Pattern-Recognition Receptors in Human Disease Synthetic agonists of Toll-like receptors 7, 8 and 9

Abstract TLRs (Toll-like receptors) are a family of innate immune receptors that induce protective immune responses against infections. Single-stranded viral RNA and bacterial DNA containing unmethylated CpG motifs are the ligands for TLR7 and TLR8 and 9 respectively. We have carried out extensive structure-activity relationship studies of DNA-and RNA-based compounds to elucidate the impact of nucleotide motifs and structures on these TLR-mediated immune responses. These studies have led us to design novel DNA-and RNA-based compounds, which act as potent agonists of TLR9 and TLR7 and 8 respectively. These novel synthetic agonists produce different immune response profiles depending on the structures and nucleotide motifs present in them. The ability to modulate TLR-mediated immune responses with these novel DNA-and RNA-based agonists in a desired fashion may allow targeting a broad range of diseases, including cancers, asthma, allergies and infections, alone or in combination with other therapeutic agents, and their use as adjuvants with vaccines. IMO-2055, our first lead candidate, is a TLR9 agonist that is currently in clinical evaluation in oncology patients. A second candidate, IMO-2125, is also a TLR9 agonist that has been shown to induce high and sustained levels of IFN (interferon) in non-human primates and is being evaluated in HepC-infected human subjects

Novel oligodeoxynucleotide agonists of TLR9 containing N3-Me-dC or N1-Me-dG modifications

Synthetic oligodeoxynucleotides containing unmethylated CpG motifs activate Toll-Like Receptor 9 (TLR9). Our previous studies have shown the role of hydrogen-bond donor and acceptor groups of cytosine and guanine in the CpG motif and identified synthetic immunostimulatory motifs. In the present study to elucidate the significance of N3-position of cytosine and N1-position of guanine in the CpG motif, we substituted C or G of a CpG dinucleotide with N3-Me-cytosine or N1-Me-guanine, respectively, in immunomodulatory oligodeoxynucleotides (IMOs). IMOs containing N-Me-cytosine or N-Me-guanine in C- or G-position, respectively, of the CpG dinucleotide showed activation of HEK293 cells expressing TLR9, but not TLR3, 7 or 8. IMOs containing N-Me-cytosine or N-Me-guanine modification showed activity in mouse spleen cell cultures, in vivo in mice, and in human cell cultures. In addition, IMOs containing N-Me-substitutions reversed antigen-induced Th2 immune responses towards a Th1-type in OVA-sensitized mouse spleen cell cultures. These studies suggest that TLR9 tolerates a methyl group at N1-position of G and a methyl group at N3-position of C may interfere with TLR9 activation to some extent. These are the first studies elucidating the role of N3-position of cytosine and N1-position of guanine in a CpG motif for TLR9 activation and immune stimulation

A Toll-Like Receptor 7, 8, and 9 Antagonist Inhibits Th1 and Th17 Responses and Inflammasome Activation in a Model of IL-23-Induced Psoriasis

Psoriasis is a chronic inflammatory skin disease that involves the induction of T-helper 1 (Th1) and T-helper 17 (Th17) cell responses and the aberrant expression of proinflammatory cytokines, including IL-1β. Copious evidence suggests that abnormal activation of Toll-like receptors (TLRs) contributes to the initiation and maintenance of psoriasis. We have evaluated an antagonist of TLR7, 8, and 9 as a therapeutic agent in an IL-23-induced psoriasis model in mice. Psoriasis-like skin lesions were induced in C57BL/6 mice by intradermal injection of IL-23 in the ear or dorsum. IL-23-induced increase in ear thickness was inhibited in a dose-dependent manner by treatment with antagonist. Histological examination of ear and dorsal skin tissues demonstrated a reduction in epidermal hyperplasia in mice treated with the antagonist. Treatment with antagonist also reduced the induction of Th1 and Th17 cytokines in skin and/or serum, as well as dermal expression of inflammasome components, NLRP3 and AIM2, and antimicrobial peptides. These results indicate that targeting TLR7, 8, and 9 may provide a way to neutralize multiple inflammatory pathways that are involved in the development of psoriasis. The antagonist has the potential for the treatment of psoriasis and other autoimmune diseases

