Centrosome- and cilia-associated proteins play crucial roles in establishing polarity and regulating intracellular transport in post-mitotic cells. Using genetic mapping and positional candidate strategy, we have identified an in-frame deletion in a novel centrosomal protein CEP290 (also called NPHP6), leading to early-onset retinal degeneration in a newly identified mouse mutant, rd16. We demonstrate that CEP290 localizes primarily to centrosomes of dividing cells and to the connecting cilium of retinal photoreceptors. We show that, in the retina, CEP290 associates with several microtubule-based transport proteins including RPGR, which is mutated in approximately 15% of patients with retinitis pigmentosa. A truncated CEP290 protein (DeltaCEP290) is detected in the rd16 retina, but in considerably reduced amounts; however, the mutant protein exhibits stronger association with specific RPGR isoform(s). Immunogold labeling studies demonstrate the redistribution of RPGR and of phototransduction proteins in the photoreceptors of rd16 retina. Our findings suggest a critical function for CEP290 in ciliary transport and provide insights into the mechanism of early-onset photoreceptor degeneration.
Summary There is pressing need to develop alternatives to annual influenza vaccines and antiviral agents licensed for mitigating influenza infection. Previous studies reported that acute lung injury (ALI) caused by chemical or microbial insults is secondary to generation of host-derived, oxidized phospholipid that potently stimulates Toll-like Receptor 4 (TLR4)-dependent inflammation1. Subsequently, we reported that TLR4−/− mice are highly refractory to influenza-induced lethality2, and hypothesized that therapeutic antagonism of TLR4 signaling would protect against influenza-induced ALI. Herein, we report that therapeutic administration of Eritoran (E5564), a potent, well-tolerated, synthetic TLR4 antagonist3,4, blocks influenza-induced lethality in mice, as well as lung pathology, clinical symptoms, cytokine and oxidized phospholipid expression, and decreases viral titers. CD14 and TLR2 are also required for Eritoran-mediated protection, and CD14 directly binds Eritoran and inhibits ligand binding to MD2. Thus, Eritoran blockade of TLR signaling represents a novel therapeutic approach for inflammation associated with influenza, and possibly other, infections.
The insect immune deficiency (IMD) pathway resembles the tumour necrosis factor receptor network in mammals and senses diaminopimelic-type peptidoglycans present in Gram-negative bacteria. Whether unidentified chemical moieties activate the IMD signalling cascade remains unknown. Here, we show that infection-derived lipids 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoglycerol (POPG) and 1-palmitoyl-2-oleoyl diacylglycerol (PODAG) stimulate the IMD pathway of ticks. The tick IMD network protects against colonization by three distinct bacteria, that is the Lyme disease spirochete Borrelia burgdorferi and the rickettsial agents Anaplasma phagocytophilum and A. marginale. Cell signalling ensues in the absence of transmembrane peptidoglycan recognition proteins and the adaptor molecules Fas-associated protein with a death domain (FADD) and IMD. Conversely, biochemical interactions occur between x-linked inhibitor of apoptosis protein (XIAP), an E3 ubiquitin ligase, and the E2 conjugating enzyme Bendless. We propose the existence of two functionally distinct IMD networks, one in insects and another in ticks.
Maintenance of membrane function is essential and regulated at the genomic, transcriptional, and translational levels. Bacterial pathogens have a variety of mechanisms to adapt their membrane in response to transmission between environment, vector, and human host. Using a well-characterized model of lipid A diversification ( Francisella ), we demonstrate temperature-regulated membrane remodeling directed by multiple alleles of the lipid A-modifying N -acyltransferase enzyme, LpxD. Structural analysis of the lipid A at environmental and host temperatures revealed that the LpxD1 enzyme added a 3-OH C18 acyl group at 37 °C (host), whereas the LpxD2 enzyme added a 3-OH C16 acyl group at 18 °C (environment). Mutational analysis of either of the individual Francisella lpxD genes altered outer membrane (OM) permeability, antimicrobial peptide, and antibiotic susceptibility, whereas only the lpxD1 -null mutant was attenuated in mice and subsequently exhibited protection against a lethal WT challenge. Additionally, growth-temperature analysis revealed transcriptional control of the lpxD genes and posttranslational control of the LpxD1 and LpxD2 enzymatic activities. These results suggest a direct mechanism for LPS/lipid A-level modifications resulting in alterations of membrane fluidity, as well as integrity and may represent a general paradigm for bacterial membrane adaptation and virulence-state adaptation.
BackgroundHepatitis B virus infection remains an important global health concern despite the availability of safe and effective prophylactic vaccines. Limitations to these vaccines include requirement for refrigeration and three immunizations thereby restricting use in the developing world. A new nasal hepatitis B vaccine composed of recombinant hepatitis B surface antigen (HBsAg) in a novel nanoemulsion (NE) adjuvant (HBsAg-NE) could be effective with fewer administrations.Methodology and Principal FindingsPhysical characterization indicated that HBsAg-NE consists of uniform lipid droplets (349+/−17 nm) associated with HBsAg through electrostatic and hydrophobic interactions. Immunogenicity of HBsAg-NE vaccine was evaluated in mice, rats and guinea pigs. Animals immunized intranasally developed robust and sustained systemic IgG, mucosal IgA and strong antigen-specific cellular immune responses. Serum IgG reached ≥106 titers and was comparable to intramuscular vaccination with alum-adjuvanted vaccine (HBsAg-Alu). Normalization showed that HBsAg-NE vaccination correlates with a protective immunity equivalent or greater than 1000 IU/ml. Th1 polarized immune response was indicated by IFN-γ and TNF-α cytokine production and elevated levels of IgG2 subclass of HBsAg-specific antibodies. The vaccine retains full immunogenicity for a year at 4°C, 6 months at 25°C and 6 weeks at 40°C. Comprehensive pre-clinical toxicology evaluation demonstrated that HBsAg-NE vaccine is safe and well tolerated in multiple animal models.ConclusionsOur results suggest that needle-free nasal immunization with HBsAg-NE could be a safe and effective hepatitis B vaccine, or provide an alternative booster administration for the parenteral hepatitis B vaccines. This vaccine induces a Th1 associated cellular immunity and also may provide therapeutic benefit to patients with chronic hepatitis B infection who lack cellular immune responses to adequately control viral replication. Long-term stability of this vaccine formulation at elevated temperatures suggests a direct advantage in the field, since potential excursions from cold chain maintenance could be tolerated without a loss in therapeutic efficacy.
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