Immunogenic Form Research Articles

Immunogenic Apoptotic Cell Death and Anticancer Immunity.

For many years it has been thought that apoptotic cells rapidly cleared by phagocytic cells do not trigger an immune response but rather have anti-inflammatory properties. However, accumulating experimental data indicate that certain anticancer therapies can induce an immunogenic form of apoptosis associated with the emission of damage-associated molecular patterns (DAMPs), which function as adjuvants to activate host antitumor immune responses. In this review, we will first discuss recent advances and the significance of danger signaling pathways involved in the emission of DAMPs, including calreticulin, ATP, and HMGB1. We will also emphasize that switching on a particular signaling pathway depends on the immunogenic cell death stimulus. Further, we address the role of ER stress in danger signaling and the classification of immunogenic cell death inducers in relation to how ER stress is triggered. In the final part, we discuss the role of radiotherapy-induced immunogenic apoptosis and the relationship of its immunogenicity to the fraction dose and concomitant chemotherapy.

The Immune System in Cancer Prevention, Development and Therapy.

The immune system plays a pivotal role in the maintenance of the integrity of an organism. Besides the protection against pathogens, it is strongly involved in cancer prevention, development and defense. This review focuses on how the immune system protects against infections and trauma and on its role in cancer development and disease. Focus is set on the interactions of the innate and adaptive immune system and tumors. The role of IFN-γ as a pleiotropic cytokine that plays a very important role at the interface of innate and adaptive immune systems in tumor development and induction of anti-tumor immune responses is outlined. Further, immune cells as prognostic and predictive markers of cancer will be discussed. Data are provided that even the brain as immune privileged organ is subjected to immune surveillance and consequently also brain tumors. Immune therapeutic approaches for glioblastoma multiforme, the most frequent and malignant brain tumor, based on vaccination with dendritic cells are outlined and application of hyperthermia in form of magnetic nanoparticles is discussed. We conclude that the immune system and developing tumors are intimately intertwined. Anti-tumor immune responses can be prominently boosted by multimodal therapies aiming on the one hand to induce immunogenic tumor cell death forms and on the other hand to actively counteract the immune suppressive microenvironment based on the tumor itself.

Current position of TNF-α in melanomagenesis.

Melanoma is one of the most heterogeneous and immunogenic forms of cancer. Both tumor and stroma cells synthesize many cytokines involved in rapid development and metastasis. One of these cytokines from the tumor milieu is tumor necrosis factor-alpha (TNF-α), which seems to have an intricate role in melanomagenesis. Initially, it was found that TNF-α can induce apoptosis of tumor cells through both extrinsic and intrinsic pathways, in contrast with later studies that revealed its protumoral activity. TNF-α is involved in inflammation, inducing the secretion of survival molecules like antiapoptotic proteins, proangiogenetic factors and metastasis markers. Although there are many therapeutic strategies against melanoma, the prognosis of advanced stages remains poor, due to several tumor resistance mechanisms. TNF seems to be a negative prognostic factor in melanoma surgery and correlates with chemotherapy resistance. However, high intratumoral levels of TNF-α might be beneficial for immunotherapy. Researchers may redirect their studies in the future by double activating of the proinflammatory molecule TNF-α and the immune cells in order to obtain an antitumoral response in metastatic melanoma.

Abstract 1319: Monitoring inflammasome activation with a bioluminescent caspase-1 assay

