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According to the hygiene hypothesis, the exposure to infectious agents in early childhood prevents the development of allergen-specific Th2 immune responses because it establishes Th1-based immunity or alternatively, induces the generation of T regulatory cells. Based on this theory, the present study pretended to identify promising microorganism-derived vaccine candidates against allergic asthma in the murine model. In the first part of this work, the efficacy of four different known Th1-inducing adjuvants, i.e. live BCG, heat-killed BCG, CpG and PPD, as components of vaccines aimed at inhibiting allergic asthma was compared. All the adjuvants were effective in inhibiting the development of allergen-induced airway eosinophilia, mucus production, and with the exception of PPD also airway hyperreactivity (AHR), when they were applied together with OVA/alum. Suppression of airway eosinophilia was not observed in IFN-gamma- or IL-12-deficient mice (hk-BCG, CpG-ODN and PPD). Interestingly, live BCG was still able to suppress allergen-induced Th2 responses in the absence of either IFN-gamma or IL-12. The effect of live BCG was also independent on IL-10-, TLR-2-, TLR-4- or MyD88-mediated signaling. When mice vaccinated with the different adjuvants together with OVA/alum were subjected to a second period of OVA/alum immunization, only live and hk-BCG were able to efficiently suppress the development of airway inflammation. This effect could be adoptively transferred by CD4+ T cells. Taken together our data suggest that live BCG>>hk-BCG>CpG>PPD are effective in suppressing allergen-induced Th2 responses. Secondly, the evaluation of a dendritic cell-based vaccination strategy leading to the induction of allergen-specific Th1 cells to protect against the development of allergen-specific Th2 responses was performed. The application of OVA-pulsed BM-DC maturated with CpG was unable to reduce airway eosinophilia and inflammation in OVA-immunized mice. OVA-specific IgG1 or IgE serum levels were also not reduced. The experiments using LC pulsed with OVA yielded similar results. However, the mice vaccinated with CpG/OVA pulsed BM-DC had greatly enhanced levels of OVA-specific IgG2a in the serum, suggesting the induction of allergen-specific Th1 responses in vivo. Thus, these data suggest that the vaccination of mice with OVA-pulsed BM-DC matured with CpG or OVA-pulsed LC did not result in a reduction of allergen-specific Th2 responses in a murine model of severe atopic asthma. Lastly, NES, an excretory/secretory product derived from the helminth Nippostrongylus brasiliensis was evaluated as a new potential adjuvant to prevent the development of allergic responses. The application of NES together with OVA/alum greatly inhibited the development of airway eosinophilia, airway goblet cell metaplasia and mucus production and the development of airway hyperreactivity after metacholine challenge. Furthermore, OVA-specific IgG1 and IgE levels in the serum were also strongly reduced. NES preparations contained small amounts of endotoxin, which may explain these results. However, the suppressive effects of NES on the development of allergen-specific Th2 responses was independent upon IFN-gamma or TLR-4 and still observed in mice treated with LPS-depleted NES. NES reduced OVA-induced Th2 responses also in a IL-10-independent manner. In addition, the digestion with proteinase K or the heat-treatment of NES did not abolish its ability to inhibit allergen-induced Th2 responses. Interestingly, NES suppress OVA-specific Th2 responses in vivo in the presence of a strong NES-specific Th2 environment. Taken together our results suggest that the helminth N. brasiliensis secretes substances which interfere with the development of allergic Th2 responses. In summary, distinct substances derived from microorganisms or helminths which may be used as potential adjuvants to prevent the development of allergic Th2 responses were identified. These findings contribute to the design of efficient vaccines protecting humans from developing allergic asthma.
