The association of Antarctic krill Euphausia superba with the under-ice habitat

The association of Antarctic krill Euphausia superba with the under-ice habitat was investigated in the Lazarev Sea (Southern Ocean) during austral summer, autumn and winter. Data were obtained using novel Surface and Under Ice Trawls (SUIT), which sampled the 0-2 m surface layer both under sea ice and in open water. Average surface layer densities ranged between 0.8 individuals m(-2) in summer and autumn, and 2.7 individuals m(-2) in winter. In summer, under-ice densities of Antarctic krill were significantly higher than in open waters. In autumn, the opposite pattern was observed. Under winter sea ice, densities were often low, but repeatedly far exceeded summer and autumn maxima. Statistical models showed that during summer high densities of Antarctic krill in the 0-2 m layer were associated with high ice coverage and shallow mixed layer depths, among other factors. In autumn and winter, density was related to hydrographical parameters. Average under-ice densities from the 0-2 m layer were higher than corresponding values from the 0-200 m layer collected with Rectangular Midwater Trawls (RMT) in summer. In winter, under-ice densities far surpassed maximum 0-200 m densities on several occasions. This indicates that the importance of the ice-water interface layer may be under-estimated by the pelagic nets and sonars commonly used to estimate the population size of Antarctic krill for management purposes, due to their limited ability to sample this habitat. Our results provide evidence for an almost year-round association of Antarctic krill with the under-ice habitat, hundreds of kilometres into the ice-covered area of the Lazarev Sea. Local concentrations of postlarval Antarctic krill under winter sea ice suggest that sea ice biota are important for their winter survival. These findings emphasise the susceptibility of an ecological key species to changing sea ice habitats, suggesting potential ramifications on Antarctic ecosystems induced by climate change.     

Induction of TRAIL- and TNF-alpha-dependent apoptosis in human monocyte-derived dendritic cells by microfilariae of Brugia malayi

Dysregulation of professional APC has been postulated as a major mechanism underlying Ag-specific T cell hyporesponsiveness in patients with patent filarial infection. To address the nature of this dysregulation, dendritic cells (DC) and macrophages generated from elutriated monocytes were exposed to live microfilariae (mf), the parasite stage that circulates in blood and is responsible for most immune dysregulation in filarial infections. DC exposed to mf for 24-96 h showed a marked increase in cell death and caspase-positive cells compared with unexposed DC, whereas mf exposure did not induce apoptosis in macrophages. Interestingly, 48-h exposure of DC to mf induced mRNA expression of the proapoptotic gene TRAIL and both mRNA and protein expression of TNF-alpha. mAb to TRAIL-R2, TNF-R1, or TNF-alpha partially reversed mf-induced cell death in DC, as did knocking down the receptor for TRAIL-R2 using small interfering RNA. The mf also induced gene expression of BH3-interacting domain death agonist and protein expression of cytochrome c in DC; mf-induced cleavage of BH3-interacting domain death agonist could be shown to induce release of cytochrome c, leading to activation of caspase 9. Our data suggest that mf induce DC apoptosis in a TRAIL- and TNF-alpha-dependent fashion.     

Identification of functional roles for both IL-17RB and IL-17RA in mediating IL-25-induced activities

IL-25 (IL-17E) is a unique IL-17 family ligand that promotes Th2-skewed inflammatory responses. Intranasal administration of IL-25 into naive mice induces pulmonary inflammation similar to that seen in patients with allergic asthma, including increases in bronchoalveolar lavage fluid eosinophils, bronchoalveolar lavage fluid IL-5 and IL-13 concentrations, goblet cell hyperplasia, and increased airway hyperresponsiveness. IL-25 has been reported to bind and signal through IL-17RB (IL-17BR, IL-17Rh1). It has been demonstrated recently that IL-17A signals through a heteromeric receptor composed of IL-17RA and IL-17RC. We sought to determine whether other IL-17 family ligands also utilize heteromeric receptor complexes. The required receptor subunits for IL-25 biological activities were investigated in vitro and in vivo using a combination of knockout (KO) mice and antagonistic Abs. Unlike wild-type mice, cultured splenocytes from either IL-17RB KO or IL-17RA KO mice did not produce IL-5 or IL-13 in response to IL-25 stimulation, and both IL-17RB KO and IL-17RA KO mice did not respond to intranasal administration of IL-25. Furthermore, treatment with antagonistic mAbs to either IL-17RB or IL-17RA completely blocked IL-25-induced pulmonary inflammation and airway hyperresponsiveness in naive BALB/c mice, similar to the effects of an antagonistic Ab to IL-25. Finally, a blocking Ab to human IL-17RA prevented IL-25 activity in a primary human cell-based assay. These data demonstrate for the first time that IL-25-mediated activities require both IL-17RB and IL-17RA and provide another example of an IL-17 family ligand that utilizes a heteromeric receptor complex.     

