Accounting Problems Under the Excess Profits Tax

DNA vaccines based on subunits from pathogens have several advantages over other vaccine strategies. DNA vaccines can easily be modified, they show good safety profiles, are stable and inexpensive to produce, and the immune response can be focused to the antigen of interest. However, the immunogenicity of DNA vaccines which is generally quite low needs to be improved. Electroporation and co-delivery of genetically encoded immune adjuvants are two strategies aiming at increasing the efficacy of DNA vaccines. Here, we have examined whether targeting to antigen-presenting cells (APC) could increase the immune response to surface envelope glycoprotein (Env) gp120 from Human Immunodeficiency Virus type 1 (HIV- 1). To target APC, we utilized a homodimeric vaccine format denoted vaccibody, which enables covalent fusion of gp120 to molecules that can target APC. Two molecules were tested for their efficiency as targeting units: the antibody-derived single chain Fragment variable (scFv) specific for the major histocompatilibility complex (MHC) class II I-E molecules, and the CC chemokine ligand 3 (CCL3). The vaccines were delivered as DNA into muscle of mice with or without electroporation. Targeting of gp120 to MHC class II molecules induced antibodies that neutralized HIV-1 and that persisted for more than a year after one single immunization with electroporation. Targeting by CCL3 significantly increased the number of HIV-1 gp120-reactive CD8(+) T cells compared to non-targeted vaccines and gp120 delivered alone in the absence of electroporation. The data suggest that chemokines are promising molecular adjuvants because small amounts can attract immune cells and promote immune responses without advanced equipment such as electroporation.Funding Agencies|Research Council of Norway; Odd Fellow</p

Reducing AD-Like Pathology in 3xTg-AD Mouse Model by DNA Epitope Vaccine — A Novel Immunotherapeutic Strategy

BACKGROUND: The development of a safe and effective AD vaccine requires a delicate balance between providing an adequate anti-Abeta antibody response sufficient to provide therapeutic benefit, while eliminating an adverse T cell-mediated proinflammatory autoimmune response. To achieve this goal we have designed a prototype chemokine-based DNA epitope vaccine expressing a fusion protein that consists of 3 copies of the self-B cell epitope of Abeta(42) (Abeta(1-11)) , a non-self T helper cell epitope (PADRE), and macrophage-derived chemokine (MDC/CCL22) as a molecular adjuvant to promote a strong anti-inflammatory Th2 phenotype. METHODS AND FINDINGS: We generated pMDC-3Abeta(1-11)-PADRE construct and immunized 3xTg-AD mouse model starting at age of 3-4 months old. We demonstrated that prophylactic immunizations with the DNA epitope vaccine generated a robust Th2 immune response that induced high titers of anti-Abeta antibody, which in turn inhibited accumulation of Abeta pathology in the brains of older mice. Importantly, vaccination reduced glial activation and prevented the development of behavioral deficits in aged animals without increasing the incidence of microhemorrhages. CONCLUSIONS: Data from this transitional pre-clinical study suggest that our DNA epitope vaccine could be used as a safe and effective strategy for AD therapy. Future safety and immunology studies in large animals with the goal to achieve effective humoral immunity without adverse effects should help to translate this study to human clinical trials

Beta defensin-2 is reduced in central but not in distal airways of smoker COPD patients

Background: Altered pulmonary defenses in chronic obstructive pulmonary disease (COPD) may promote distal airways bacterial colonization. The expression/activation of Toll Like receptors (TLR) and beta 2 defensin (HBD2) release by epithelial cells crucially affect pulmonary defence mechanisms. Methods: The epithelial expression of TLR4 and of HBD2 was assessed in surgical specimens from current smokers COPD (s-COPD; n = 17), ex-smokers COPD (ex-s-COPD; n = 8), smokers without COPD (S; n = 12), and from non-smoker non-COPD subjects (C; n = 13). Results: In distal airways, s-COPD highly expressed TLR4 and HBD2. In central airways, S and s-COPD showed increased TLR4 expression. Lower HBD2 expression was observed in central airways of s-COPD when compared to S and to ex-s-COPD. s-COPD had a reduced HBD2 gene expression as demonstrated by real-time PCR on micro-dissected bronchial epithelial cells. Furthermore, HBD2 expression positively correlated with FEV1/FVC ratio and inversely correlated with the cigarette smoke exposure. In a bronchial epithelial cell line (16 HBE) IL-1β significantly induced the HBD2 mRNA expression and cigarette smoke extracts significantly counteracted this IL-1 mediated effect reducing both the activation of NFkB pathway and the interaction between NFkB and HBD2 promoter. Conclusions: This study provides new insights on the possible mechanisms involved in the alteration of innate immunity mechanisms in COPD. © 2012 Pace et al