Interleukin-4 (IL-4) is an anti-inflammatory and analgesic cytokine that induces opioid receptor transcription. We investigated IL-4 knockout (ko) mice to characterize their pain behavior before and after chronic constriction injury (CCI) of the sciatic nerve as a model for neuropathic pain. We investigated opioid responsivity and measured cytokine and opioid receptor gene expression in the peripheral and central nervous system (PNS, CNS) of IL-4 ko mice in comparison with wildtype (wt) mice. Naïve IL-4 ko mice displayed tactile allodynia (wt: 0.45 g; ko: 0.18 g; p<0.001), while responses to heat and cold stimuli and to muscle pressure were not different. No compensatory changes in the gene expression of tumor necrosis factor-alpha (TNF), IL-1β, IL-10, and IL-13 were found in the PNS and CNS of naïve IL-4 ko mice. However, IL-1β gene expression was stronger in the sciatic nerve of IL-4 ko mice (p<0.001) 28 days after CCI and only IL-4 ko mice had elevated IL-10 gene expression (p = 0.014). Remarkably, CCI induced TNF (p<0.01), IL-1β (p<0.05), IL-10 (p<0.05), and IL-13 (p<0.001) gene expression exclusively in the ipsilateral spinal cord of IL-4 ko mice. The compensatory overexpression of the anti-inflammatory and analgesic cytokines IL-10 and IL-13 in the spinal cord of IL-4 ko mice may explain the lack of genotype differences for pain behavior after CCI. Additionally, CCI induced gene expression of μ, κ, and δ opioid receptors in the contralateral cortex and thalamus of IL-4 ko mice, paralleled by fast onset of morphine analgesia, but not in wt mice. We conclude that a lack of IL-4 leads to mechanical sensitivity; the compensatory hyperexpression of analgesic cytokines and opioid receptors after CCI, in turn, protects IL-4 ko mice from enhanced pain behavior after nerve lesion.
Background
Oncolytic viruses, including vaccinia virus (VACV), are a promising alternative to classical mono-cancer treatment methods such as surgery, chemo- or radiotherapy. However, combined therapeutic modalities may be more effective than mono-therapies. In this study, we enhanced the effectiveness of oncolytic virotherapy by matrix metalloproteinase (MMP-9)-mediated degradation of proteins of the tumoral extracellular matrix (ECM), leading to increased viral distribution within the tumors.
Methods
For this study, the oncolytic vaccinia virus GLV-1h255, containing the mmp-9 gene, was constructed and used to treat PC-3 tumor-bearing mice, achieving an intra-tumoral over-expression of MMP-9. The intra-tumoral MMP-9 content was quantified by immunohistochemistry in tumor sections. Therapeutic efficacy of GLV-1h255 was evaluated by monitoring tumor growth kinetics and intra-tumoral virus titers. Microenvironmental changes mediated by the intra-tumoral MMP-9 over-expression were investigated by microscopic quantification of the collagen IV content, the blood vessel density (BVD) and the analysis of lymph node metastasis formation.
Results
GLV-1h255-treatment of PC-3 tumors led to a significant over-expression of intra-tumoral MMP-9, accompanied by a marked decrease in collagen IV content in infected tumor areas, when compared to GLV-1h68-infected tumor areas. This led to considerably elevated virus titers in GLV-1h255 infected tumors, and to enhanced tumor regression. The analysis of the BVD, as well as the lumbar and renal lymph node volumes, revealed lower BVD and significantly smaller lymph nodes in both GLV-1h68- and GLV-1h255- injected mice compared to those injected with PBS, indicating that MMP-9 over-expression does not alter the metastasis-reducing effect of oncolytic VACV.
Conclusions
Taken together, these results indicate that a GLV-1h255-mediated intra-tumoral over-expression of MMP-9 leads to a degradation of collagen IV, facilitating intra-tumoral viral dissemination, and resulting in accelerated tumor regression. We propose that approaches which enhance the oncolytic effect by increasing the intra-tumoral viral load, may be an effective way to improve therapeutic outcome.
Physiological Notch Signaling Maintains Bone Homeostasis via RBPjk and Hey Upstream of NFATc1
(2012)
Notch signaling between neighboring cells controls many cell fate decisions in metazoans both during embryogenesis and in postnatal life. Previously, we uncovered a critical role for physiological Notch signaling in suppressing osteoblast differentiation in vivo. However, the contribution of individual Notch receptors and the downstream signaling mechanism have not been elucidated. Here we report that removal of Notch2, but not Notch1, from the embryonic limb mesenchyme markedly increased trabecular bone mass in adolescent mice. Deletion of the transcription factor RBPjk, a mediator of all canonical Notch signaling, in the mesenchymal progenitors but not the more mature osteoblast-lineage cells, caused a dramatic high-bone-mass phenotype characterized by increased osteoblast numbers, diminished bone marrow mesenchymal progenitor pool, and rapid age-dependent bone loss. Moreover, mice deficient in Hey1 and HeyL, two target genes of Notch-RBPjk signaling, exhibited high bone mass. Interestingly, Hey1 bound to and suppressed the NFATc1 promoter, and RBPjk deletion increased NFATc1 expression in bone. Finally, pharmacological inhibition of NFAT alleviated the high-bone-mass phenotype caused by RBPjk deletion. Thus, Notch-RBPjk signaling functions in part through Hey1-mediated inhibition of NFATc1 to suppress osteoblastogenesis, contributing to bone homeostasis in vivo.