Human papillomavirus type 16 and 18 E7-pulsed dendritic cell vaccination of stage IB or IIA cervical cancer patients: a phase I escalating-dose trial

The safety and immunogenicity of the human papillomavirus type 16 (HPV16) or HPV18 (HPV16/18) E7 antigen-pulsed mature dendritic cell (DC) vaccination were evaluated for patients with stage IB or IIA cervical cancer. Escalating doses of autologous DC (5, 10, and 15 × 10⁶ cells for injection) were pulsed with recombinant HPV16/18 E7 antigens and keyhole limpet hemocyanin (KLH; an immunological tracer molecule) and delivered in five subcutaneous injections at 21-day intervals to 10 cervical cancer patients with no evidence of disease after they underwent radical surgery. Safety, toxicity, delayed-type hypersensitivity (DTH) reaction, and induction of serological and cellular immunity against HPV16/18 E7 and KLH were monitored. DC vaccination was well tolerated, and no significant toxicities were recorded. All patients developed CD4⁺ T-cell and antibody responses to DC vaccination, as detected by enzyme-linked immunosorbent spot (ELISpot) and enzyme-linked immunosorbent assays (ELISA), respectively, and 8 out of 10 patients demonstrated levels of E7-specific CD8⁺ T-cell counts, detected by ELISpot during or immediately after immunization, that were increased compared to prevaccination baseline levels. The vaccine dose did not predict the magnitude of the antibody or T-cell response or the time to detection of HPV16/18 E7-specific immunity. DTH responses to intradermal injections of HPV E7 antigen and KLH were detected for all patients after vaccination. We conclude that HPV E7-loaded DC vaccination is safe and immunogenic for stage IB or IIA cervical cancer patients. Phase II E7-pulsed DC-based vaccination trials with cervical cancer patients harboring a limited tumor burden, or who are at significant risk of tumor recurrence, are warranted.     

Systematic mutagenesis of the murine gammaherpesvirus 68 M2 protein identifies domains important for chronic infection

Murine gammaherpesvirus 68 (MHV68) infection of inbred mice represents a genetically tractable small-animal model for assessing the requirements for the establishment of latency, as well as reactivation from latency, within the lymphoid compartment. By day 16 postinfection, MHV68 latency in the spleen is found in B cells, dendritic cells, and macrophages. However, as with Epstein-Barr virus, by 3 months postinfection MHV68 latency is predominantly found in isotype-switched memory B cells. The MHV68 M2 gene product is a latency-associated antigen with no discernible homology to any known cellular or viral proteins. However, depending on experimental conditions, the M2 protein has been shown to play a critical role in both the efficient establishment of latency in splenic B cells and reactivation from latently infected splenic B cells. Inspection of the sequence of the M2 protein reveals several hallmarks of a signaling molecule, including multiple PXXP motifs and two potential tyrosine phosphorylation sites. Here, we report the generation of a panel of recombinant MHV68 viruses harboring mutations in the M2 gene that disrupt putative functional motifs. Subsequent analyses of the panel of M2 mutant viruses revealed a functionally important cluster of PXXP motifs in the C-terminal region of M2, which have previously been implicated in binding Vav proteins (P. A. Madureira, P. Matos, I. Soeiro, L. K. Dixon, J. P. Simas, and E. W. Lam, J. Biol. Chem. 280:37310-37318, 2005; L. Rodrigues, M. Pires de Miranda, M. J. Caloca, X. R. Bustelo, and J. P. Simas, J. Virol. 80:6123-6135, 2006). Further characterization of two adjacent PXXP motifs in the C terminus of the M2 protein revealed differences in the functions of these domains in M2-driven expansion of primary murine B cells in culture. Finally, we show that tyrosine residues 120 and 129 play a critical role in both the establishment of splenic latency and reactivation from latency upon explant of splenocytes into tissue culture. Taken together, these analyses will aide future studies for identifying M2 interacting partners and B-cell signaling pathways that are manipulated by the M2 protein.     