Abstract Inflammatory responses and immune modulation play important and complex roles in cancer development and therapy, but methods to monitor caspase-1 activity associated with inflammasome activation have been limited. Inflammasomes are protein complexes induced by diverse inflammatory stimuli. Caspase-1, an essential component of the inflammasome, is activated in response to these stimuli, resulting in the processing and release of cytokines, IL-1β and IL-18, and pyroptosis, an immunogenic form of cell death. Western blots and ELISA are the primary, but indirect, methods for monitoring caspase-1 activity currently in use. To simplify and provide a more direct means of detecting cell-based caspase-1 activity, we developed a sensitive, homogeneous, plate-based assay that eliminates the need for significant sample processing. The assay employs a single-step, bioluminescent format combining a caspase-1 substrate, Z-WEHD-aminoluciferin, with a thermostable luciferase in an optimized reagent subsequently added to treated cells in an assay well. The coupled-enzyme system quickly reaches a steady-state between caspase cleavage of the substrate and luciferase conversion of the aminoluciferin, with light generated proportional to the amount of caspase-1 activity present in the sample. In addition to substrate selection, assay specificity for caspase-1 is conferred by inclusion of a proteasome inhibitor, MG132, in the reagent and by the subsequent use of a caspase-1 inhibitor, Ac-YVAD-CHO, to confirm activity. This approach enables clear determination of caspase-1 activity even in the context of apoptotic cells. Studies with Casp1−/− cells further demonstrate the effectiveness of this assay system to specifically detect cell-based, caspase-1 activity. Using this novel assay system, caspase-1 activation has been quantitatively determined in THP-1 cells following treatment with α-hemolysin, LPS, nigericin, monosodium urate crystals, R-848, Pam3CSK4, and flagellin. Caspase-1 activation has also been demonstrated in treated J774A.1 mouse macrophages, bone marrow-derived macrophages from mice, as well as in human primary monocytes. Of note, caspase-1 activity can be monitored either directly in cells or released into the culture medium following cell treatment with various inflammatory stimuli. Monitoring released caspase-1 activity in supernatants is fast, sensitive, and nondestructive, thereby enabling subsequent multiplexing of the biological sample with other assays to monitor additional cell parameters, such as IL-1β release or cell death. Therefore, this assay system provides a rapid, convenient, and flexible method to specifically and quantitatively monitor caspase-1 activation in cells in a plate-based format. This will allow a more efficient and effective assessment of inflammasome activation as well as enable high-throughput screening for inflammasome modulators. Citation Format: Martha O'Brien, Danielle Moehring, Raúl Muñoz-Planillo, Gabriel Núñez, Justin Callaway, Jenny Ting, Mike Scurria, Tim Ugo, Laurent Bernad, James Cali, Dan Lazar. Monitoring inflammasome activation with a bioluminescent caspase-1 assay. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1319. doi:10.1158/1538-7445.AM2015-1319

A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27) Protein as an HIV Immunogen.

In the HIV vaccine field, there is a need to produce highly immunogenic forms of the Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K) by fusing gp120 from clade B with the vaccinia virus (VACV) 14K oligomeric protein (derived from A27L gene). Stable CHO cell lines expressing HIV-1 gp120-14K protein were generated and the protein purified was characterized by size exclusion chromatography, electron microscopy and binding to anti-Env antibodies. These approaches indicate that gp120-14K protein is oligomeric and reacts with a wide spectrum of HIV-1 neutralizing antibodies. Furthermore, in human monocyte-derived dendritic cells (moDCs), gp120-14K protein upregulates the levels of several proinflammatory cytokines and chemokines associated with Th1 innate immune responses (IL-1β, IFN-γ, IL-6, IL-8, IL-12, RANTES). Moreover, we showed in a murine model, that a heterologous prime/boost immunization protocol consisting of a DNA prime with a plasmid expressing gp120-14K protein followed by a boost with MVA-B [a recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 gp120, Gag, Pol and Nef antigens from clade B], generates stronger, more polyfunctional, and greater effector memory HIV-1-specific CD4+ and CD8+ T-cell immune responses, than immunization with DNA-gp120/MVA-B. The DNA/MVA protocol was superior to immunization with the combination of protein/MVA and the latter was superior to a prime/boost of MVA/MVA or protein/protein. In addition, these immunization protocols enhanced antibody responses against gp120 of the class IgG2a and IgG3, together favoring a Th1 humoral immune response. These results demonstrate that fusing HIV-1 gp120 with VACV 14K forms an oligomeric protein which is highly antigenic as it activates a Th1 innate immune response in human moDCs, and in vaccinated mice triggers polyfunctional HIV-1-specific adaptive and memory T-cell immune responses, as well as humoral responses. This novel HIV-1 gp120-14K immunogen might be considered as an HIV vaccine candidate for broad T and B-cell immune responses.

Genome-wide DNA hypomethylation and RNA:DNA hybrid accumulation in Aicardi-Goutières syndrome.

Aicardi-Goutières syndrome (AGS) is a severe childhood inflammatory disorder that shows clinical and genetic overlap with systemic lupus erythematosus (SLE). AGS is thought to arise from the accumulation of incompletely metabolized endogenous nucleic acid species owing to mutations in nucleic acid-degrading enzymes TREX1 (AGS1), RNase H2 (AGS2, 3 and 4), and SAMHD1 (AGS5). However, the identity and source of such immunogenic nucleic acid species remain undefined. Using genome-wide approaches, we show that fibroblasts from AGS patients with AGS1-5 mutations are burdened by excessive loads of RNA:DNA hybrids. Using MethylC-seq, we show that AGS fibroblasts display pronounced and global loss of DNA methylation and demonstrate that AGS-specific RNA:DNA hybrids often occur within DNA hypomethylated regions. Altogether, our data suggest that RNA:DNA hybrids may represent a common immunogenic form of nucleic acids in AGS and provide the first evidence of epigenetic perturbations in AGS, furthering the links between AGS and SLE.