β-defensin 2 as an Adjuvant Promotes Anti-Melanoma Immune Responses and Inhibits the Growth of Implanted Murine Melanoma In Vivo

β-defensin 2 is a small antimicrobial peptide of the innate immune system and has been thought to regulate anti-tumor immunity. However, little is known on whether β-defensin 2 could modulate melanoma-specific NK and T cell responses. In this study, we first cloned the murine β-defensin 2 gene by RT-PCR and generated the β-defensin 2 stably expressing B16 cells (B16-mBD2). Subsequently, we evaluated whether vaccination with irradiated B16-mBD2 could modulate the growth of implanted B16 cells and determined the potential mechanisms underlying the action of B16-mBD2 vaccine in modulating the growth of B16 tumors in C57BL/6. We found that vaccination with irradiated B16-mBD2, but not with control B16-p or parental B16, inhibited the development and progression of B16 tumors, and prolonged the survival of tumor-bearing mice. However, vaccination with irradiated B16-mBD2 failed to inhibit the development of B16 tumors in the CD4+- or CD8+-depleted recipients. Furthermore, vaccination with irradiated B16-mBD2 stimulated strong NK activity and promoted potent B16-specific CTL responses, accompanied by augmenting IFN-γ and IL-12, but not IL-4, responses in the recipient mice. Moreover, vaccination with irradiated B16-mBD2 promoted the infiltration of CD8+ and CD4+ T, NK cells and macrophages in the tumor tissues. These data suggest β-defensin 2 may act as a positive regulator, promoting anti-tumor NK and T cell responses in vivo. Therefore, β-defensin 2 may be used for the development of immunotherapy for the intervention of melanoma

β-Defensin-2 Protein Is a Serum Biomarker for Disease Activity in Psoriasis and Reaches Biologically Relevant Concentrations in Lesional Skin

BACKGROUND: Previous studies have extensively documented antimicrobial and chemotactic activities of beta-defensins. Human beta-defensin-2 (hBD-2) is strongly expressed in lesional psoriatic epidermis, and recently we have shown that high beta-defensin genomic copy number is associated with psoriasis susceptibility. It is not known, however, if biologically and pathophysiologically relevant concentrations of hBD-2 protein are present in vivo, which could support an antimicrobial and proinflammatory role of beta-defensins in lesional psoriatic epidermis. METHODOLOGY/PRINCIPAL FINDINGS: We found that systemic levels of hBD-2 showed a weak but significant correlation with beta defensin copy number in healthy controls but not in psoriasis patients with active disease. In psoriasis patients but not in atopic dermatitis patients, we found high systemic hBD-2 levels that strongly correlated with disease activity as assessed by the PASI score. Our findings suggest that systemic levels in psoriasis are largely determined by secretion from involved skin and not by genomic copy number. Modelling of the in vivo epidermal hBD-2 concentration based on the secretion rate in a reconstructed skin model for psoriatic epidermis provides evidence that epidermal hBD-2 levels in vivo are probably well above the concentrations required for in vitro antimicrobial and chemokine-like effects. CONCLUSIONS/SIGNIFICANCE: Serum hBD-2 appears to be a useful surrogate marker for disease activity in psoriasis. The discrepancy between hBD-2 levels in psoriasis and atopic dermatitis could explain the well known differences in infection rate between these two diseases

Sphingosine Kinase-1 Is Required for Toll Mediated β-Defensin 2 Induction in Human Oral Keratinocytes

Host defense against invading pathogens is triggered by various receptors including toll-like receptors (TLRs). Activation of TLRs is a pivotal step in the initiation of innate, inflammatory, and antimicrobial defense mechanisms. Human beta-defensin 2 (HBD-2) is a cationic antimicrobial peptide secreted upon gram-negative bacterial perturbation in many cells. Stimulation of various TLRs has been shown to induce HBD-2 in oral keratinocytes, yet the underlying cellular mechanisms of this induction are poorly understood.Here we demonstrate that HBD-2 induction is mediated by the Sphingosine kinase-1 (Sphk-1) and augmented by the inhibition of Glycogen Synthase Kinase-3beta (GSK-3beta) via the Phosphoinositide 3-kinase (PI3K) dependent pathway. HBD-2 secretion was dose dependently inhibited by a pharmacological inhibitor of Sphk-1. Interestingly, inhibition of GSK-3beta by SB 216763 or by RNA interference, augmented HBD-2 induction. Overexpression of Sphk-1 with concomitant inhibition of GSK-3beta enhanced the induction of beta-defensin-2 in oral keratinocytes. Ectopic expression of constitutively active GSK-3beta (S9A) abrogated HBD-2 whereas kinase inactive GSK-3beta (R85A) induced higher amounts of HBD-2.These data implicate Sphk-1 in HBD-2 regulation in oral keratinocytes which also involves the activation of PI3K, AKT, GSK-3beta and ERK 1/2. Thus we reveal the intricate relationship and pathways of toll-signaling molecules regulating HBD-2 which may have therapeutic potential

