatives, and chemical synthesis of references compounds.     

Expression of a 91-kilodalton PEA3-binding protein is down-regulated during differentiation of F9 embryonal carcinoma cells.

Proteins binding to the PEA3 enhancer motif (AGGAAG) activate the polyomavirus early promoter and help comprise the viral late mRNA initiator element (W. Yoo, M. E. Martin, and W. R. Folk, J. Virol. 65:5391-5400, 1991). Because many developmentally regulated cellular genes have PEA3 motifs near their promoter sequences, and because Ets family gene products activate the PEA3 motif, we have studied the expression of PEA3-binding proteins and Ets-related proteins during differentiation of F9 embryonal carcinoma cells. An approximately 91-kDa protein (PEA3-91) was identified in F9 cell nuclear extracts by UV cross-linking to a radiolabeled PEA3 oligonucleotide probe, and expression of PEA3-91 was down-regulated after differentiation of F9 cells to parietal endoderm. The c-ets-1 gene product binds to a sequence in the murine sarcoma virus long terminal repeat that is similar to the PEA3 motif (cGGAAG), but PEA3-91 was not cross-linked to this Ets-1-binding motif, nor did antiserum which recognizes murine c-ets-1 and c-ets-2 proteins have any effect on PEA3-binding activity in mobility shift assays. Furthermore, c-ets-1 mRNA was not detected in undifferentiated or differentiated F9 cells, and c-ets-2 mRNA levels remained high after differentiation. Antiserum against the Drosophila Ets-related E74A protein, however, recognized an approximately 92-kDa protein in F9 cells whose expression during differentiation varied in a manner identical to that of PEA3-91. These data suggest that PEA3-91 is not the product of the ets-1 or ets-2 genes but is likely to be the product of a murine homolog of the Drosophila E74 gene.     

Gene expression in visceral endoderm: a comparison of mutant and wild- type F9 embryonal carcinoma cell differentiation

We have examined the abundance and cell specificity of several mRNAs that are regulated during the retinoic acid (RA)-induced differentiation of F9 embryonal carcinoma cells to visceral endoderm. The experiments confirmed the multistep nature of this process by demonstrating the expression of the ERA-1/Hox 1.6 message within 6 h after RA addition; the expression of messages specific for the extracellular matrix proteins laminin B1 and B2, and collagen IV(alpha 1) between days 4 and 12; and the expression of two visceral endoderm markers, alpha-fetoprotein (AFP) and H19, by days 8-15. In situ hybridization experiments revealed that the collagen IV(alpha 1) mRNA is restricted to the outer cell layer of F9 cell aggregates regardless of the presence or absence of RA. Laminin B1 and B2 mRNAs are concentrated in the outer cell layer of RA-treated aggregates although significant levels of message are also observed within the interior cells of the aggregates. Unexpectedly, AFP mRNA is detectable in only a subset of the outer cells of F9 cell aggregates grown 15 d in the presence of RA. The results obtained from wild-type F9 cells were compared with those from a mutant F9 cell line, RA-5-1, which was previously shown to synthesize collagen IV containing six- to ninefold less 4-hydroxyproline than that in wild-type F9 cells. RA-5-1 cells exhibit four- to sixfold less of the mRNAs encoding two visceral endoderm proteins, AFP and H19, than wild-type F9 cells after RA treatment of RA-5-1 aggregates. RA-5-1 cells, however, do exhibit an RA-associated increase in the level of ERA-1/Hox 1.6 mRNA within 6 h after adding RA. Although the collagen IV protein level is similar in wild-type F9 and RA-5-1 aggregates, the collagen IV(alpha 1) message level is 6-20-fold greater in aggregates of mutant cells than in aggregates of wild-type cells. Moreover, in situ hybridizations showed that this message is evenly distributed throughout the RA-5-1 aggregates rather than restricted to the outer cell layers as it is in wild-type F9 aggregates. These results suggest that abnormal collagen IV expression and localization are associated with decreased expression of the visceral endoderm markers, AFP and H19, in RA-5-1 cell aggregates.     

SufA of the Opportunistic Pathogen Finegoldia magna Modulates Actions of the Antibacterial Chemokine MIG/CXCL9, Promoting Bacterial Survival during Epithelial Inflammation