Modulation of radiochemoimmunotherapy-induced B16 melanoma cell death by the pan-caspase inhibitor zVAD-fmk induces anti-tumor immunity in a HMGB1-, nucleotide- and T-cell-dependent manner.

One prerequisite that radiotherapy (RT) and chemotherapy (CT) result in anti-tumor immune responses is triggering of immunogenic cell death forms such as necroptosis. The latter is inducible by inhibition of apoptosis with the pan-caspase inhibitor zVAD-fmk. The design of multimodal therapies that overcome melanoma's resistance to apoptosis is a big challenge of oncoimmunology. As hints exist that immune stimulation by hyperthermia (HT) augments the efficacy of melanoma therapies and that tumors can be sensitized for RT with zVAD-fmk, we asked whether combinations of RT with dacarbazine (DTIC) and/or HT induce immunogenic melanoma cell death and how this is especially influenced by zVAD-fmk. Necroptosis was inducible in poorly immunogenic B16-F10 melanoma cells and zVAD-fmk generally increased melanoma cell necrosis concomitantly with the release of HMGB1. Supernatants (SNs) of melanoma cells whose cell death was modulated with zVAD-fmk induced an upregulation of the activation markers CD86 and MHCII on macrophages. The same was seen on dendritic cells (DCs), but only when zVAD-fmk was added to multimodal tumor treatments including DTIC. DCs of MyD88 KO mice and DCs incubated with SNs containing apyrase did not increase the expression of these activation markers on their surface. The in vivo experiments revealed that zVAD-fmk decreases the tumor growth significantly and results in a significantly reduced tumor infiltration of Tregs when added to multimodal treatment of the tumor with RT, DTIC and HT. Further, a significantly increased DC and CD8+ T-cell infiltration into the tumor and in the draining lymph nodes was induced, as well as an increased expression of IFNγ by CD8+ T cells. However, zVAD-fmk did not further reduce tumor growth in MyD88 KO mice, mice treated with apyrase or RAG KO mice. We conclude that HMGB1, nucleotides and CD8+ T cells mediate zVAD-fmk induced anti-melanoma immune reactions in multimodal therapy settings.

Modulation of inflammation by low and high doses of ionizing radiation: Implications for benign and malign diseases

Inflammation is a homeostatic mechanism aiming to maintain tissue integrity. The underlying immunological mechanisms and the interrelationship between ionizing radiation and inflammation are complex and multifactorial on cellular and chemical levels. On the one hand, radiation with single doses exceeding 1 Gy might initiate inflammatory reactions and thereby impact on tumor development. On the other hand, radiation is capable of attenuating an established inflammatory process, which is clinically used for the treatment of inflammatory and degenerative diseases with low-dose radiotherapy (single dose <1 Gy). At higher doses, ionizing radiation, especially in combination with additional immune stimulation, fosters the induction of immunogenic forms of tumor cell death and shifts the tumor microenvironment as well as the infiltration of immune cells from an anti- to a pro-inflammatory state. Distinct tumor infiltrating immune cells predict the response to radiochemotherapy in a multitude of tumor entities. While a high tumor infiltration of these adaptive immune cells mostly predicts a favorable disease outcome, a high infiltration of tumor-associated macrophages predicts an unfavorable response. Pro-inflammatory events should dominate over anti-inflammatory ones in this scenario. This review focuses on how ionizing radiation modulates inflammatory events in benign inflammatory and in malign diseases. A special focus is set on the role of tumor infiltrating lymphocytes and macrophages as biomarkers to predict treatment response and anti-tumor immunity and on mechanisms implicated in the anti-inflammatory effects of low-dose radiation therapy.

Apoptotic cell clearance and its role in the origin and resolution of chronic inflammation.

Apoptotic cell clearance and its role in the origin and resolution of chronic inflammation.

Per-oral immunization with antigen-conjugated nanoparticles followed by sub-cutaneous boosting immunization induces long-lasting mucosal and systemic antibody responses in mice.

