A tetravalent dengue nanoparticle stimulates antibody production in mice

Background

Dengue is a major public health problem worldwide, especially in the tropical and subtropical regions of the world. Infection with a single Dengue virus (DENV) serotype causes a mild, self-limiting febrile illness called dengue fever. However, a subset of patients experiencing secondary infection with a different serotype progresses to the severe form of the disease, dengue hemorrhagic fever/dengue shock syndrome. Currently, there are no licensed vaccines or antiviral drugs to prevent or treat dengue infections. Biodegradable nanoparticles coated with proteins represent a promising method for in vivo delivery of vaccines.

Findings

Here, we used a murine model to evaluate the IgG production after administration of inactivated DENV corresponding to all four serotypes adsorbed to bovine serum albumin nanoparticles. This formulation induced a production of anti-DENV IgG antibodies (p < 0.001). However, plaque reduction neutralization assays with the four DENV serotypes revealed that these antibodies have no neutralizing activity in the dilutions tested.

Conclusions

Our results show that while the nanoparticle system induces humoral responses against DENV, further investigation with different DENV antigens will be required to improve immunogenicity, epitope specicity, and functional activity to make this platform a viable option for DENV vaccines.     

Protease-activated Receptor 2 (PAR2) Protein and Transient Receptor Potential Vanilloid 4 (TRPV4) Protein Coupling Is Required for Sustained Inflammatory Signaling

G protein-coupled receptors of nociceptive neurons can sensitize transient receptor potential (TRP) ion channels, which amplify neurogenic inflammation and pain. Protease-activated receptor 2 (PAR₂), a receptor for inflammatory proteases, is a major mediator of neurogenic inflammation and pain. We investigated the signaling mechanisms by which PAR₂ regulates TRPV4 and determined the importance of tyrosine phosphorylation in this process. Human TRPV4 was expressed in HEK293 cells under control of a tetracycline-inducible promoter, allowing controlled and graded channel expression. In cells lacking TRPV4, the PAR₂ agonist stimulated a transient increase in [Ca²⁺]_i. TRPV4 expression led to a markedly sustained increase in [Ca²⁺]_i. Removal of extracellular Ca²⁺ and treatment with the TRPV4 antagonists Ruthenium Red or HC067047 prevented the sustained response. Inhibitors of phospholipase A₂ and cytochrome P450 epoxygenase attenuated the sustained response, suggesting that PAR₂ generates arachidonic acid-derived lipid mediators, such as 5′,6′-EET, that activate TRPV4. Src inhibitor 1 suppressed PAR₂-induced activation of TRPV4, indicating the importance of tyrosine phosphorylation. The TRPV4 tyrosine mutants Y110F, Y805F, and Y110F/Y805F were expressed normally at the cell surface. However, PAR₂ was unable to activate TRPV4 with the Y110F mutation. TRPV4 antagonism suppressed PAR₂ signaling to primary nociceptive neurons, and TRPV4 deletion attenuated PAR₂-stimulated neurogenic inflammation. Thus, PAR₂ activation generates a signal that induces sustained activation of TRPV4, which requires a key tyrosine residue (TRPV4-Tyr-110). This mechanism partly mediates the proinflammatory actions of PAR₂.     

TRPV1 induction in airway vagal low-threshold mechanosensory neurons by allergen challenge and neurotrophic factors

We addressed the hypothesis that allergic inflammation in guinea pig airways leads to a phenotypic switch in vagal tracheal cough-causing, low-threshold mechanosensitive Aδ neurons, such that they begin expressing functional transient receptor potential vanilloid (TRPV1) channels. Guinea pigs were actively sensitized to ovalbumin (OVA) and beginning 21 days later exposed via aerosol to OVA daily for 3 days. Tracheal-specific neurons were identified in the nodose ganglion using retrograde tracing techniques. Tracheal specific neurons were isolated, and mRNA expression was evaluated at the single-neuron level using RT-PCR analysis. Electrophysiological studies have revealed that the vast majority of vagal nodose afferent nerves innervating the trachea are capsaicin-insensitive Aδ-fibers. Consistent with this, we found <20% of these neurons express TRPV1 mRNA or respond to capsaicin in a calcium assay. Allergen exposure induced de novo TRPV1 mRNA in a majority of the tracheal-specific nodose neurons (P < 0.05). The allergen-induced TRPV1 induction was mimicked by applying either brain-derived neurotrophic factor (BDNF) or glial-derived neurotrophic factor (GDNF) to the tracheal lumen. The BDNF-induced phenotypic change observed at the level of mRNA expression was mimicked using a calcium assay to assess functional TRPV1 ion channels. Finally, OVA exposure induced BDNF and GDNF production in the tracheal epithelium, the immediate vicinity of the nodose Aδ -fibers terminations. The induction of TRPV1 in nodose tracheal Aδ -fibers would substantively expand the nature of stimuli capable of activating these cough-causing nerves.     