We earlier established a model of a persistent viral CNS infection using two week old immunologically normal (genetically unmodified) mice and recombinant measles virus (MV). Using this model infection we investigated the role of regulatory T cells (Tregs) as regulators of the immune response in the brain, and assessed whether the persistent CNS infection can be modulated by manipulation of Tregs in the periphery. CD4\(^+\) CD25\(^+\) Foxp3\(^+\) Tregs were expanded or depleted during the persistent phase of the CNS infection, and the consequences for the virus-specific immune response and the extent of persistent infection were analyzed. Virus-specific CD8\(^+\) T cells predominantly recognising the H-2D(b)-presented viral hemagglutinin epitope MV-H22-30 (RIVINREHL) were quantified in the brain by pentamer staining. Expansion of Tregs after intraperitoneal (i.p.) application of the superagonistic anti-CD28 antibody D665 inducing transient immunosuppression caused increased virus replication and spread in the CNS. In contrast, depletion of Tregs using diphtheria toxin (DT) in DEREG (depletion of regulatory T cells)-mice induced an increase of virus-specific CD8\(^+\) effector T cells in the brain and caused a reduction of the persistent infection. These data indicate that manipulation of Tregs in the periphery can be utilized to regulate virus persistence in the CNS.
Background: The angiotensin II receptor subtype 2 (AT2 receptor) is ubiquitously and highly expressed in early postnatal life. However, its role in postnatal cardiac development remained unclear.
Methodology/Principal Findings: Hearts from 1, 7, 14 and 56 days old wild-type (WT) and AT2 receptor-deficient (KO) mice were extracted for histomorphometrical analysis as well as analysis of cardiac signaling and gene expression. Furthermore, heart and body weights of examined animals were recorded and echocardiographic analysis of cardiac function as well as telemetric blood pressure measurements were performed. Moreover, gene expression, sarcomere shortening and calcium transients were examined in ventricular cardiomyocytes isolated from both genotypes. KO mice exhibited an accelerated body weight gain and a reduced heart to body weight ratio as compared to WT mice in the postnatal period. However, in adult KO mice the heart to body weight ratio was significantly increased most likely due to elevated systemic blood pressure. At postnatal day 7 ventricular capillarization index and the density of \(\alpha\)-smooth muscle cell actin-positive blood vessels were higher in KO mice as compared to WT mice but normalized during adolescence. Echocardiographic assessment of cardiac systolic function at postnatal day 7 revealed decreased contractility of KO hearts in response to beta-adrenergic stimulation. Moreover, cardiomyocytes from KO mice showed a decreased sarcomere shortening and an increased peak Ca\(^{2+}\) transient in response to isoprenaline when stimulated concomitantly with angiotensin II.
Conclusion: The AT2 receptor affects postnatal cardiac growth possibly via reducing body weight gain and systemic blood pressure. Moreover, it moderately attenuates postnatal vascularization of the heart and modulates the beta adrenergic response of the neonatal heart. These AT2 receptor-mediated effects may be implicated in the physiological maturation process of the heart.
Parent of origin imprints on the genome have been implicated in the regulation of neural cell type differentiation. The ability of human parthenogenetic (PG) embryonic stem cells (hpESCs) to undergo neural lineage and cell type-specific differentiation is undefined. We determined the potential of hpESCs to differentiate into various neural subtypes. Concurrently, we examined DNA methylation and expression status of imprinted genes. Under culture conditions promoting neural differentiation, hpESC-derived neural stem cells (hpNSCs) gave rise to glia and neuron-like cells that expressed subtype-specific markers and generated action potentials. Analysis of imprinting in hpESCs and in hpNSCs revealed that maternal-specific gene expression patterns and imprinting marks were generally maintained in PG cells upon differentiation. Our results demonstrate that despite the lack of a paternal genome, hpESCs generate proliferating NSCs that are capable of differentiation into physiologically functional neuron-like cells and maintain allele-specific expression of imprinted genes. Thus, hpESCs can serve as a model to study the role of maternal and paternal genomes in neural development and to better understand imprinting-associated brain diseases.