Food or water-borne enteric pathogens invade their hosts via intestinal mucosal surfaces, thus developing effective oral vaccines would greatly reduce the burden of infectious diseases. The nature of the antigen, as well as the mode of its internalization in the intestinal mucosa affects the ensuing immune response. We show that model protein antigen ovalbumin (Ova) given per-orally (p.o.) induces oral tolerance (OT), characterized by systemic IgG1—dominated antibody response, which cannot be boosted by sub-cutaneous (s.c.) immunization with Ova in complete Freund’s adjuvant (CFA). Intestinal IgA generated in response to Ova feeding diminished over time and was abrogated by s.c. immunization with Ova+CFA. Humoral response to Ova was altered by administering Ova conjugated to 20 nm nanoparticles (NP-Ova). P.o. administration of NP-Ova induced systemic IgG1/IgG2c, and primed the intestinal mucosa for secretion of IgA. These responses were boosted by secondary s.c. immunization with Ova+CFA or p.o. immunization with NP-Ova. However, only in s.c.-boosted mice serum and mucosal antibody titers remained elevated for 6 months after priming. In contrast, s.c. priming with NP-Ova induced IgG1-dominated serum antibodies, but did not prime the intestinal mucosa for secretion of IgA, even after secondary p.o. immunization with NP-Ova. These results indicate that Ova conjugated to NPs reaches the internal milieu in an immunogenic form and that mucosal immunization with NP-Ova is necessary for induction of a polarized Th1/Th2 immune response, as well as intestinal IgA response. In addition, mucosal priming with NP-Ova, followed by s.c. boosting induces superior systemic and mucosal memory responses. These findings are important for the development of efficacious mucosal vaccines.

Glycosylation and disulfide bond analysis of transiently and stably expressed clade C HIV-1 gp140 trimers in 293T cells identifies disulfide heterogeneity present in both proteins and differences in O-linked glycosylation.

The HIV-1 envelope protein (Env) mediates viral entry into host cells to initiate infection and is the sole target of antibody-based vaccine development. Significant efforts have been made toward the design, engineering, and expression of various soluble forms of HIV Env immunogen, yet a highly effective immunogen remains elusive. One of the key challenges in the development of an effective HIV vaccine is the presence of the complex set of post-translational modifications (PTMs) on Env, namely, glycosylation and disulfide bonds, that affect protein folding, epitope accessibility, and immunogenecity. Although these PTMs vary with expression systems, variations in Env’s PTMs due to changes in the expression method are not yet well established. In this study, we compared the disulfide bond network and glycosylation profiles of clade C recombinant HIV-1 Env trimers, C97ZA012 gp140, expressed by stable and transient transfections using an integrated mass mapping workflow that combines collision induced dissociation (CID) and electron transfer dissociation (ETD). Site-specific analysis of the N- and O-glycosylation profiles revealed that C97ZA012 gp140 produced by both transfection methods displayed a high degree of similarity in N-glycosylation profiles and site occupancy except for one site. By contrast, different O-glycosylation profiles were detected. Analysis of the disulfide bond networks of the Env revealed that both transfection methods yielded C97ZA012 gp140 adopting the expected disulfide bond pattern identified for the monomeric gp120 and gp41 as well as alternative disulfide bond patterns in the C1, V1/V2, and C2 regions. The finding that disulfide bonding is consistently heterogeneous in these proteins is perhaps the most significant outcome of these studies; this disulfide heterogeneity has been reported for multiple other recombinant gp140s, and it is likely present in most recombinantly expressed Env immunogens.

RIG-I-like helicases induce immunogenic cell death of pancreatic cancer cells and sensitize tumors toward killing by CD8(+) T cells.

Pancreatic cancer is characterized by a microenvironment suppressing immune responses. RIG-I-like helicases (RLH) are immunoreceptors for viral RNA that induce an antiviral response program via the production of type I interferons (IFN) and apoptosis in susceptible cells. We recently identified RLH as therapeutic targets of pancreatic cancer for counteracting immunosuppressive mechanisms and apoptosis induction. Here, we investigated immunogenic consequences of RLH-induced tumor cell death. Treatment of murine pancreatic cancer cell lines with RLH ligands induced production of type I IFN and proinflammatory cytokines. In addition, tumor cells died via intrinsic apoptosis and displayed features of immunogenic cell death, such as release of HMGB1 and translocation of calreticulin to the outer cell membrane. RLH-activated tumor cells led to activation of dendritic cells (DCs), which was mediated by tumor-derived type I IFN, whereas TLR, RAGE or inflammasome signaling was dispensable. Importantly, CD8α(+) DCs effectively engulfed apoptotic tumor material and cross-presented tumor-associated antigen to naive CD8(+) T cells. In comparison, tumor cell death mediated by oxaliplatin, staurosporine or mechanical disruption failed to induce DC activation and antigen presentation. Tumor cells treated with sublethal doses of RLH ligands upregulated Fas and MHC-I expression and were effectively sensitized towards Fas-mediated apoptosis and cytotoxic T lymphocyte (CTL)-mediated lysis. Vaccination of mice with RLH-activated tumor cells induced protective antitumor immunity in vivo. In addition, MDA5-based immunotherapy led to effective tumor control of established pancreatic tumors. In summary, RLH ligands induce a highly immunogenic form of tumor cell death linking innate and adaptive immunity.