Impaired adult myeloid progenitor CMP and GMP cell function in conditional c-myb-knockout mice

The differentiation of myeloid progenitors to mature, terminally differentiated cells is a highly regulated process. Here, we showed that conditional disruption of the c-myb proto-oncogene in adult mice resulted in dramatic reductions in CMP, GMP and MEP myeloid progenitors, leading to a reduction of neutrophils, basophils, monocytes and platelets in peripheral blood. In addition, c-myb plays a critical role at multiple stages of myeloid development, from multipotent CMP and bipotent GMP to unipotent CFU-G and CFU-M progenitor cells. c-myb controls the differentiation of these cells and is required for the proper commitment, maturation and normal differentiation of CMPs and GMPs. Specifically, c-myb regulates the precise commitment to the megakaryocytic and granulo-monocytic pathways and governs the granulocytic-monocytic lineage choice. c-myb is also required for the commitment along the granulocytic pathway for early myeloid progenitor cells and for the maturation of committed precursor cells along this pathway. On the other hand, disruption of the c-myb gene favors the commitment to the monocytic lineage, although monocytic development was abnormal with cells appearing more mature with atypical CD41 surface markers. These results demonstrate that c-myb plays a pivotal role in the regulation of multiple stages in adult myelogenesis.     

The trans-Golgi network golgin, GCC185, is required for endosome-to-Golgi transport and maintenance of Golgi structure

Four mammalian golgins are specifically targeted to the trans-Golgi network (TGN) membranes via their C-terminal GRIP domains. The TGN golgins, p230/golgin-245 and golgin-97, are recruited via the GTPase Arl1, whereas the TGN golgin GCC185 is recruited independently of Arl1. Here we show that GCC185 is localized to a region of the TGN distinct from Arl1 and plays an essential role in maintaining the organization of the Golgi apparatus. Using both small interfering RNA (siRNA) and microRNA (miRNA), we show that depletion of GCC185 in HeLa cells frequently resulted in fragmentation of the Golgi apparatus. Golgi apparatus fragments were dispersed throughout the cytoplasm and contained both cis and trans markers. Trafficking of anterograde and retrograde cargo was analysed over an extended period following GCC185 depletion. Early effects of GCC185 depletion included a perturbation in the distribution of the mannose-6-phosphate receptor and a block in shiga toxin trafficking to the Golgi apparatus, which occurred in parallel with the fragmentation of the Golgi ribbon. Internalized shiga toxin accumulated in Rab11-positive endosomes, indicating GCC185 is essential for transport between the recycling endosome and the TGN. In contrast, the plasma membrane-TGN recycling protein TGN38 was efficiently transported into GCC185-depleted Golgi apparatus fragments throughout a 96-h period, and anterograde transport of E-cadherin was functional until a late stage of GCC185 depletion. This study demonstrated (i) a more effective long-term depletion of GCC185 using miRNA than siRNA and (ii) a dual role for the GCC185 golgin in the regulation of endosome-to-TGN membrane transport and in the organization of the Golgi apparatus.     

Role of matrix metalloproteinase 13 in both endochondral and intramembranous ossification during skeletal regeneration

Extracellular matrix (ECM) remodeling is important during bone development and repair. Because matrix metalloproteinase 13 (MMP13, collagenase-3) plays a role in long bone development, we have examined its role during adult skeletal repair. In this study we find that MMP13 is expressed by hypertrophic chondrocytes and osteoblasts in the fracture callus. We demonstrate that MMP13 is required for proper resorption of hypertrophic cartilage and for normal bone remodeling during non-stabilized fracture healing, which occurs via endochondral ossification. However, no difference in callus strength was detected in the absence of MMP13. Transplant of wild-type bone marrow, which reconstitutes cells only of the hematopoietic lineage, did not rescue the endochondral repair defect, indicating that impaired healing in Mmp13-/- mice is intrinsic to cartilage and bone. Mmp13-/- mice also exhibited altered bone remodeling during healing of stabilized fractures and cortical defects via intramembranous ossification. This indicates that the bone phenotype occurs independently from the cartilage phenotype. Taken together, our findings demonstrate that MMP13 is involved in normal remodeling of bone and cartilage during adult skeletal repair, and that MMP13 may act directly in the initial stages of ECM degradation in these tissues prior to invasion of blood vessels and osteoclasts.     

Aging and respiratory muscle metabolic plasticity: effects of endurance training.

We examined the oxidative and antioxidant enzyme activities in respiratory and locomotor muscles in response to endurance training in young and aging rats. Young adult (4-mo-old) and old (24-mo-old) female Fischer 344 rats were divided into four groups: 1) young trained (n = 12), 2) young untrained (n = 12), 3) old trained (n = 10), and 4) old untrained (n = 6). Both young and old endurance-trained animals performed the same training protocol during 10 wk of continuous treadmill exercise (60 min/day, 5 days/wk). Compared with young untrained animals, the young trained group had significantly elevated (P less than 0.05) activities of 3-hydroxyacyl-CoA dehydrogenase (HADH), glutathione peroxidase (GPX), and citrate synthase (CS) in both the costal diaphragm and the plantaris muscle. In contrast, training had no influence (P greater than 0.05) on the activity of lactate dehydrogenase within the costal diaphragm in young animals. In the aging animals, training did not alter (P greater than 0.05) activities of CS, HADH, GPX, or lactate dehydrogenase in the costal diaphragm but significantly (P less than 0.05) increased CS, HADH, and GPX activities in the plantaris muscle. Furthermore, training resulted in higher activities of CS and HADH in the intercostal muscles in the old trained than in the old untrained animals. Finally, activities of CS, HADH, and GPX were significantly (P less than 0.05) lower in the plantaris in the old untrained than in the young untrained animals; however, CS, HADH, and GPX activities were greater (P less than 0.05) in the costal diaphragm in the old sedentary than in the young untrained animals.(ABSTRACT TRUNCATED AT 250 WORDS)