TLR9 Agonist Protects Mice from Radiation-Induced Gastrointestinal Syndrome

Radiation-induced gastrointestinal syndrome (RIGS) is due to the clonogenic loss of crypt cells and villi depopulation, resulting in disruption of mucosal barrier, bacterial invasion, inflammation and sepsis. Intestinal macrophages could recognize invading bacterial DNA via TLR9 receptors and transmit regenerative signals to the neighboring crypt. We therefore investigated whether systemic administration of designer TLR9 agonist could ameliorate RIGS by activating TLR9.Male C57Bl6 mice were distributed in four experimental cohorts, whole body irradiation (WBI) (8.4-10.4 Gy), TLR9 agonist (1 mg/kg s.c.), 1 h pre- or post-WBI and TLR9 agonist+WBI+iMyd88 (pretreatment with inhibitory peptide against Myd88). Animals were observed for survival and intestine was harvested for histological analysis. BALB/c mice with CT26 colon tumors in abdominal wall were irradiated with 14 Gy single dose of whole abdominal irradiation (AIR) for tumor growth study.Mice receiving pre-WBI TLR9 agonist demonstrated improvement of survival after 10.4 Gy (p<0.03), 9.4 Gy (p<0.008) and 8.4 Gy (p<0.002) of WBI, compared to untreated or iMyd88-treated controls. Post-WBI TLR9 agonist mitigates up to 8.4 Gy WBI (p<0.01). Histological analysis and xylose absorption test demonstrated significant structural and functional restitution of the intestine in WBI+TLR9 agonist cohorts. Although, AIR reduced tumor growth, all animals died within 12 days from RIGS. TLR9 agonist improved the survival of mice beyond 28 days post-AIR (p<0.008) with significant reduction of tumor growth (p<0.0001).TLR9 agonist treatment could serve both as a prophylactic or mitigating agent against acute radiation syndrome and also as an adjuvant therapy to increase the therapeutic ratio of abdominal Radiation Therapy for Gastro Intestinal malignancies

An In Situ Autologous Tumor Vaccination with Combined Radiation Therapy and TLR9 Agonist Therapy

PURPOSE:Recent studies have shown that a new generation of synthetic agonist of Toll-like receptor (TLR) 9 consisting a 3'-3'-attached structure and a dCp7-deaza-dG dinucultodie shows more potent immunostimulatory effects in both mouse and human than conventional CpG oligonucleotides. Radiation therapy (RT) provides a source of tumor antigens that are released from dying, irradiated, tumor cells without causing systemic immunosuppression. We, therefore, examined effect of combining RT with a designer synthetic agonist of TLR9 on anti-tumoral immunity, primary tumor growth retardation and metastases in a murine model of lung cancer. METHODS:Grouped C57BL/6 and congenic B cell deficient mice (B(-/-)) bearing footpad 3LL tumors were treated with PBS, TLR9 agonist, control oligonucelotide, RT or the combination of RT and TLR9 agonist. Immune phenotype of splenocytes and serum IFN-γ and IL-10 levels were analyzed by FACS and ELISA, 24 h after treatment. Tumor growth, lung metastases and survival rate were monitored and tumor specific antibodies in serum and deposition in tumor tissue were measured by ELISA and immunofluorescence. RESULTS:TLR9 agonist expanded and activated B cells and plasmacytoid dendritic cells in wild-type mice and natural killer DCs (NKDCs) in B cell-deficient (B(-/-)) mice bearing ectopic Lewis lung adenocarcinoma (3LL). Combined RT with TLR9 agonist treatment inhibited 3LL tumor growth in both wild type and B(-/-) mice. A strong tumor-specific humoral immune response (titer: 1/3200) with deposition of mouse IgG auto-antibodies in tumor tissue were found in wildtype mice, whereas the number of tumor infiltrating NKDCs increased in B(-/-) mice following RT+ TLR9 agonist therapy. Furthermore, mice receiving combination therapy had fewer lung metastases and a higher survival than single treatment cohorts. CONCLUSIONS:Combination therapy with TLR9 agonist and RT induces systemic anti-tumoral humoral response, augments tumoral infiltration of NKDCs, reduces pulmonary metastases and improves survival in a murine model of 3LL cancer

The Use of Molecular Analyses in Voided Urine for the Assessment of Patients with Hematuria