NEXT-GENERATION RECOMBINANT IMMUNOTOXINS FOR GLIOBLASTOMAS AND MELANOMAS TREATMENT

BACKGROUND: Glioblastoma (GBM) remains uniformly lethal despite the progress of conventional therapies, which lack specificity and thus result in high toxicity to normal cells. Immunotargeting therapy such as recombinant immunotoxin (RIT) is an alternative and a Phase I clinical trial of MR1-1-PE38KDEL is undergoing at Duke University Medical Center. To avoid the production of neutralizing antibodies against toxin portion and to allow for repeat treatment cycles, a less immunogenic form of PE, LR-LO10, has been generated by silencing the human B cell epitopes on PE38 and removing the immunodominant T cell epitopes. We have previously developed an affinity-matured glycoprotein NMB (GPNMB)-specific RIT, F6V-PE38, which exhibits significant in vitro and anti-tumor activity against GBM and melanomas. Here we report a newly engineered F6V-LR-LO10 RIT for efficient, targeted therapy of GBM and other GPNMB-expressing cancer patients with normal immunity. METHODS: We have constructed an RIT, F6V-LR-LO10, by fusing a fully human F6V scFv, as VH-(G4S)3-VL, with a truncated form of Pseudomonas exotoxin A, LR-LO10. F6V-LR-LO10 was expressed in E. coli BL21 (DE3), and the inclusion bodies were reduced, refolded, and purified by ion exchange chromatography and gel filtration. The antigen binding of F6V-LR-LO10 was assessed by surface plasmon resonance and flow cytometric analysis. The cytotoxicity was measured by protein inhibition assay on GPNMB-expressing GBM and melanoma cells. The in vivo animal toxicity was tested in nude mice and anti-tumor activity will be assessed in human xenograft mouse models. RESULTS: The F6V-LR-LO10 protein was purified as a monomer of 51 kDa to 95% homogeneity. The F6V-LR-LO10 bound to GPNMB-coated chips with high affinity at KD = 1.1e-8 M. The F6V-LR-LO10 also bound to native GPNMB protein via flow cytometric analysis on GPNMB positive cell lines. We tested the in vitro activity of F6V-LR-LO10 on various glioma and melanoma xenograft cells and demonstrated cell-killing activity. The IC50 of F6V-LR-LO10 on GBM D392 cells was 1.5 ng/mL; the IC50 was 10 ng/mL and 15 ng/mL on melanoma DM443 and DM440 cells, respectively, and that was comparable to the IC50 of F6V-PE38, while negative control RIT did not kill those cells. The animal toxicity (MTD) was greatly reduced by >17 fold for F6V-LR-LO10 than F6V-PE38. CONCLUSIONS: Our experiments showed that a new fully human anti-GPNMB RIT, F6V-LR-LO10, is highly active with higher therapeutic index and is predicted to have low immunogenicity in humans. This next-generation RIT is suitable for further clinical development in therapy of GBM, melanoma, and other GPNMB-expressing malignancies. SECONDARY CATEGORY: Preclinical Experimental Therapeutics.

Mo1949 Rapamycin Protects Intestinal Ischemia/Reperfusion-Induced Remote Lung Injury in Mice