Uterine Epithelial Cell Regulation of DC-SIGN Expression Inhibits Transmitted/Founder HIV-1 Trans Infection by Immature Dendritic Cells

Sexual transmission accounts for the majority of HIV-1 infections. In over 75% of cases, infection is initiated by a single variant (transmitted/founder virus). However, the determinants of virus selection during transmission are unknown. Host cell-cell interactions in the mucosa may be critical in regulating susceptibility to infection. We hypothesized in this study that specific immune modulators secreted by uterine epithelial cells modulate susceptibility of dendritic cells (DC) to infection with HIV-1.Here we report that uterine epithelial cell secretions (i.e. conditioned medium, CM) decreased DC-SIGN expression on immature dendritic cells via a transforming growth factor beta (TGF-β) mechanism. Further, CM inhibited dendritic cell-mediated trans infection of HIV-1 expressing envelope proteins of prototypic reference. Similarly, CM inhibited trans infection of HIV-1 constructs expressing envelopes of transmitted/founder viruses, variants that are selected during sexual transmission. In contrast, whereas recombinant TGF- β1 inhibited trans infection of prototypic reference HIV-1 by dendritic cells, TGF-β1 had a minimal effect on trans infection of transmitted/founder variants irrespective of the reporter system used to measure trans infection.Our results provide the first direct evidence for uterine epithelial cell regulation of dendritic cell transmission of infection with reference and transmitted/founder HIV-1 variants. These findings have immediate implications for designing strategies to prevent sexual transmission of HIV-1

Induction of beta defensin 2 by NTHi requires TLR2 mediated MyD88 and IRAK-TRAF6-p38MAPK signaling pathway in human middle ear epithelial cells

<p>Abstract</p> <p>Background</p> <p>All mucosal epithelia, including those of the tubotympanium, are secreting a variety of antimicrobial innate immune molecules (AIIMs). In our previous study, we showed the bactericidal/bacteriostatic functions of AIIMs against various otitis media pathogens. Among the AIIMs, human β-defensin 2 is the most potent molecule and is inducible by exposure to inflammatory stimuli such as bacterial components or proinflammatory cytokines. Even though the β-defensin 2 is an important AIIM, the induction mechanism of this molecule has not been clearly established. We believe that this report is the first attempt to elucidate NTHi induced β-defensin expression in airway mucosa, which includes the middle ear.</p> <p>Methods</p> <p>Monoclonal antibody blocking method was employed in monitoring the TLR-dependent NTHi response. Two gene knock down methods – dominant negative (DN) plasmid and small interfering RNA (siRNA) – were employed to detect and confirm the involvement of several key genes in the signaling cascade resulting from the NTHi stimulated β-defensin 2 expression in human middle ear epithelial cell (HMEEC-1). The student's <it>t</it>-test was used for the statistical analysis of the data.</p> <p>Results</p> <p>The experimental results showed that the major NTHi-specific receptor in HMEEC-1 is the Toll-like receptor 2 (TLR2). Furthermore, recognition of NTHi component(s)/ligand(s) by TLR2, activated the Toll/IL-1 receptor (TIR)-MyD88-IRAK1-TRAF6-MKK3/6-p38 MAPK signal transduction pathway, ultimately leading to the induction of β-defensin 2.</p> <p>Conclusion</p> <p>This study found that the induction of β-defensin 2 is highest in whole cell lysate (WCL) preparations of NTHi, suggesting that the ligand(s) responsible for this up-regulation may be soluble macromolecule(s). We also found that this induction takes place through the TLR2 dependent MyD88-IRAK1-TRAF6-p38 MAPK pathway, with the primary response occurring within the first hour of stimulation. In combination with our previous studies showing that IL-1α-induced β-defensin 2 expression takes place through a MyD88-independent Raf-MEK1/2-ERK MAPK pathway, we found that both signaling cascades act synergistically to up-regulate β-defensin 2 levels. We propose that this confers an essential evolutionary advantage to the cells in coping with infections and may serve to amplify the innate immune response through paracrine signaling.</p