The anaerobic bacterium Finegoldia magna is part of the human commensal microbiota, but is also an important opportunistic pathogen. This bacterium expresses a subtilisin-like serine proteinase, SufA, which partially degrade the antibacterial chemokine MIG/CXCL9. Here, we show that MIG/CXCL9 is produced by human keratinocytes in response to inflammatory stimuli. In contrast to the virulent human pathogen Streptococcus pyogenes, the presence of F. magna had no enhancing effect on the MIG/CXCL9 expression by keratinocytes, suggesting poor detection of the latter by pathogen-recognition receptors. When MIG/CXCL9 was exposed to SufA-expressing F. magna, the molecule was processed into several smaller fragments. Analysis by mass spectrometry showed that SufA cleaves MIG/CXCL9 at several sites in the COOH-terminal region of the molecule. At equimolar concentrations, SufA-generated MIG/CXCL9 fragments were not bactericidal against F. magna, but retained their ability to kill S. pyogenes. Moreover, the SufA-generated MIG/CXCL9 fragments were capable of activating the angiostasis-mediating CXCR3 receptor, which is expressed on endothelial cells, in an order of magnitude similar to that of intact MIG/CXCL9. F. magna expresses a surface protein called FAF that is released from the bacterial surface by SufA. Soluble FAF was found to bind and inactivate the antibacterial activity of MIG/CXCL9, thereby further potentially promoting the survival of F. magna. The findings suggest that SufA modulation of the inflammatory response could be a mechanism playing an important role in creating an ecologic niche for F. magna, decreasing antibacterial activity and suppressing angiogenesis, thus providing advantage in survival for this anaerobic opportunist compared with competing pathogens during inflammation.The mucosal surfaces and skin of the human body are colonized by a large number of bacterial species constituting the normal microbiota. In contrast to pathogens, these commensals usually do not elicit any inflammatory responses in epithelial tissues of the healthy host (1). The Gram-positive coccus Finegoldia magna is part of the anaerobic normal microbiota associated with the skin (2), but it also inhabits the oro-pharynx, gastrointestinal, and urogenital tracts (3). During disturbed homeostasis, this bacterium becomes an important opportunistic pathogen; associated with several clinical conditions, such as soft tissue infections, wound infections, bone/joint infections, and vaginosis (3–5). Among anaerobic cocci of the normal microbiota, F. magna is the species most commonly isolated from clinical conditions (3).Recognition of bacteria and their products by cells residing in the submucosal tissues, for example dendritic cells, triggers an inflammatory response leading to production of host defense molecules, including chemokines. Chemokines comprise a large family of peptides that are key players in inflammation by regulating leukocyte trafficking and activation. They are divided into four groups, XC, CC, CXC, and CX₃C, depending on the arrangement of conserved cysteine residues in their NH₂ terminus (6). The CXC subfamily can be further divided into ELR-positive and ELR-negative respectively, based on the presence or absence of the sequence motif glutamic acid-leucine-arginine (ELR) NH₂ terminal to the first cysteine. IFN-γ, a key cytokine produced during bacterial infection, induces expression of the ELR-negative CXC-chemokine MIG/CXCL9 (Monokine Induced by Gamma-interferon)³ (7). MIG/CXCL9 binds and activates a G-protein-coupled seven transmembrane receptor, CXCR3, which is present on eosinophils, activated T cells (CD8+), and NK cells (8). In addition to its ability to recruit and activate leukocytes, MIG/CXCL9 possesses angiostatic properties through activation of CXCR3 expressed on endothelial cells, and it also exerts potent antibacterial properties (9–11). Upon IFN-dependent inflammation, for example during bacterial infection, this chemokine is produced by epithelial cells and participates in activities of both innate and adaptive immunity (10, 12–14).The finding that epithelial cells recognize important human pathogens, such as Streptococcus pyogenes, leading to an increased MIG/CXCL9 production (10, 12) raised the question whether an opportunistic pathogen like F. magna could be recognized in a similar fashion. In skin F. magna is localized to the epidermis where they adhere to basement membranes through an interaction with the basement membrane protein BM-40 (15). Binding to BM-40 is mediated by the surface protein FAF (F. magna adhesion factor) that is expressed by more than 90% of F. magna isolates (15). Bacteria, both commensals and pathogens, express proteases that are important both during colonization and invasion (16). In the case of F. magna, most strains express a subtilisin-like enzyme, SufA (Subtilase of Finegoldia magna), which is associated with the bacterial surface, but also secreted in substantial amounts during bacterial growth (17). Studies on the proteolytic activity of SufA demonstrated that the enzyme cleaves and inactivates antibacterial molecules like LL-37 and MIG/CXCL9 (17). Here, we show that MIG/CXCL9, produced by human keratinocytes in response to inflammatory stimuli, is degraded by SufA-expressing F. magna. The generated MIG/CXCL9 fragments are still able to activate the MIG/CXCL9 receptor, CXCR3 and kill S. pyogenes, while F. magna is left unaffected. This modulation of the MIG/CXCL9 activities promotes the survival of F. magna during inflammation.     

Vaccinia Virus Protein F1L Is a Caspase-9 Inhibitor

Apoptosis plays important roles in host defense, including the elimination of virus-infected cells. The executioners of apoptosis are caspase family proteases. We report that vaccinia virus-encoded F1L protein, previously recognized as anti-apoptotic viral Bcl-2 family protein, is a caspase-9 inhibitor. F1L binds to and specifically inhibits caspase-9, the apical protease in the mitochondrial cell death pathway while failing to inhibit other caspases. In cells, F1L inhibits apoptosis and proteolytic processing of caspases induced by overexpression of caspase-9 but not caspase-8. An N-terminal region of F1L preceding the Bcl-2-like fold accounts for caspase-9 inhibition and significantly contributes to the anti-apoptotic activity of F1L. Viral F1L thus provides the first example of caspase inhibition by a Bcl-2 family member; it functions both as a suppressor of proapoptotic Bcl-2 family proteins and as an inhibitor of caspase-9, thereby neutralizing two sequential steps in the mitochondrial cell death pathway.