Background: Enterotoxigenic Escherichia coli (ETEC), defined by their production of heat labile (LT) and/or heat stable (ST) toxins, are major causes of infantile diarrhea in developing countries and of travelers' diarrhea in visitors to the developing world. Despite our understanding of ETEC pathogenesis, no broadly protective ETEC vaccine is available. This is largely a result of the difficulty in inducing immunity to the small ST molecule. Our goal is to develop a broadly protective live attenuated vaccine against ETEC by taking advantage of the recent demonstration (Zhang et al.,2013,PLoSONe 8 (10):e77386) that genetically produced fusions of mutant ST with LT components can induce effective immunity against both toxins. Methods: To achieve this aim, we engineered an attenuated strain of E. coli, which we have developed as a vaccine vector for mucosal antigen delivery, to express immunogenic fusion constructs of LT and mutant ST. Our vaccine vector strain ZCR533, is a derivative of O157:H7 enterohemorrhagic E. coli (EHEC) strain 86-24 attenuated by deleting its genes for Shiga toxin production and truncating its major adhesin, intimin, such that it can no longer adhere intimately, but can still induce anti-intimin antibody. To express LT-ST fusions in immunogenic form in our vector strain, we used restriction-free cloning technology to incorporate them into the passenger domain of an autotransporter protein (EspP) expressed on a medium copy number plasmid. This system is designed to express the toxin antigens in either surface-bound or secreted forms, and to provide effective delivery to the mucosal immune system as we have done for other toxin antigens. Results: The incorporation of the LT-ST fusions into the passenger domain of the EspP autotransporter plasmids was confirmed by PCR and DNA sequencing. Protein expression by ZCR533 containing the autotransporter plasmids was confirmed by SDS-PAGE, and Western blot of whole cell lysates and culture supernates, using poly-clonal antisera to the LT component. Moreover, appropriate expression of the LT-ST fusions on the bacterial surface of ZCR533 was confirmed by immuno-fluorescent staining with the specific antisera. Conclusions: These studies show that immunogenic fusions of LT and mutant ST can be expressed on the surface, and in the culture supernates, of our attenuated E. coli vaccine vector ZCR533 via the EspP autotransporter. Since our recent studies using the similarly-sized Shiga toxin B subunit antigen have indicated the validity of this approach to mucosal immunization against toxin antigens, these studies could lead to the development of broadly protective vaccines directed against both LTand ST-producing ETEC, as well as against attaching/ effacing E. coli strains expressing intimin.

Mo1948 Toward a Broadly Protective Vaccine Against Enterotoxigenic E.coli (ETEC) Infections

Background: Enterotoxigenic Escherichia coli (ETEC), defined by their production of heat labile (LT) and/or heat stable (ST) toxins, are major causes of infantile diarrhea in developing countries and of travelers' diarrhea in visitors to the developing world. Despite our understanding of ETEC pathogenesis, no broadly protective ETEC vaccine is available. This is largely a result of the difficulty in inducing immunity to the small ST molecule. Our goal is to develop a broadly protective live attenuated vaccine against ETEC by taking advantage of the recent demonstration (Zhang et al.,2013,PLoSONe 8 (10):e77386) that genetically produced fusions of mutant ST with LT components can induce effective immunity against both toxins. Methods: To achieve this aim, we engineered an attenuated strain of E. coli, which we have developed as a vaccine vector for mucosal antigen delivery, to express immunogenic fusion constructs of LT and mutant ST. Our vaccine vector strain ZCR533, is a derivative of O157:H7 enterohemorrhagic E. coli (EHEC) strain 86-24 attenuated by deleting its genes for Shiga toxin production and truncating its major adhesin, intimin, such that it can no longer adhere intimately, but can still induce anti-intimin antibody. To express LT-ST fusions in immunogenic form in our vector strain, we used restriction-free cloning technology to incorporate them into the passenger domain of an autotransporter protein (EspP) expressed on a medium copy number plasmid. This system is designed to express the toxin antigens in either surface-bound or secreted forms, and to provide effective delivery to the mucosal immune system as we have done for other toxin antigens. Results: The incorporation of the LT-ST fusions into the passenger domain of the EspP autotransporter plasmids was confirmed by PCR and DNA sequencing. Protein expression by ZCR533 containing the autotransporter plasmids was confirmed by SDS-PAGE, and Western blot of whole cell lysates and culture supernates, using poly-clonal antisera to the LT component. Moreover, appropriate expression of the LT-ST fusions on the bacterial surface of ZCR533 was confirmed by immuno-fluorescent staining with the specific antisera. Conclusions: These studies show that immunogenic fusions of LT and mutant ST can be expressed on the surface, and in the culture supernates, of our attenuated E. coli vaccine vector ZCR533 via the EspP autotransporter. Since our recent studies using the similarly-sized Shiga toxin B subunit antigen have indicated the validity of this approach to mucosal immunization against toxin antigens, these studies could lead to the development of broadly protective vaccines directed against both LTand ST-producing ETEC, as well as against attaching/ effacing E. coli strains expressing intimin.

Kill and spread the word: stimulation of antitumor immune responses in the context of radiotherapy.