Introduction:Patients presenting with painless hematuria form a large part of the urological patient population. In many cases, especially in younger patients, the cause of hematuria is harmless. Nonetheless, hematuria could be a symptom of malignant disease and hence most patients will be subject to cystoscopy. In this study, we aimed to develop a prediction model based on methylation markers in combination with clinical variables, in order to stratify patients with high risk for bladder cancer.Material and Methods:Patients (n=169) presenting with painless hematuria were included. 54 patients were diagnosed with bladder cancer. In the remaining 115 patients, the cause of hematuria was non-malignant. Urine samples were collected prior to cystoscopy. Urine DNA was analyzed for methylation of OSR1, SIM2, OTX1, MEIS1 and ONECUT2. Methylation percentages were calculated and were combined with clinical variables into a logistic regression model.Results:Logistic regression analysis based on the five methylation markers, age, gender and type of hematuria resulted in an area under the curve (AUC) of 0.88 and an optimism corrected AUC of 0.84 after internal validation by bootstrapping. Using a cut-off value of 0.307 allowed stratification of patients in a low-risk and high-risk group, resulting in a sensitivity of 82% (44/54) and a specificity of 82% (94/115). Most aggressive tumors were found in patients in the high-risk group. The addition of cytology to the prediction model, improved the AUC from 0.88 to 0.89, with a sensitivity and specificity of 85% (39/46) and 87% (80/92), retrospectively.Conclusions:This newly developed prediction model could be a helpful tool in risk stratification of patients presenting with painless hematuria. Accurate risk prediction might result in less extensive examination of low risk patients and thereby, reducing patient burden and costs. Further validation in a large prospective patient cohort is necessary to prove the true clinical value of this model

Modeling Personalized Adjuvant TreaTment in EaRly stage coloN cancer (PATTERN)

Aim: To develop a decision model for the population-level evaluation of strategies to improve the selection of stage II colon cancer (CC) patients who benefit from adjuvant chemotherapy. Methods: A Markov cohort model with a one-month cycle length and a lifelong time horizon was developed. Five health states were included; diagnosis, 90-day mortality, death other causes, recurrence and CC death. Data from the Netherlands Cancer Registry were used to parameterize the model. Transition probabilities were estimated using parametric survival models including relevant clinical and pathological covariates. Subsequently, biomarker status was implemented using external data. Treatment effect was incorporated using pooled trial data. Model development, data sources used, parameter estimation, and internal and external validation are described in detail. To illustrate the use of the model, three example strategies were evaluated in which allocation of treatment was based on (A) 100% adherence to the Dutch guidelines, (B) observed adherence to guideline recommendations and (C) a biomarker-driven strategy. Results: Overall, the model showed good internal and external validity. Age, tumor growth, tumor sidedness, evaluated lymph nodes, and biomarker status were included as covariates. For the example strategies, the model predicted 83, 87 and 77 CC deaths after 5 years in a cohort of 1000 patients for strategies A, B and C, respectively. Conclusion: This model can be used to evaluate strategies for the allocation of adjuvant chemotherapy in stage II CC patients. In future studies, the model will be used to estimate population-level long-term health gain and cost-effectiveness of biomarker-based selection strategies

Mechanism of Neuronal versus Endothelial Cell Uptake of Alzheimer's Disease Amyloid β Protein

Alzheimer's disease (AD) is characterized by significant neurodegeneration in the cortex and hippocampus; intraneuronal tangles of hyperphosphorylated tau protein; and accumulation of β-amyloid (Aβ) proteins 40 and 42 in the brain parenchyma as well as in the cerebral vasculature. The current understanding that AD is initiated by the neuronal accumulation of Aβ proteins due to their inefficient clearance at the blood-brain-barrier (BBB), places the neurovascular unit at the epicenter of AD pathophysiology. The objective of this study is to investigate cellular mechanisms mediating the internalization of Aβ proteins in the principle constituents of the neurovascular unit, neurons and BBB endothelial cells. Laser confocal micrographs of wild type (WT) mouse brain slices treated with fluorescein labeled Aβ40 (F-Aβ40) demonstrated selective accumulation of the protein in a subpopulation of cortical and hippocampal neurons via nonsaturable, energy independent, and nonendocytotic pathways. This groundbreaking finding, which challenges the conventional belief that Aβ proteins are internalized by neurons via receptor mediated endocytosis, was verified in differentiated PC12 cells and rat primary hippocampal (RPH) neurons through laser confocal microscopy and flow cytometry studies. Microscopy studies have demonstrated that a significant proportion of F-Aβ40 or F-Aβ42 internalized by differentiated PC12 cells or RPH neurons is located outside of the endosomal or lysosomal compartments, which may accumulate without degradation. In contrast, BBME cells exhibit energy dependent uptake of F-Aβ40, and accumulate the protein in acidic cell organelle, indicative of endocytotic uptake. Such a phenomenal difference in the internalization of Aβ40 between neurons and BBB endothelial cells may provide essential clues to understanding how various cells can differentially regulate Aβ proteins and help explain the vulnerability of cortical and hippocampal neurons to Aβ toxicity