Four molecules of the tumor suppressor p53 assemble to cooperatively bind proapoptotic target genes. The structural basis for cooperativity consists of interactions between adjacent DNA binding domains. Mutations at the interaction interface that compromise cooperativity were identified in cancer patients, suggesting a requirement of cooperativity for tumor suppression. We report on an analysis of cooperativity mutant p53(E177R) mice. Apoptotic functions of p53 triggered by DNA damage and oncogenes were abolished in these mice, whereas functions in cell-cycle control, senescence, metabolism, and antioxidant defense were retained and were sufficient to suppress development of spontaneous T cell lymphoma. Cooperativity mutant mice are nevertheless highly cancer prone and susceptible to different oncogene-induced tumors. Our data underscore the relevance of DNA binding cooperativity for p53-dependent apoptosis and tumor suppression and highlight cooperativity mutations as a class of p53 mutations that result in a selective loss of apoptotic functions due to an altered quaternary structure of the p53 tetramer.
Desmosomes provide intercellular adhesive strength required for integrity of epithelial and some non-epithelial tissues. Within the epidermis, the cadherin-type adhesion molecules desmoglein (Dsg) 1-4 and desmocollin (Dsc) 1-3 build the adhesive core of desmosomes. In keratinocytes, several isoforms of these proteins are co-expressed. However, the contribution of specific isoforms to overall cell cohesion is unclear. Therefore, in this study we investigated the roles of Dsg2 and Dsg3, the latter of which is known to be essential for keratinocyte adhesion based on its autoantibody-induced loss of function in the autoimmune blistering skin disease pemphigus vulgaris (PV). The pathogenic PV antibody AK23, targeting the Dsg3 adhesive domain, led to profound loss of cell cohesion in human keratinocytes as revealed by the dispase-based dissociation assays. In contrast, an antibody against Dsg2 had no effect on cell cohesion although the Dsg2 antibody was demonstrated to interfere with Dsg2 transinteraction by single molecule atomic force microscopy and was effective to reduce cell cohesion in intestinal epithelial Caco-2 cells which express Dsg2 as the only Dsg isoform. To substantiate these findings, siRNA-mediated silencing of Dsg2 or Dsg3 was performed in keratinocytes. In contrast to Dsg3-depleted cells, Dsg2 knockdown reduced cell cohesion only under conditions of increased shear. These experiments indicate that specific desmosomal cadherins contribute differently to keratinocyte cohesion and that Dsg2 compared to Dsg3 is less important in this context.
The purpose of this study was to evaluate whether spatial hippocampus-dependent learning is affected by the serotonergic system and stress. Therefore, 5-HTT knockout (-/-), heterozygous (+/-) and wildtype (+/+) mice were subjected to the Barnes maze (BM) and the Morris water maze (WM), the latter being discussed as more aversive. Additionally, immediate early gene (IEG) expression, hippocampal adult neurogenesis (aN), and blood plasma corticosterone were analyzed.
While the performance of 5-HTT-/- mice in the BM was undistinguishable from both other genotypes, they performed worse in the WM. However, in the course of the repeated WM trials 5-HTT-/- mice advanced to wildtype level. The experience of a single trial of either the WM or the BM resulted in increased plasma corticosterone levels in all genotypes. After several trials 5-HTT-/- mice exhibited higher corticosterone concentrations compared with both other genotypes in both tests. Corticosterone levels were highest in 5-HTT-/- mice tested in the WM indicating greater aversiveness of the WM and a greater stress sensitivity of 5-HTT deficient mice.
Quantitative immunohistochemistry in the hippocampus revealed increased cell counts positive for the IEG products cFos and Arc as well as for proliferation marker Ki67 and immature neuron marker NeuroD in 5-HTT-/- mice compared to 5-HTT+/+ mice, irrespective of the test. Most differences were found in the suprapyramidal blade of the dentate gyrus of the septal hippocampus. Ki67-immunohistochemistry revealed a genotype x environment interaction with 5-HTT genotype differences in naïve controls and WM experience exclusively yielding more Ki67-positive cells in 5-HTT+/+ mice. Moreover, in 5-HTT-/- mice we demonstrate that learning performance correlates with the extent of aN.
Overall, higher baseline IEG expression and increased an in the hippocampus of 5-HTT-/- mice together with increased stress sensitivity may constitute the neurobiological correlate of raised alertness, possibly impeding optimal learning performance in the more stressful WM.