Besides the direct, targeted effects of ionizing irradiation (x-ray) on cancer cells, namely DNA damage and cell death induction, indirect, nontargeted ones exist, which are mediated in large part by the immune system. Immunogenic forms of tumor cell death induced by x-ray, including immune modulating danger signals like the heat shock protein 70, adenosine triphosphate, and high-mobility group box 1 protein are presented. Further, antitumor effects exerted by cells of the innate (natural killer cells) as well as adaptive immune system (T cells activated by dendritic cells) are outlined. Tumor cell death inhibiting molecules such as survivin are introduced as suitable target for molecularly tailored therapies in combination with x-ray. Finally, reasonable combinations of immune therapies with radiotherapy are discussed.

Novel Role of the Serine Protease Inhibitor Elafin in Gluten-Related Disorders

Elafin, an endogenous serine protease inhibitor, modulates colonic inflammation. We investigated the role of elafin in celiac disease (CD) using human small intestinal tissues and in vitro assays of gliadin deamidation. We also investigated the potential beneficial effects of elafin in a mouse model of gluten sensitivity. Epithelial elafin expression in the small intestine of patients with active CD, treated CD, and controls without CD was determined by immunofluorescence. Interaction of elafin with human tissue transglutaminase-2 (TG-2) was investigated in vitro. The 33-mer peptide, a highly immunogenic gliadin peptide, was incubated with TG-2 and elafin at different concentrations. The degree of deamidation of the 33-mer peptide was analyzed by liquid chromatography-mass spectrometry. Elafin was delivered to the intestine of gluten-sensitive mice using a recombinant Lactococcus lactis vector. Small intestinal barrier function, inflammation, proteolytic activity, and zonula occludens-1 (ZO-1) expression were assessed. Elafin expression in the small intestinal epithelium was lower in patients with active CD compared with control patients. In vitro, elafin significantly slowed the kinetics of the deamidation of the 33-mer peptide to its more immunogenic form. Treatment of gluten-sensitive mice with elafin delivered by the L. lactis vector normalized inflammation, improved permeability, and maintained ZO-1 expression. The decreased elafin expression in the small intestine of patients with active CD, the reduction of 33-mer peptide deamidation by elafin, coupled to the barrier enhancing and anti-inflammatory effects observed in gluten-sensitive mice, suggest that this molecule may have pathophysiological and therapeutic importance in gluten-related disorders.

Occurrence of transient back pain among teachers, researchers and students in Kharkiv

In modern medicine the problem of diagnosing and treatment of dorsal pain is one of the most relevant which is supported by the global statistics. Dorsal pain is included in list of diseases and pathological conditions (together with osteoporosis, osteoarthritis, rheumatoid arthritis, injuries of the musculoskeletal system) which has essential medical and social value and cause significant economic losses to society according to opinion of foreign experts. Purpose: to determine peculiarities of transitory dorsalgia manifestations in mental work individuals with theoretical analysis of possible pathophysiological mechanisms of this pain development. Methods: we c onducted a questionnaire survey of 765 persons (age 21-70 years) in 3 scientific research and 7 educational institutions in Kharkiv. Results: an analysis of the completed questionnaires showed that in the evaluated sample women were predominant (401 / 55.39 %), and the same was in age group of young people up to 30 years (261 / 36.05 %). Transient dorsal pain was noted in 483 people (66.71 ± 1.75) %. Teachers have had recurrent dorsal pain significantly more often (p = 0.0001) than research staff and students. According to gender characteristics manifestations of dorsalgia were prevalent in women (p = 0.005). Statistically, tight correlation between transient manifestations of dorsalgia was detected with sleep disturbances and psycho emotional stresses. We have shown that dorsal pain was diagnosed in 57.22 % individuals whose work according to them was calm, and in 68.42% individuals who described their work as hard but without stresses, and in 74.50 % persons whose work is accompanied by psycho emotional stresses. Dorsal pain was noted in (56.25 ± 3.44) % persons who reported daily deep and prolonged (> 8 hours) sleeping. Among those with a deep but not long (< 8 hours) sleeping pain was noted in (63.12 ± 2.78)% cases. If cases of not deep sleeping with bout of insomnia dorsal pain was noted in (79.77 ± 3.05) % of respondents. And finally, in the group of persons who have suffered from insomnia transitory dorsal pain was noted in (92.68 ± 4.07) % cases. In vast majority of people with transitory dorsal pain its manifestations did not have any seasonal dependence. The questionnaire identified the most common pain patterns: aching, burning, cutting, stitching, and patient’s own version. An analysis of the data showed that the vast majority of people (71.84 %) have had aching, 9.94 % - stabbing, 8.9 % - cutting, 6.63 % - burning, and 3.11% have had other type of pain. In fact pain spectrum was limited by these four types, and manifestations of dull pain primarily dominated. Conclusion : in-depth research of transitory dorsal pain pathogenesis and improvement of differential diagnostics of various forms of this disease is necessary. Obtained data represent an important basis for further study of transitory dorsal pain pathogenesis and for deepening of possibilities for differential diagnostics between spinal, neurogenic, immunogenic and other forms of dorsalgia.