Optical measurement of mechanical forces inside short DNA loops

Knowledge of the mechanical properties of double-stranded DNA (dsDNA) is essential to understand the role of dsDNA looping in gene regulation and the mechanochemistry of molecular machines that operate on dsDNA. Here, we use a newly developed tool, force sensors with optical readout, to measure the forces inside short, strained loops composed of both dsDNA and single-stranded DNA. By varying the length of the loops and their proportion of dsDNA, it was possible to vary their internal forces from 1 pN to >20 pN. Surprisingly, internal loop forces changed erratically as the amount of dsDNA was increased for a given loop length, with the effect most notable in the smallest loop (57 nucleotides). Monte Carlo simulations based on the helical wormlike chain model accurately predict internal forces when more than half of the loop is dsDNA but fail otherwise. Mismatches engineered into the double-stranded regions increased flexibility, suggesting that Watson-Crick basepaired dsDNA can withstand high compressive forces without recourse to multibase melts. Fluorescence correlation spectroscopy further excluded transient melting (microsecond to millisecond duration) as a mechanism for relief of compressive forces in the tested dsDNAs. DNA loops with integrated force sensors may allow the comprehensive mapping of the elasticity of short dsDNAs as a function of both sequence and salt.     

Immunolocalization of MMP-19 in the human intervertebral disc: implications for disc aging and degeneration

Matrix metalloproteinases (MMPs) degrade components of the extracellular matrix of the disc, but the presence of MMP-19 has not been explored. In other tissues, MMP-19 is known to act in proteolysis of the insulin-like growth factor (IGF) binding protein-3, thereby exposing this protein to make it available to influence cell behavior. MMP-19 also has been shown to inhibit capillary-like formation and thus play a role in the avascular nature of the disc. Using immunohistochemistry, normal discs from six subjects aged newborn through 10 years and 20 disc specimens from control donors or surgical patients aged 15-76 (mean age 40.2 years) were examined for immunolocalization of MMP-19; six Thompson grade I discs, five Thompson grade II, eight Thompson grade III, five Thompson grade IV, and one Thompson grade V discs were analyzed. The results indicate that in discs from young subjects, MMP-19 was uniformly localized in the outer annulus. In discs from adult donors and surgical patients, outer and inner annulus cells only occasionally showed MMP-19 localization. The greatest expression of MMP-19 was observed in young discs, and little expression was seen in older or degenerating discs. Because MMP-19 has been shown to regulate IGF-mediated proliferation in other tissues, its decline in the aging/degenerating disc may contribute to the age-related decrease in disc cell numbers.     

Protection against Tetanus by Needle-Free Inoculation of Adenovirus-Vectored Nasal and Epicutaneous Vaccines

The effectiveness of vaccination programs would be enhanced greatly through the availability of vaccines that can be administered simply and, preferably, painlessly without the need for timed booster injections. Tetanus is a prime example of a disease that is readily preventable by vaccination but remains a major threat to public health due to the problems associated with administration of the present vaccine. Here we show that a protective immune response against live Clostridium tetani infection in mice can be elicited by an adenovirus vector encoding the tetanus toxin C fragment when administered as a nasal or epicutaneous vaccine. The results suggest that these vaccination modalities would be effective needle-free alternatives. This is the first demonstration that absorption of a small number of vectored vaccines into the skin following topical application of a patch can provide protection against live bacteria in a disease setting.