Fractionated radiotherapy is the main stimulus for the induction of cell death and of Hsp70 release of p53 mutated glioblastoma cell lines

BackgroundGlioblastoma multiforme (GBM) is the most common primary brain tumor in adults. Despite a multimodal therapy consisting of resection followed by fractionated radiotherapy (RT) combined with the chemotherapeutic agent (CT) temozolomide (TMZ), its recurrence is almost inevitable. Since the immune system is capable of eliminating small tumor masses, a therapy should also aim to stimulate anti-tumor immune responses by induction of immunogenic cell death forms. The histone deacetylase inhibitor valproic acid (VPA) might foster this.MethodsReflecting therapy standards, we applied in our in vitro model fractionated RT with a single dose of 2Gy and clinically relevant concentrations of CT. Not only the impact of RT and/or CT with TMZ and/or VPA on the clonogenic potential and cell cycle of the glioblastoma cell lines T98G, U251MG, and U87MG was analyzed, but also the resulting cell death forms and release of danger signals such as heat-shock protein70 (Hsp70) and high-mobility group protein B1 (HMGB1).ResultsThe clonogenic assays revealed that T98G and U251MG, having mutated tumor suppressor protein p53, are more resistant to RT and CT than U87MG with wild type (WT) p53. In all glioblastoma cells lines, fractionated RT induced a G2 cell cycle arrest, but only in the case of U87MG, TMZ and/or VPA alone resulted in this cell cycle block. Further, fractionated RT significantly increased the number of apoptotic and necrotic tumor cells in all three cell lines. However, only in U87MG, the treatment with TMZ and/or VPA alone, or in combination with fractionated RT, induced significantly more cell death compared to untreated or irradiated controls. While necrotic glioblastoma cells were present after VPA, TMZ especially led to significantly increased amounts of U87MG cells in the radiosensitive G2 cell cycle phase. While CT did not impact on the release of Hsp70, fractionated RT resulted in significantly increased extracellular concentrations of Hsp70 in p53 mutated and WT glioblastoma cells.ConclusionsOur results indicate that fractionated RT is the main stimulus for induction of glioblastoma cell death forms with immunogenic potential. The generated tumor cell microenvironment might be beneficial to include immune therapies for GBM in the future.

First Evidence for a Covalent Linkage between Enterobacterial Common Antigen and Lipopolysaccharide in Shigella sonnei Phase II ECALPS

Enterobacterial common antigen (ECA) is expressed by Gram-negative bacteria belonging to Enterobacteriaceae, including emerging drug-resistant pathogens such as Escherichia coli, Klebsiella pneumoniae, and Proteus spp. Recent studies have indicated the importance of ECA for cell envelope integrity, flagellum expression, and resistance of enteric bacteria to acetic acid and bile salts. ECA, a heteropolysaccharide built from the trisaccharide repeating unit, →3)-α-D-Fucp4NAc-(1→4)-β-D-ManpNAcA-(1→4)-α-D-GlcpNAc-(1→, occurs as a cyclic form (ECA(CYC)), a phosphatidylglycerol (PG)-linked form (ECA(PG)), and an endotoxin/lipopolysaccharide (LPS)-associated form (ECA(LPS)). Since the discovery of ECA in 1962, the structures of ECA(PG) and ECA(CYC) have been completely elucidated. However, no direct evidence has been presented to support a covalent linkage between ECA and LPS; only serological indications of co-association have been reported. This is paradoxical, given that ECA was first identified based on the capacity of immunogenic ECA(LPS) to elicit antibodies cross-reactive with enterobacteria. Using a simple isolation protocol supported by serological tracking of ECA epitopes and NMR spectroscopy and mass spectrometry, we have succeeded in the first detection, isolation, and complete structural analysis of poly- and oligosaccharides of Shigella sonnei phase II ECA(LPS). ECA(LPS) consists of the core oligosaccharide substituted with one to four repeating units of ECA at the position occupied by the O-antigen in the case of smooth S. sonnei phase I. These data represent the first structural evidence for the existence of ECA(LPS) in the half-century since it was first discovered and provide insights that could prove helpful in further structural analyses and screening of ECA(LPS) among Enterobacteriaceae species.